Targeted disruption of either c-Src or TNFR-associated factor 6 (TRAF6) in mice causes osteoclast dysfunction and an osteopetrotic phenotype, suggesting that both molecules play important roles in osteoclastic bone resorption. We previously demonstrated that IL-1 induces actin ring formation and osteoclast activation. In this study, we examined the relationship between IL-1/TRAF6-dependent and c-Src-mediated pathways in the activation of osteoclast-like cells (prefusion cells (pOCs); multinucleated cells) formed in the murine coculture system. In normal pOCs, IL-1 induces actin ring formation and tyrosine phosphorylation of p130Cas, a known substrate of c-Src. However, in Src-deficient pOCs, p130Cas was not tyrosine phosphorylated following IL-1 treatment. In normal pOCs treated with IL-1, anti-TRAF6 Abs coprecipitate p130Cas, protein tyrosine kinase 2, and c-Src. In Src-deficient pOCs, this molecular complex was not detected, suggesting that c-Src is required for formation of the TRAF6, p130Cas, and protein tyrosine kinase 2 complex. Moreover, an immunocytochemical analysis revealed that in osteoclast-like multinucleated cells, IL-1 induced redistribution of TRAF6 to actin ring structures formed at the cell periphery, where TRAF6 also colocalized with c-Src. Taken together, these data suggest that IL-1 signals feed into the tyrosine kinase pathways through a TRAF6-Src molecular complex, which regulates the cytoskeletal reorganization essential for osteoclast activation.

Interleukin-1 is a multifunctional cytokine that regulates various cellular and tissue functions (1). Bone is one of the most sensitive tissues to IL-1 (2, 3, 4). IL-1 exhibits potent bone-resorbing activity in vivo (3) and in vitro (2, 4), and the natural inhibitory protein, IL-1R antagonist, blocks the ability of IL-1 to stimulate bone resorption in organ culture (5) and in ovariectomized rats in vivo (6). Several lines of evidence suggest that IL-1 is also involved in the bone destruction associated with multiple myeloma, rheumatoid arthritis, and osteoporosis (1).

Osteoclasts, the bone-resorbing cells, are macrophage-related multinucleated cells that play a critical role in bone remodeling (7, 8). Osteoclastic bone resorption consists of multiple steps: proliferation of osteoclast progenitors, differentiation of progenitors into mononuclear prefusion osteoclasts (pOCs),3 fusion of pOCs into osteoclast-like multinucleated cells (OCLs), sealing zone (actin ring) and ruffled border formation, the active resorption, and eventually apoptosis. In vitro evidence indicates that IL-1 participates in the following steps: 1) IL-1 stimulates osteoclast formation indirectly by stimulating PGE2 synthesis in osteoblasts/stromal cells (9); 2) IL-1 induces fusion of mononuclear osteoclasts, leading to multinucleation (10); 3) IL-1 potentiates osteoclast function (pit-forming activity) directly (10) or indirectly via osteoblasts (11); 4) and finally, IL-1 is directly involved in prolonging osteoclast life span (12, 13, 14).

The objective of this study was to investigate the intracellular signaling involved in osteoclast activation by IL-1. After IL-1 binding, the IL-1R-associated kinase (IRAK), a serine/threonine kinase, becomes autophosphorylated and is recruited to the receptor complex by binding to MyD88. Another adapter, TNFR-associated factor 6 (TRAF6), then interacts with IRAK (15). Interestingly, targeted disruption of TRAF6 in mice results in osteoclast dysfunction and in osteopetrotic phenotype (16), which is similar to that in c-Src-deficient mice (17, 18), suggesting that both molecules play important roles in osteoclastic bone resorption. In this study, we examined the relationship between IL-1/TRAF6-dependent and c-Src-mediated pathways in osteoclast-like cells derived from the in vitro coculture system, and showed that IL-1 cross-regulates the tyrosine kinase pathway via the association of TRAF6 and c-Src, leading to osteoclast cytoskeletal rearrangement and activation.

Vitronectin (Vn) and poly(l-lysine) (PL) were from Life Technologies (Grand Island, NY) and Sigma-Aldrich (St. Louis, MO), respectively. Abs specific to c-Src (N-16) and TRAF6 (H-274 and D-10) were from Santa Cruz Biotechnology (Santa Cruz, CA); p130Cas (mAb 21) and protein tyrosine kinase 2 (PYK2) (mAb 11) from BD Transduction Laboratories (Lexington, KY); phosphotyrosine (4G10) and c-Src (GD11) from Upstate Biotechnology (Lake Placid, NY); and c-Src (mAb 327) from Oncogene Research Products (Cambridge, MA). Other conjugated secondary Abs were from Jackson ImmunoResearch Laboratories (West Grove, PA) and Amersham (Arlington Heights, IL). The 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and collagenase were from WAKO (Dallas, TX), and dispase was from Boehringer Mannheim (Indianapolis, IN). Human rIL-1α and murine M-CSF were from R&D Systems (Minneapolis, MN).

BALB/c mice were obtained from Taconic Farms (Germantown, NY). Heterozygote Src+/− mice obtained from The Jackson Laboratory (Bar Harbor, ME) were mated in our laboratory, and Src−/− mice were phenotypically distinguished from their Src+/? siblings by lack of tooth eruption. All animals were cared for according to the Institutional Animal Care and Use Committee Guide.

pOCs were prepared as described previously, with slight modifications (19). Briefly, spleen cells isolated from 2- to 3-wk-old Src−/− or their normal littermates were cocultured with osteoblastic MB1.8 cells for 5–6 days in the presence of 10 nM 1α,25(OH)2D3. pOCs were released from dishes with 10 mM EDTA after removing MB1.8 cells with collagenase-dispase.

Murine OCLs were prepared using BALB/c mice, as described previously (20). Primary osteoblastic cells were obtained from newborn mouse calvaria, and bone marrow cells were obtained from tibiae of 7- to 9-wk-old male mice. Cells were cocultured in α-MEM containing 10% FBS and 10 nM 1α,25(OH)2D3 on culture dishes precoated with 5 ml 0.2% collagen gel matrix (Nitta Gelatin, Osaka, Japan). OCLs were formed within 7 days of culture, and were released from the dishes by treatment with 5 ml 0.2% collagenase, before being collected by centrifugation at 250 × g for 5 min (crude OCL preparation). Crude OCL preparations were cultured on culture dishes or glass coverslips for 4 h and then purified by collagenase and dispase (purified OCL preparation).

After isolation, pOCs (8 × 105cells/condition) were washed twice with serum-free α-MEM medium containing 0.1% BSA (Sigma-Aldrich) and allowed to attach to polystyrene dishes coated with Vn (20 μg/ml) or PL (50 μg/ml). After culture for 60 min, cells were treated with IL-1 (10 ng/ml) for the indicated periods, and an equal volume of 2× TNE lysis buffer (20 mM Tris (pH 7.8), 300 mM NaCl, 2 mM EDTA, 2% Nonidet P-40, 2 mM Na3VO4, 20 mM NaF, 20 μg/ml leupeptin, 10 μg/ml aprotinin, and 2 mM PMSF) was added to the plates. For coimmunoprecipitation, 1.5 × 106 cells/condition and 1× TNE lysis buffer with 10% glycerol (10 nM Tris (pH 7.8), 300 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 10 μg/ml leupeptin, 0.5 TIU/ml aprotinin, 1 mM PMSF, and 10% glycerol) were used. Clarified lysates were subjected to immunoprecipitation and immunoblotting. Alternatively, cells were fixed and stained for tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts, and F-actin (20).

Osteoclast-like cells were prepared as described previously, with slight modifications (21). After the removal of bone marrow stromal cells, nonadherent bone marrow cells (2.5 × 105/well in six-well plates) were cultured in α-MEM containing 10% FBS and 10 ng/ml M-CSF for 3 days, and adherent cells were subsequently used as bone marrow monocyte/macrophage precursor cells after washing out the nonadherent cells, including lymphocytes. The bone marrow monocyte/macrophage precursor cells were further cultured in the presence of 100 ng/ml soluble receptor activator of NF-κB (RANK) ligand (Peprotech, Rocky Hill, NJ) and 10 ng/ml M-CSF to generate murine osteoclast-like cells. After 3 days, the medium was changed to serum-free α-MEM for 2 h before stimulation with 10 ng/ml IL-1. At the indicated time points, whole cell lysates were prepared and assayed for the kinase activity using poly(Glu-Tyr) as a substrate after immunoprecipitation with anti-v-Src Ab by universal tyrosine kinase assay kit (Takara, Tokyo, Japan), according to the manufacturer’s protocol. A unit of c-Src kinase activity is defined as an activity to incorporate 1 pmol phosphate into a substrate (KVEKIGEGTYGVVYK) per minute.

Immunoprecipitation and immunoblotting were performed as previously described (22). Briefly, lysates were precipitated with anti-p130Cas, TRAF6, or c-Src Abs (2 μg) for 2 h at 4°C, followed by protein G-Sepharose for 1 h at 4οC. After washing with lysis buffer (four times), proteins were separated on an 8% SDS-PAGE and blotted onto Immobilon-P membrane (Millipore, Bedford, MA). After blocking with 100 mM NaCl, 10 mM Tris, 0.1% Tween 20, and 2% BSA, the membrane was incubated with primary Abs, followed by HRP-conjugated secondary Abs, and detected with the ECL system (Amersham).

Crude preparations of OCLs were seeded on glass coverslips. After culture for 4 h, osteoclasts were purified as described above, serum starved for 4 h, and then treated with or without 10 ng/ml IL-1 for 30 min. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, and incubated for 30 min at 37°C with rabbit anti-TRAF6 polyclonal (H-274) or anti-c-Src mAb 327 Abs. Cells were washed with PBS and incubated for 30 min at 37°C with Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories), FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), or Oregon Green 488 phalloidin (Molecular Probes, Eugene, OR). Samples were viewed with a confocal microscope (Radiance; Bio-Rad, Hercules, CA).

Purified OCLs treated with or without IL-1 for 30 min were washed, collected with 1 ml 0.1% BSA in PBS, and spinned down for 2 min at 3000 rpm. The pellet was resuspended in 200 μl ice-cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1 mM PMSF, 1 TIU/ml aprotinin, 10 mM NaF, and 1 mM DTT) and left to swell on ice for 10 min. After the addition of 1% Nonidet P-40 detergent (10 μl), lysates were subjected to vortexing for 30 s and centrifuged for 5 min at 3000 rpm. The pellets were washed once in buffer A (100 μl), centrifuged at 1.5 min at 3000 rpm, replaced with 50 μl buffer B (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM PMSF, 10 μm/ml aprotinin, 10 mM NaF, and 1 mM DTT), and then shaken vigorously at 4°C for >15 min. Subsequently, the mixture was centrifuged for 20 min at 14,000 rpm. Finally, the supernatant was recovered and used as source for nuclear proteins. To determine the purity of the nuclear extract preparations, PYK2 was used as an abundant cytosolic marker in osteoclasts. When proteins (25 μg) isolated from nuclear and cytosolic fractions were subjected to Western blotting for PYK2, the level of PYK2 detected in nuclear extract was about 5% of that in the cytosol (data not shown), suggesting that the purity of the nuclear extracts was >90%.

It was previously shown that IL-1 induces actin ring formation, leading to osteoclast activation (10). In this study, we confirmed these findings in a serum-free system. pOCs were prepared from the murine coculture system and plated on Vn-coated dishes in the absence of serum. pOCs spread on Vn-coated surfaces 60 min after plating (Fig. 1,A), whereas on PL they remain rounded (Fig. 1,I). Initial adhesion to Vn did not induce actin ring formation (Fig. 1,B); however, after treatment with IL-1, these cells started to form actin rings in a time-dependent manner (Fig. 1 C–H).

FIGURE 1.

Effect of IL-1 on actin ring formation of pOCs. Src+/? pOCs were plated on Vn (20 μg/ml) (A–H)- or PL (50 μg/ml) (I)-coated dishes in the absence of serum. After culture for 60 min, cells were treated with IL-1 (10 ng/ml) for 0 (A, B, and I), 5 (C and D), 15 (E and F), 30 (G), and 60 (H) min. Cells were fixed and stained for tartrate-resistant acid phosphatase (A, C, E and I) or F-actin (B, D, F, G and H). Bar = 20 μm.

FIGURE 1.

Effect of IL-1 on actin ring formation of pOCs. Src+/? pOCs were plated on Vn (20 μg/ml) (A–H)- or PL (50 μg/ml) (I)-coated dishes in the absence of serum. After culture for 60 min, cells were treated with IL-1 (10 ng/ml) for 0 (A, B, and I), 5 (C and D), 15 (E and F), 30 (G), and 60 (H) min. Cells were fixed and stained for tartrate-resistant acid phosphatase (A, C, E and I) or F-actin (B, D, F, G and H). Bar = 20 μm.

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We previously reported on the role of p130Cas and PYK2 in the αvβ3 integrin-mediated signaling pathways that lead to actin ring formation in osteoclasts (22, 23, 24, 25). We therefore examined the involvement of p130Cas in IL-1-induced actin ring formation. Tyrosine phosphorylation of p130Cas and PYK2 following cell adhesion peaks at 30–60 min after plating (22, 24, 25). pOCs were therefore plated on PL- or Vn-coated dishes for 60 min and then treated with or without IL-1 for 30 min. Cell lysates from these samples were immunoprecipitated with anti-p130Cas Abs and analyzed by Western blotting with anti-phosphotyrosine Abs. As reported previously (24, 25), cell adhesion resulted in tyrosine phosphorylation of p130Cas (Fig. 2,A, left upper panel). Interestingly, IL-1 increased the tyrosine phosphorylation of p130Cas (Fig. 2 A, left upper panel), suggesting that p130Cas might be a downstream mediator of the IL-1R-dependent signaling pathway. PYK2 was also tyrosine phosphorylated not only by cell adhesion to Vn, but also by IL-1 treatment (data not shown).

FIGURE 2.

Effects of IL-1 on tyrosine phosphorylation of p130Cas and c-Src kinase activity in pOCs. A, Src+/? or Src−/− pOCs were plated on Vn (20 μg/ml)- or PL (50 μg/ml)-coated dishes in the absence of serum. After culture for 60 min, cells were treated with or without IL-1 (10 ng/ml) for 30 min. Total cell lysates were immunoprecipitated with anti-p130Cas Ab, followed by immunoblotting with anti-phosphotyrosine (pTyr) Ab (upper panels). The same membranes were stripped and blotted with anti-Cas Ab (middle panels). Total cell lysates were also subjected to immunoblotting with anti-c-Src Ab (lower panels). ECM, IB, IP, and TCL: extracellular matrix, total cell lysate, immunoblotting, and immunoprecipitation, respectively. B, Osteoclast-like cells were prepared as described in Materials and Methods. Cells were serum starved for 2 h before treating with 10 ng/ml IL-1. After the indicated periods, whole cell lysates were extracted and assayed for the kinase activity using poly(Glu-Tyr) as a substrate after immunoprecipitation with anti-v-Src Ab by universal tyrosine kinase assay kit. A unit of c-Src kinase activity is defined as an activity to incorporate 1 pmol phosphate into a substrate (KVEKIGEGTYGVVYK) per minute.

FIGURE 2.

Effects of IL-1 on tyrosine phosphorylation of p130Cas and c-Src kinase activity in pOCs. A, Src+/? or Src−/− pOCs were plated on Vn (20 μg/ml)- or PL (50 μg/ml)-coated dishes in the absence of serum. After culture for 60 min, cells were treated with or without IL-1 (10 ng/ml) for 30 min. Total cell lysates were immunoprecipitated with anti-p130Cas Ab, followed by immunoblotting with anti-phosphotyrosine (pTyr) Ab (upper panels). The same membranes were stripped and blotted with anti-Cas Ab (middle panels). Total cell lysates were also subjected to immunoblotting with anti-c-Src Ab (lower panels). ECM, IB, IP, and TCL: extracellular matrix, total cell lysate, immunoblotting, and immunoprecipitation, respectively. B, Osteoclast-like cells were prepared as described in Materials and Methods. Cells were serum starved for 2 h before treating with 10 ng/ml IL-1. After the indicated periods, whole cell lysates were extracted and assayed for the kinase activity using poly(Glu-Tyr) as a substrate after immunoprecipitation with anti-v-Src Ab by universal tyrosine kinase assay kit. A unit of c-Src kinase activity is defined as an activity to incorporate 1 pmol phosphate into a substrate (KVEKIGEGTYGVVYK) per minute.

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Several lines of evidence, including our previous findings, have shown that tyrosine phosphorylation of p130Cas is mediated by Src kinase and that c-Src deficiency abrogated adhesion-induced p130Cas phosphorylation in fibroblasts and osteoclasts (23, 24, 25, 26, 27). We examined therefore the involvement of c-Src in IL-1-induced tyrosine phosphorylation of p130Cas, using Src-deficient pOCs. As shown in Fig. 2 A (right upper panel), neither cell adhesion to Vn nor IL-1 treatment induced tyrosine phosphorylation of p130Cas. Furthermore, nor did IL-1 induce actin ring formation and pit-forming activity in Src−/− pOCs (data not shown). These data suggest that c-Src may play an important role in IL-1-induced tyrosine phosphorylation of p130Cas and its osteoclast activation.

To support the above notion, we also examined the effect of IL-1 on c-Src kinase activity using poly(Glu-Tyr) as a substrate. As shown in Fig. 2 B, IL-1 induces activation of c-Src within 5 min after treatment, suggesting that c-Src activity might be directly regulated by the IL-1 signaling pathway, leading to the tyrosine phosphorylation of downstream mediators such as p130Cas and PYK2.

Among members of the TRAF family adapter proteins, TRAF6 is to date the only one mediating IL-1 signaling, through its interaction with the IRAK (28). We therefore examined the relationship of TRAF6 with IL-1-induced tyrosine-phosphorylated proteins in osteoclasts. Src+/? pOCs were plated on PL- or Vn-coated dishes for 60 min and then treated with or without IL-1 for 30 min. Cell lysates were immunoprecipitated with anti-TRAF6 Ab and analyzed by Western blotting with anti-phosphotyrosine Ab. Interestingly, from lysates of cells plated on Vn and treated with IL-1, anti-TRAF6 Abs coprecipitated at least three tyrosine-phosphorylated proteins with molecular mass values of about 130, 120, and 60 kDa (Fig. 3,A, lanes 1–3, arrowheads). Western blotting of the same membrane revealed that anti-TRAF6 Ab coprecipitated c-Src (∼60 kDa), PYK2 (∼110–120 kDa), and p130Cas (∼130 kDa) (Fig. 3 A, lanes 4–9), suggesting that these three proteins could be the major tyrosine-phosphorylated proteins in IL-1-treated cells. In contrast, we could not rule out additional unidentified proteins with similar molecular masses could be tyrosine phosphorylated in IL-1-treated osteoclasts. These observations indicate that IL-1 treatment induces the formation of a complex containing TRAF6, c-Src, PYK2, and p130Cas in osteoclasts on Vn. Adhesion appears to be a prerequisite for IL-1/TRAF6-dependent association with c-Src, PYK2, and p130Cas, because IL-1 treatment of pOCs on PL or in suspension did not result in the same complex formation (data not shown).

FIGURE 3.

Association of TRAF6 with c-Src, PYK2, and p130Cas in pOCs. A, Src+/? pOCs (1.5 × 106 cells/condition) were plated on Vn (20 μg/ml)- or PL (50 μg/ml)-coated dishes in the absence of serum. After culture for 60 min, cells were treated with or without IL-1 (10 ng/ml) for 30 min. Total cell lysates (>1 mg/ml) were immunoprecipitated with anti-TRAF6 Ab (H-274) (lanes 1–12), followed by immunoblotting with anti-phosphotyrosine Ab (lanes 1–3). The same membrane was sequentially stripped and blotted with anti-PYK2 and c-Src (lanes 4–6), anti-p130Cas (lanes 7–9), and anti-TRAF6 (D-10) (lanes 10–12) Abs. Total cell lysates were also subjected to immunoblotting with anti-c-Src and PYK2 Abs (lanes 13–15). B, Src+/? (lanes 1, 4, 7, 10, 13, and 16) and Src−/− (lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, and 18) pOCs were plated on Vn- or PL-coated dishes in the absence of serum. After culture for 60 min, cells were treated with or without IL-1 for 30 min. Total cell lysates (>1 mg/ml) were immunoprecipitated with anti-TRAF6 Ab (H-274) (lanes 1–15), followed by immunoblotting with anti-phosphotyrosine Ab (lanes 1–3). The same membrane was sequentially stripped and blotted with anti-c-Src (lanes 4–6), anti-PYK2 (lanes 7–9), anti- p130Cas (lanes 10–12), and anti-TRAF6 (D-10) (lanes 13–15) Abs. Total cell lysates were also subjected to immunoblotting with anti-c-Src and PYK2 Abs (lanes 16–18). The molecular masses of marker proteins are indicated in kDa on the left. ECM, IB, IP, TCL, and pTyr: extracellular matrix, immunoblotting, immunoprecipitation, total cell lysate, and phosphotyrosine, respectively.

FIGURE 3.

Association of TRAF6 with c-Src, PYK2, and p130Cas in pOCs. A, Src+/? pOCs (1.5 × 106 cells/condition) were plated on Vn (20 μg/ml)- or PL (50 μg/ml)-coated dishes in the absence of serum. After culture for 60 min, cells were treated with or without IL-1 (10 ng/ml) for 30 min. Total cell lysates (>1 mg/ml) were immunoprecipitated with anti-TRAF6 Ab (H-274) (lanes 1–12), followed by immunoblotting with anti-phosphotyrosine Ab (lanes 1–3). The same membrane was sequentially stripped and blotted with anti-PYK2 and c-Src (lanes 4–6), anti-p130Cas (lanes 7–9), and anti-TRAF6 (D-10) (lanes 10–12) Abs. Total cell lysates were also subjected to immunoblotting with anti-c-Src and PYK2 Abs (lanes 13–15). B, Src+/? (lanes 1, 4, 7, 10, 13, and 16) and Src−/− (lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, and 18) pOCs were plated on Vn- or PL-coated dishes in the absence of serum. After culture for 60 min, cells were treated with or without IL-1 for 30 min. Total cell lysates (>1 mg/ml) were immunoprecipitated with anti-TRAF6 Ab (H-274) (lanes 1–15), followed by immunoblotting with anti-phosphotyrosine Ab (lanes 1–3). The same membrane was sequentially stripped and blotted with anti-c-Src (lanes 4–6), anti-PYK2 (lanes 7–9), anti- p130Cas (lanes 10–12), and anti-TRAF6 (D-10) (lanes 13–15) Abs. Total cell lysates were also subjected to immunoblotting with anti-c-Src and PYK2 Abs (lanes 16–18). The molecular masses of marker proteins are indicated in kDa on the left. ECM, IB, IP, TCL, and pTyr: extracellular matrix, immunoblotting, immunoprecipitation, total cell lysate, and phosphotyrosine, respectively.

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We next asked which molecule is essential for the formation of this complex with TRAF6. To address this question, we plated Src−/− pOCs on Vn, treated with IL-1, and found that anti-TRAF6 Abs did precipitate neither PYK2 nor p130Cas (Fig. 3,B, lanes 7–12), suggesting that c-Src may play an important role in the association of PYK2 and p130Cas with TRAF6. IL-1-mediated association of TRAF6 and c-Src was further confirmed in OCLs, in which IL-1 induced the coimmunoprecipitation of c-Src with TRAF6, using anti-TRAF6 Abs (Fig. 4,A, lanes 1 and 2). Furthermore, anti-c-Src polyclonal Abs reciprocally pulled down TRAF6 from IL-1-treated OCLs (Fig. 4,B, lane 1). We also tried two different anti-c-Src mAbs only to fail to coprecipitate TRAF6 under the same condition (Fig. 4 B, lanes 2 and 3), probably because these mAbs might interfere with the TRAF6 and Src association.

FIGURE 4.

Association of TRAF6 and c-Src in OCLs. Purified OCLs were prepared as described in Materials and Methods. A, After culture for 4 h in the absence of serum, cells were treated with (lanes 2 and 4) or without 10 ng/ml IL-1 (lanes 1 and 3) for 30 min. Total cell lysates (>1 mg/ml) were immunoprecipitated with anti-TRAF6 (H-274) Ab, followed by immunoblotting with anti-c-Src (GD11). The same membrane was stripped and blotted with anti-TRAF6 Ab (D-10). B, Purified OCLs were treated with IL-1 (10 ng/ml) for 30 min. Total cell lysates (>1 mg/ml) were immunoprecipitated with anti-c-Src (lane 1, N-16; lane 2, mAb327; lane 3, GD11) Abs, followed by immunoblotting with anti-TRAF6 Ab (H-274). The same membrane was stripped and blotted with anti-c-Src Ab (GD11). The molecular masses of marker proteins are indicated in kDa on the left. TCL, IB, and IP: total cell lysate, immunoblotting, and immunoprecipitation, respectively. Arrows and arrowheads show the position of c-Src and TRAF6, respectively.

FIGURE 4.

Association of TRAF6 and c-Src in OCLs. Purified OCLs were prepared as described in Materials and Methods. A, After culture for 4 h in the absence of serum, cells were treated with (lanes 2 and 4) or without 10 ng/ml IL-1 (lanes 1 and 3) for 30 min. Total cell lysates (>1 mg/ml) were immunoprecipitated with anti-TRAF6 (H-274) Ab, followed by immunoblotting with anti-c-Src (GD11). The same membrane was stripped and blotted with anti-TRAF6 Ab (D-10). B, Purified OCLs were treated with IL-1 (10 ng/ml) for 30 min. Total cell lysates (>1 mg/ml) were immunoprecipitated with anti-c-Src (lane 1, N-16; lane 2, mAb327; lane 3, GD11) Abs, followed by immunoblotting with anti-TRAF6 Ab (H-274). The same membrane was stripped and blotted with anti-c-Src Ab (GD11). The molecular masses of marker proteins are indicated in kDa on the left. TCL, IB, and IP: total cell lysate, immunoblotting, and immunoprecipitation, respectively. Arrows and arrowheads show the position of c-Src and TRAF6, respectively.

Close modal

Protein-protein interaction of TRAF6 and c-Src in osteoclasts led us to examining the intracellular localization of these molecules. Few actin rings were observed in purified OCLs that were seeded and serum starved for 4 h on glass coverslips (Fig. 5,Ba). In these OCLs, c-Src and TRAF6 were distributed throughout the cytoplasm (Fig. 5,A, a and b; Bb). However, when cells were treated with IL-1, both TRAF6 and c-Src were redistributed to the cell periphery, where they were colocalized (Fig. 5,Af). Moreover, the ring-like distribution of TRAF6 at the cell periphery overlapped with that of F-actin (Fig. 5 Bf), suggesting that TRAF6-Src complex may be involved in actin ring formation in osteoclasts.

FIGURE 5.

Effect of IL-1 on intracellular localization of TRAF6 in OCLs. Purified OCLs were prepared on glass coverslips, as described in Materials and Methods. After purification, cells were serum starved for 4 h, and then treated with (A, df; B, df) or without 10 ng/ml IL-1 (A, ac; B, ac) for 30 min. Cells were fixed and double immunostained for TRAF6 (A, b and e; B, b and e; red) and c-Src (A, a and d; green) or F-actin (B, a and d; green). Their colocalization is shown in yellow (A, c and f; B, c and f). Bars = 20 μm.

FIGURE 5.

Effect of IL-1 on intracellular localization of TRAF6 in OCLs. Purified OCLs were prepared on glass coverslips, as described in Materials and Methods. After purification, cells were serum starved for 4 h, and then treated with (A, df; B, df) or without 10 ng/ml IL-1 (A, ac; B, ac) for 30 min. Cells were fixed and double immunostained for TRAF6 (A, b and e; B, b and e; red) and c-Src (A, a and d; green) or F-actin (B, a and d; green). Their colocalization is shown in yellow (A, c and f; B, c and f). Bars = 20 μm.

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An interesting phenomenon observed in this study was the translocation of TRAF6 to nucleus following IL-1 treatment (Fig. 5, Ae and Be), whereas c-Src was distributed only to perinuclear regions (Fig. 5,Ad). As shown in Fig. 6,A, TRAF6 started to traffic to nucleus 5 min after IL-1 treatment. The translocation of TRAF6 to nucleus and cell periphery seems to occur almost simultaneously. Furthermore, the translocation of TRAF6 to nucleus was also confirmed biochemically, as shown in Fig. 6 B.

FIGURE 6.

Effect of IL-1 on translocation of TRAF6 to nucleus in OCLs. A, Purified OCLs were prepared on glass coverslips, as described in Materials and Methods. After purification, cells were serum starved for 4 h, and then treated with 10 ng/ml IL-1 for 0 (a), 5 (b), 10 (c), and 30 (d) min. Cells were fixed and immunostained for TRAF6. Bars = 20 μm. B, Purified OCLs were serum starved for 4 h, and then treated with or without IL-1 (10 ng/ml) for 30 min. Nuclear proteins, prepared as described in Materials and Methods, were immunoblotted with anti-TRAF6 Ab. IB, immunoblotting.

FIGURE 6.

Effect of IL-1 on translocation of TRAF6 to nucleus in OCLs. A, Purified OCLs were prepared on glass coverslips, as described in Materials and Methods. After purification, cells were serum starved for 4 h, and then treated with 10 ng/ml IL-1 for 0 (a), 5 (b), 10 (c), and 30 (d) min. Cells were fixed and immunostained for TRAF6. Bars = 20 μm. B, Purified OCLs were serum starved for 4 h, and then treated with or without IL-1 (10 ng/ml) for 30 min. Nuclear proteins, prepared as described in Materials and Methods, were immunoblotted with anti-TRAF6 Ab. IB, immunoblotting.

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In the previous study, we have shown that IL-1 is an important cytokine for osteoclastic bone resorption (10). In this study, therefore, we examined the mechanism of IL-1-induced osteoclast activation and the involvement of c-Src in IL-1 signal transduction in the regulation of osteoclast function. Using Src+/? and Src−/− osteoclast-like cells, we first demonstrated that c-Src is required for IL-1-induced tyrosine phosphorylation of p130Cas, which was previously shown to take part in actin ring formation in osteoclasts in vitro (23). Second, we showed that IL-1 induces the complex formation of TRAF6, an adapter molecule for IL-1 signaling, c-Src, PYK2, and p130Cas in osteoclasts. c-Src is, also in this case, required for formation of this heteromeric complex, because IL-1 treatment of Src-deficient pOCs did not result in the association of TRAF6 with PYK2 and p130Cas. Third, IL-1 induced the colocalization of TRAF6 and c-Src in the ring-like structures at the periphery of spreading osteoclasts. These ring structures also contained F-actin, a prerequisite for sealing zone formation during osteoclast activation (29). Our previous studies have documented the important role of c-Src and its association with PYK2 and p130Cas in the maintenance of the ring structure of F-actin (actin rings) and bone resorption (22, 24). Data presented in this work, therefore, might clarify another interesting role of c-Src as a transducer of IL-1 signaling to tyrosine phosphorylation pathway, leading to cytoskeletal reorganization and osteoclast activation.

These findings also support the in vivo findings showing that targeted disruption of either c-Src or TRAF6 in mice results in a similar osteopetrotic phenotype, caused by osteoclast dysfunction without change in osteoclast number (16, 17). In contrast, Naito et al. (30) reported that TRAF6 is an essential transducer for osteoclast differentiation, because TRAF6-deficient mice are defective in osteoclast formation and exhibit severe osteopetrosis. Although TRAF6 may be involved in both osteoclast differentiation and osteoclast function, in this study, we focused on the role of TRAF6 in osteoclast function.

During the course of this study, Wong et al. (31) reported that RANK ligand (TNF-related activation-induced cytokine/osteoclast differentiation factor/osteoprotegerin ligand), a TNF family member that stimulates osteoclast differentiation and function, activates Akt/protein kinase B through a signaling complex that includes TRAF6 and c-Src. In their study, the significance of this TRAF6-Src complex lies in RANK ligand-induced cell survival through the activation of Akt/protein kinase B, whereas our data suggest that this molecular complex is involved in the IL-1-induced cytoskeletal rearrangement and osteoclast activation via c-Src-mediated tyrosine phosphorylation of PYK2 and p130Cas. Wong et al. also showed the direct interaction of TRAF6 and c-Src mediated by the RPTIPRNPK motif (aa 469–477) in TRAF6 and the Src homology 3 (SH3) domain in c-Src. The findings obtained to date and previous reports including ours suggest the sequence of the heteromeric molecular complex containing TRAF6, c-Src, PYK2, and p130Cas, as follows: 1) association of TRAF6 and c-Src, mediated by the RPTIPRNPK motif in TRAF6 and the SH3 domain in c-Src (31); 2) interaction of c-Src and PYK2, mediated by the SH2 domain in c-Src and phosphotyrosine in PYK2 (22); 3) constitutive association of PYK2 and p130Cas, mediated by proline-rich regions in PYK2 and an SH3 domain in p130Cas (24, 32). This study points to the significance of the TRAF6 and c-Src interaction in osteoclast activation and presents evidence for cross-talk between IL-1 signaling and tyrosine kinase pathways. In contrast, previous reports have demonstrated that IL-1-mediated cytosolic and nuclear signaling pathways require appropriate assembly of cell-matrix adhesion complexes and organization of actin cytoskeleton (33, 34, 35). Therefore, we cannot rule out the possibility that the molecular complex, including TRAF6, c-Src, p130Cas, and PYK2, shown in this work is indirectly involved in IL-1 signaling.

An additional novel observation reported in this study is the intracellular localization of TRAF6 in osteoclasts. Before IL-1 stimulation, TRAF6 is distributed throughout the cytoplasm; IL-1 induces a redistribution of TRAF6 to the cell periphery, where TRAF6 is colocalized with c-Src and F-actin. We have previously reported that both p130Cas and PYK2 colocalize to the ring-like structure of F-actin (24). The biochemical and morphological evidence presented in this study suggests the involvement of a complex containing TRAF6, c-Src, PYK2, and p130Cas in actin ring formation, leading to osteoclast activation by IL-1. We find in this study, using both morphological and biochemical methods, that upon IL-1 treatment, TRAF6 translocates also to the nucleus. IL-1 thus appears to induce translocation of TRAF6 into two subcellular localizations in osteoclasts, coincident with cell spreading: one pool of TRAF6 that localizes to actin rings in a c-Src-dependent manner, and the other that translocates to the nucleus. The requirement of c-Src for TRAF6 nuclear translocation remains unclear, because IL-1 treatment did not induce cell spreading and actin ring formation in Src-deficient osteoclasts. Biochemical analyses of TRAF6 translocation into the nuclear fraction of Src-deficient cells under treatments with various cytokines will be a subject of our future study. In addition, while TRAF4 was also reported to localize to cell nuclei (36), the physiological significance of these two cellular localizations of TRAF6 induced by cytokines requires further study.

In summary, we show that IL-1 induces, in Src-dependent manner, tyrosine phosphorylation of PYK2 and p130Cas, known downstream mediators of the adhesion-dependent signaling pathway. Furthermore, IL-1 regulates the tyrosine kinase pathway, possibly by inducing the association of TRAF6 and c-Src, leading to further recruitment of PYK2 and p130Cas. Finally, IL-1 induces osteoclast spreading and physical recruitment of the TRAF6/Src-dependent complex to the actin ring adhesion structures, a prerequisite for sealing zone formation, essential for osteoclast activation.

We thank Drs. Ai-ichiro Yamamoto, Toshiki Miura, Toru Akiyama, and Toru Ogata (University of Tokyo) for fruitful suggestions.

1

This work was in part supported by Takeda Science Foundation (to I.N.), Health Science Research Grants from Ministry of Health and Welfare (to S.T.), and Uehara Memorial Foundation (to S.T.).

3

Abbreviations used in this paper: pOC, prefusion osteoclast-like cell; 1α,25(OH)2D3, 1α,25-dihydroxyvitamin D3; Cas, Crk-associated substrate; IRAK, IL-1R-associated kinase; OCL, osteoclast-like multinucleated cell; PL, poly(l-lysine); PYK2, protein tyrosine kinase 2; RANK, receptor activator of NF-κB; SH, Src homology; TRAF6, TNFR-associated factor 6; Vn, vitronectin.

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