A RANK/TRAF6-DEPENDENT SIGNAL TRANSDUCTION PATHWAY IS ESSENTIAL FOR OSTEOCLAST CYTOSKELETAL ORGANIZATION AND RESORPTIVE FUNCTION

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1 JBC Papers in Press. Published on August 15, 2002 as Manuscript M A RANK/TRAF6-DEPENDENT SIGNAL TRANSDUCTION PATHWAY IS ESSENTIAL FOR OSTEOCLAST CYTOSKELETAL ORGANIZATION AND RESORPTIVE FUNCTION Allison P. Armstrong*, Mark E. Tometsko*, Moira Glaccum#, Claire L. Sutherland*, David Cosman* and William C. Dougall* Departments of Molecular Biology* and Molecular Immunology#, Immunex Corporation, Seattle, Washington, 98101, USA Address correspondence to William C. Dougall, Department of Molecular Biology, Immunex Corporation, 51 University Street, Seattle, WA Phone: ; FAX: ; wdougall@immunex.com Running title: RANK mediated osteoclast differentiation and activation 1 Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

2 2 SUMMARY Signaling through RANK (receptor activator of NF-κB) is essential for the differentiation and activation of osteoclasts, the cell principally responsible for bone resorption. Animals genetically deficient in RANK or the cognate ligand RANKL are profoundly osteopetrotic due to the lack of bone resorption and remodeling. RANK provokes biochemical signaling via the recruitment of intracellular TNFR-associated factors (TRAFs) after ligand binding and receptor oligomerization. To understand the RANK-mediated signal transduction mechanism in osteoclastogenesis, we have designed a system to recapitulate osteoclast differentiation and activation in vitro by transfer of the RANK cdna into hematopoietic precursors genetically deficient in RANK. Gene transfer of RANK constructs that are selectively incapable of binding different TRAF proteins revealed that TRAF pathways downstream of RANK that affect osteoclast differentiation are functionally redundant. In contrast, the interaction of RANK with TRAF6 is absolutely required for the proper formation of cytoskeletal structures and functional resorptive activity of osteoclasts. Moreover, signaling via the IL-1 receptor, which also utilizes TRAF6, rescues the osteoclast activation defects observed in the absence of RANK/TRAF6 interactions. These studies are the first to define the functional domains of the RANK cytoplasmic tail that control specific differentiation and activation pathways in osteoclasts. 2

3 3 INTRODUCTION The structural and metabolic integrity of bone is maintained through the dynamic process of bone remodeling that results from the coordinate action of bone resorption by osteoclasts and the formation of new bone by osteoblasts. Osteoclasts are large, multinuclear cells that develop from a hematopoietic progenitor and are highly specialized for the resorptive process (Teitelbaum, 2000). Regulation of bone remodeling occurs through multiple mechanisms that ultimately converge on the interaction of osteoclasts or their precursors with osteoblasts and bone marrow stromal cells. Two key factors supplied by the stromal environment are CSF-1 and the TNF family member, RANKL (also called TRANCE, ODF, OPGL) (Riggs et al., 2000) as confirmed by the osteopetrotic phenotypes of the op/op mice that are mutated in the CSF-1 gene (Yoshida et al., 1990) and the RANKL/OPGL knockout mice (Kong et al., 1999). It is now widely accepted that most osteotropic agents including IL-1, IL-6, IL-11, IL-17, TNFα, PGE2, parathyroid hormone, and 1,25 dihydroxy vitamin D3 (Hofbauer et al., 2000) affect bone resorption primarily by enhancing stromal cell production of RANKL. RANKL affects bone resorption and bone density by influencing the osteoclast population at multiple stages. Not only does RANKL drive the differentiation of osteoclasts from multipotential progenitors thereby expanding the pool of osteoclasts available for bone resorption (Yasuda et al., 1998), but RANKL also activates resorption and enhances survival of existing mature osteoclasts in vitro (Burgess et al, 1999; Lacey et al., 1998) and in vivo (Lacey et al., 2000). An essential role for the RANKL receptor, RANK, in osteoclast differentiation in vivo is demonstrated by the lack of osteoclasts and resulting osteopetrosis in RANK-/- animals (Dougall et al., 1999; Li et al., 2000). Thus, RANK plays a key role in bone metabolism as the critical signaling receptor responsible for osteoclast differentiation, activation and survival. 3

4 4 RANK, like many of the TNFR family proteins, transmits biochemical signals after recruitment of intracellular adaptor TNF receptor associated factor (TRAF) proteins. Activation of NF-κB, JNK and MAPK pathways by RANK occurs via TRAFs 2, 5 and 6 (reviewed in Inoue et al., 2000). The relevance of each of these biochemical signals in osteoclast biology is underscored by the osteopetrotic phenotypes of the p50/p52 NF-κB double knockout mouse (Franzoso et al., 1997; Iotsova et al., 1997) and the fos-/- mouse (Grigoriadis et al., 1994) as well as the inhibition of in vitro osteoclast differentiation after blockade of p38 MAPK activity (Matsumoto et al., 2000). TRAF6, which binds to a RANK structural motif distinct from the region that binds TRAFs1, 2, 3 and 5, plays an essential role in RANKL-mediated NF-κB activation in transfected cell systems (Darnay et al., 1999; Galibert et al., 1998). TRAF6 also provides a functional connection between RANK signaling and the activation of c-src-, AKT-, and PI-3-kinases (Wong et al., 1999). Knockouts of c-src (Soriano et al., 1991) and TRAF6 (Lomaga et al., 1999) are also osteopetrotic, although the bone defects in these animals are not due to lack of osteoclasts as seen in the p50/p52 NF-κB double knockout, RANK-/-, and the fos- /- mice, but rather are due to defective osteoclast activity. RANK signaling is essential for the initiation of osteoclast differentiation. However, transfection studies in heterologous cells have demonstrated that RANK signal transduction is mediated via multiple distinct and selective TRAF-binding motifs (reviewed in Inoue et al., 2000). In order to define the relevance of these distinct RANK cytoplasmic motifs and downstream biochemical pathways in the context of each stage of osteoclast differentiation and function, we have established an experimental system in which transfer of the RANK gene into RANK-/- hematopoietic precursors will restore the formation and activation of osteoclasts. Using a panel of RANK mutants selectively incapable of binding distinct TRAF proteins, we have 4

5 5 established that there are redundant RANK signaling paths necessary for the differentiation of hematopoietic precursors into multinuclear osteoclasts and for the upregulation of genes which function during osteoclastogenesis. In contrast to RANK-dependent differentiation signals, the sequence elements in the RANK cytoplasmic domain necessary for TRAF6 binding are nonredundant and critical for the proper cytoskeletal organization and resorptive activities of osteoclasts. 5

6 6 MATERIALS AND METHODS Retroviral Production Each of the human RANK cdna constructs has been previously described (Galibert et al., 1998) except for RANK / Full-length and mutated human RANK constructs were subcloned into the LZRS-pBMNZ vector (Kinsella and Nolan, 1996) and production of infectious retroviral vector particles was performed in 293-E Phoenix packaging cells as described. Normalized expression of each of the RANK constructs was confirmed by analysis of RANK surface expression using an antibody against human RANK (mab M330) (Anderson et al., 1997) on infected cells. NIH3T3 cells were infected with varying amounts of each RANK retroviral construct in the presence of 5 µg/ml polybrene. Forty-eight hours post infection, cells were lifted with Versene and stained with an antibody to hrank (2.5 µg/ml), and FITCconjugated anti-mouse IgG1 secondary (1:500) and positive cells were analyzed by flow cytometry. Retroviral Transduction of Hematopoietic Progenitors and Osteoclast Differentiation Assays The generation of the RANK-/- mice has been described previously (Dougall et al., 1999). Spleen cells were isolated from 3-6 week old RANK-/- mice and T cells, erythroid cells, and neutrophils were depleted using rat antibodies against CD3, Ter-119, and GR-1, (Pharmingen, San Diego, CA) and sheep anti-rat conjugated magnetic beads (Dynabeads M-450; DYNAL, Oslo, Norway). Cells were incubated for 48 h in 40 ng/ml hcsf-1 (R&D Systems, Minneapolis, MN) and infected using retroviral supernatants (MOI of 5) in the presence of recombinant fibronectin fragments (Retronectin, PanVera Corp., Madison, WI) for 48 h in 40 ng/ml CSF-1. 6

7 7 Following infection, spleen cells were harvested and plated in α-mem/10%fbs containing 40ng/ml CSF-1 and the presence or absence of 200 ng/ml mrankl as indicated. Recombinant murine RANKL is an NH2-terminal fusion of a leucine zipper trimerization domain (Fanslow et al., 1994) with residues of murine RANKL (Anderson et al., 1997). TRAP staining was done at 37º C using the leukocyte acid phosphatase kit (Sigma; KIT 387-A) in the presence of tartrate as described (Dougall et al., 1999). Calcitonin binding was performed by blocking cells in 5% non-fat dry milk followed by incubation with 1 µci/ml 125 I salmon calcitonin (Amersham; sp activity=2000ci/mmol). After cells were washed and solubilized in 0.5 M NaOH, total bound counts measured using a gamma counter. The specificity of the calcitonin radio-ligand binding assay was determined by competition of the signal using 100X molar excess of unlabeled salmon calcitonin (Calbiochem, San Diego, CA). Resorption of the CaPO 4 matrix (BioCoat TM Osteologic TM Discs; Becton Dickinson, Bedford, MA) was measured after treatment of slides in bleach and staining with 0.5% alizarin red. Quantitation of the resorbed area was performed by measuring the surface area of cleared CaPO4 resorption pits using Image Pro Plus 4.1 software from Media Cybernetics (Silver Spring, MD). Values are expressed as relative resorbed area in percent compared to that measured in cells transduced with full-length hrank and treated with RANKL and were measured from wells in triplicate for each experiment and expressed as mean +/- standard deviation. For the pit formation/resorption lacunae assay, transduced cells were cultured in α-mem/10%fbs containing 40ng/ml CSF-1 and with either RANKL (200 ng/ml), IL-1β (20ng/ml) or a combination of RANKL and IL-1β. After culture for a period of 7 d, cells were stained for TRAP, removed from the dentin slices and the resorption pits were visualized by staining with 1% Toluidine blue. RT-PCR 7

8 8 RNA was isolated from infected splenic cultures 24, 48, 72 and 120 h after retroviral infection. Treatment of cultures with RANKL was begun at the 48 h timepoint after the initiation of retroviral infection. cdna corresponding to 10 ng of total RNA was used per reaction in quantitative PCR performed on a GeneAmp 9600 thermocycler in the presence of the dye SYBR Green, and analyzed by the GeneAmp 5700 sequence detection system (Applied Biosystems, Foster City, CA). We used primers corresponding to cathepsin K, TRAP, OC-116kDa, c-src, MMP-9, HPRT, carbonic anhydrase II, calcitonin receptor, hrank, and CD61; the correct sequence of the reaction products was confirmed by DNA sequencing. All samples were normalized for equivalent cdna amounts using the HPRT signal. Fluorescence Microscopy Cells were cultured on coverslip-thick microscope slides. After incubation for 5 d the media was aspirated, and cells were washed twice in PBS and then fixed in a solution of 3% paraformaldehyde for 10 minutes followed by quenching in 50 mm NH 4 Cl. Cells were blocked using 5% normal goat sera in PBS. Cells were then incubated with antibodies against human RANK (M330), mouse CD61/β3 integrin (clone 2C9.G2, Pharmingen, San Diego, CA) and the appropriate fluorescent secondary antibodies. Alternatively, cells were permeabilized in 0.1% Triton X-100, 5% normal goat sera in PBS and incubated with antibodies against hrank, c-src (M327v-src, Ab-1 Calbiochem), c-cbl (clone C-15, Santa Cruz Biotechnology) and the appropriate fluorescent secondary antibodies. For visualization of the F-actin, cells were permeabilized as above and then incubated with fluorescently conjugated Phallodin (Molecular Probes, Eugene, OR) at 1:100. To visualize nuclei, cells were permeablized as described in the 8

9 9 presence of RNAse A at 100 µg/ml and incubated with Sytox (Molecular Probes) at 1:20,000 in 5% NGS and 0.1%Triton X. RESULTS Confirmation of Retroviral mediated Gene Transfer of RANK Constructs The complete absence of osteoclasts and resulting osteopetrotic phenotype of the RANK knockout mice revealed an obligate role for RANK in normal bone remodeling (Dougall et al., 1999; Li et al., 2000). However, it is not clear how the distinct TRAF-binding domains of the RANK cytoplasmic domain contribute to osteoclast formation. The C-terminal 72 amino acids of the human RANK cytoplasmic domain are capable of binding to multiple TRAFs (TRAF1, 2, 3 and 5) and transmit signaling through NF-κB and JNK-dependent pathways (Darnay et al., 1998; Galibert et al., 1998). A separate region of the human RANK sequence (aa # ) mediates direct binding to TRAF6 only and is both necessary and sufficient for NF-κB activation in transfected 293 cells (Darnay et al. 1999; Galibert et al., 1998). In order to define the functional role of these RANK cytoplasmic domains within the physiological context of the osteoclast, we reconstituted osteoclast differentiation and activation of RANK-/- hematopoietic progenitors using retroviral gene transfer of various human RANK cdna constructs that selectively lack the ability to bind specific TRAF proteins. We confirmed that infection of fibroblasts with each of the different RANK retroviral supernatants leads to equivalent levels of surface expression (Figure 1a). Splenic cells purified from RANK-/- animals and enriched for hematopoietic progenitors cannot form osteoclasts in vitro after addition of RANKL and CSF-1 (Dougall et al., 1999) and therefore provide a null background for the osteoclast rescue experiments. These cells were infected with retroviruses encoding the different RANK constructs or a control lacz virus, 9

10 10 and cultured using osteoclastogenic conditions (RANKL + CSF-1, see Materials and Methods). Specific expression of the transgenic mrnas (Figure 1b) and total or surface protein level (Figure 1c and 1d) could be detected for each of the RANK constructs only in infected cells. The expression of the full-length RANK transgene mrna and protein was consistently lower by a factor of 2 to 2.5 fold relative to the other RANK construct transgenes (Figure 1b, and 1c). Thus, any deficiency in osteoclast differentiation or function was not due to lack of expression of the mutated RANK constructs. Evaluation of the Osteoclast Differentiation Phenotype After Expression of RANK Transgenes The formation of differentiated osteoclasts was monitored in situ by expression of the enzyme marker tartrate resistant acid phosphatase (TRAP) in multinuclear cells. Osteoclasts failed to form from RANK-/- progenitors that were infected with a control virus encoding lacz and then cultured with RANKL and CSF-1 (Figure 2). These cells expressed CD11b +, Mac-2 +, F4/80 + (not shown) consistent with a macrophage phenotype. Retroviral-mediated expression of the full-length human RANK gene efficiently rescued multinuclear TRAP + osteoclast differentiation even in the absence of exogenously added RANKL (Figure 2 a and b). Addition of RANKL to the culture clearly increased the size of TRAP + osteoclasts. RANK constructs lacking the C-terminal TRAF binding domain (RANK or RANK ) also promoted the differentiation of large TRAP + osteoclasts in the presence of RANKL (Figures 2a and b) although to a lesser extent than full-length RANK. The absence of significant numbers of TRAP + cells without addition of RANKL and the reduced number of multinuclear TRAP + cells overall suggests that the differentiation signal from these RANK constructs is less potent. Cells transduced with a RANK construct lacking the entire cytoplasmic 10

11 11 domain (RANK ) were completely incapable of osteoclast differentiation (Figure 2a and b). A RANK construct lacking all TRAF binding sites (RANK / ) was also completely incapable of forming TRAP + osteoclasts in the presence of RANKL and CSF-1 (Figure 2) and was indistinguishable from the C-terminal deletion RANK To specifically address the role of RANK/TRAF6 signaling in osteoclast differentiation, RANK-/- cells were infected with the RANK retroviral construct and cultured under the same conditions as described above. TRAP + osteoclast formation was observed at equal efficiencies as that observed with the C-terminal RANK deletions (Figure 2b), although the cells were clearly more compact than, and morphologically distinct from, cells derived from the transduction of wild-type RANK or C-terminal RANK deletions. Higher magnification of these cells revealed that they contained more dendritic- or lamellipodia-like processes and were less spread than TRAP + osteoclasts derived from the full-length RANK (Figures 5 and 6). Importantly, cells derived from the C-terminal deletion RANK constructs or the internal deletion of the TRAF6 binding site (RANK ) were frequently multinuclear (Figure 2b). Quantitation of the nuclei in TRAP + osteoclasts transduced and differentiated with the various RANK constucts indicated that there was no significant difference in the mean nuclei number in each case. The reduced efficiencies of TRAP + osteoclast formation mediated by the different RANK/TRAF-binding mutants indicate that both the RANK C-terminal (TRAF 1, 2, 3 and 5) binding element and the membrane proximal TRAF 6 binding element contribute to osteoclast differentiation, but in the absence of either individual TRAF-binding site, osteoclast differentiation still proceeds. This compensatory signaling toward osteoclast differentiation is completely abrogated upon removal of all TRAF binding elements. The relative differences in 11

12 12 osteoclast differentiation did not vary substantially over several independent viral transductions with the RANK constructs. Analysis of Osteoclast Marker Expression In addition to multinuclearity and TRAP staining, we have monitored the expression of multiple mrnas and proteins expressed specifically during osteoclast differentiation to confirm that cells infected with RANK constructs lacking either the TRAF6 binding site or the separate TRAF 1,2, 3, 5 binding site still commit and differentiate into the osteoclast lineage. Expression of the osteoclast marker calcitonin receptor was detected using a radio-ligand binding assay. We found that expression of the full-length RANK transgene led to specific 125 I-calcitonin binding that was enhanced further by the addition of RANKL (Figure 3). The expression of calcitonin receptor was also confirmed in cultures infected with either the C-terminal RANK deletions (RANK or RANK ) or the internal TRAF6 deletion (RANK However, similar to the reduced efficiency of TRAP positive osteoclast differentiation, these cultures exhibited a reduction in overall 125 I-calcitonin binding as compared to osteoclasts differentiated via the full-length RANK protein. Quantitative RT-PCR analysis of several osteoclast differentiation marker mrnas (MMP-9, cathepsin K, β3 integrin, carbonic anhydrase II, c-src, calcitonin receptor, TRAP) demonstrated significant increases of each of these mrnas in cells cultured in the presence of RANKL after infection with the full-length RANK (Figure 4). In addition, we confirmed protein expression of c-src (see below, Figure 5) as well as surface expression of CD61/β3 integrin, a subunit of the vitronectin receptor (data not shown). Osteoclast marker mrnas were not detected in cells infected with the control lacz virus or cells infected with the RANK constructs 12

13 13 that cannot bind TRAFs (RANK / and deletion RANK ). Similar to the TRAP staining and calcitonin receptor expression, RANK constructs unable to bind TRAF6 (RANK ) or TRAFs 1,2,3 and 5 (RANK or RANK ) still promoted osteoclast marker expression but to a lesser degree than the full-length RANK (Figure 4). These data are consistent with the TRAP/multinuclear osteoclast potential (Figure 2) and suggest that TRAF/RANK interactions lead to a similar capacity for osteoclast differentiation. The c-src kinase is highly expressed in osteoclasts and serves as a specific determinant of osteoclast differentiation from macrophages (Horne et al., 1992). In order to confirm c-src expression and to better visualize the osteoclast morphology of cells differentiated with various RANK constructs, we have used immunofluorescent/confocal microscopic analysis of infected cells. Expression analysis of c-src confirmed that c-src protein is abundantly expressed in large multinuclear osteoclasts and that overall cell morphology is not appreciably different in cells derived from wild type RANK or the C-terminal RANK deletions, RANK or RANK (Figure 5). C-src protein was absent in RANK-/- cells infected with the control lacz virus or the RANK constructs that cannot bind TRAFs (RANK / and RANK ) confirming the specificity and sensitivity of this measure of osteoclast formation. In cells derived from the internal RANK/TRAF6 binding deletion (RANK ), c-src was also expressed at high levels in multinuclear cells; however, this analysis makes apparent the reduced size and altered, more-compact morphology of these cells. Taken together with the osteoclast differentiation marker data, these results indicate that osteoclast differentiation is retained in the absence of a direct signal transduction between RANK and TRAF6; however, there may be a selective cytoskeletal defect leading to the altered morphology in these cells. 13

14 14 Deletion of the TRAF6 Binding Domain of RANK Leads to Disruptions of the Osteoclast Cytoskeleton The formation of F-actin rings is a significant measure of the integrity of the osteoclast cytoskeleton and is a marker of functional, resorbing osteoclasts (Lakkakorpi and Vaananen, 1996). Using phalloidin/fluorescent microscopy, we analyzed F-actin in cells differentiated in the presence of each RANK retroviral construct. Distinct F-actin rings were apparent at the periphery of multinuclear osteoclasts derived from either full-length RANK or the C-terminal TRAF binding mutants (RANK or RANK ) (Figure 6). However, the actin cytoskeleton was not organized into peripheral bands or rings in osteoclasts derived from the RANK/TRAF6 binding mutant (RANK ). Higher magnification confirmed that this is clearly not a defect of multinuclearity or cell fusion (Figure 6b), but rather a specific disruption of the normally organized F-actin rings resulting in a more punctate or focal pattern of actin localization within the lamellipodia-like structures observed in these cells. Because the involvement of c-cbl and c-src in osteoclast cytoskeletal organization and function is well established (Tanaka et al., 1996), we analyzed the expression and localization of these proteins. Analysis of osteoclasts derived from full-length RANK by dual color immunofluorescence reveals a characteristic double-ring pattern of c-src at the periphery with c-cbl intercalated between these rings (Figure 7). In RANK-/- cells infected with the control lacz virus, c-src is absent and c-cbl is not obviously organized into cytoskeletal structures. Similar to the actin cytoskeleton of osteoclasts derived from the RANK/TRAF6 binding mutant (RANK ), c-cbl and c-src localization was also disrupted and was frequently concentrated in foci at the cell 14

15 15 periphery (Figure 7). These observations support a distinct role for the RANK/TRAF6 linkage in the cytoskeleletal organization of osteoclasts. RANK/TRAF6 Signal Transduction is Necessary for Functional Resorption of Bone/CaPO 4 In order to test the functional capabilities of osteoclasts derived from RANK-/- cells infected and differentiated with the different RANK constructs, we cultured transduced cells on a synthetic matrix of CaPO 4 for quantitative measurement of resorption and then confimed resorptive activity on authentic bone/dentin fragments. Resorption of CaPO 4 was only evident in cultures of cells infected with the full-length RANK or the C-terminal RANK deletions (RANK or RANK ) (Figure 8), suggesting that this assay selectively monitors the activation of existing osteoclasts. Interestingly, although TRAP + osteoclasts were formed in the absence of RANKL (see above, Figure 2), resorption was only observed in these cells when cultured with exogenous RANKL (Figure 8). The area of resorption pits generated by cultures transduced with the C-terminal RANK deletions (RANK or RANK ) was reduced approximately 25-50% compared to the full-length RANK consistent with the reduction in osteoclast number/well inthese cultures (Figure 8). The relative resorption activity of cultures treated with RANKL and transduced with RANK was reduced to 80% +/-21% while the resorption activity of RANK cultures was reduced to 72% +/- 27% compared to full-length RANK transduced cells (100% resorption activity). Cells transduced with the fulllength RANK also generated resorptive lacunae when cultured on dentin slices (Figure 10). Uniformly similar results on dentin resorption were observed with cells infected with either the C-terminal RANK deletions (RANK or RANK ) (data not shown). Differentiated osteoclasts derived from the RANK construct (TRAF6 binding mutant) 15

16 16 were incapable of resorbing CaPO 4 (Figure 8) or forming resorption lacunae on dentin slices (Figure 10) even in the presence of RANKL. The absence of resorptive activity is observed although there were approximately equal numbers of osteoclasts generated from the RANK construct as compared to the C-terminal RANK deletions (RANK or RANK ) (Figure 2b). These data suggest that in the absence of coupling of RANK signaling to the TRAF6 protein, early steps in osteoclast differentiation can proceed giving rise to TRAP + multinucleated cells, but later events related to osteoclast cytoskeletal formation and functional activation are compromised. IL-1b Rescues the Activation/Resorption Defect of Osteoclast Derived From the RANK/TRAF6 Binding Mutant Previous studies have shown that IL-1 influences osteoclast differentiation by promoting the synthesis of RANKL (reviewed in Hofbauer et al., 2000), however, direct exposure of osteoclasts to IL-1 fails to result in differentiation, but rather leads to enhancement of osteoclast survival and activity (Jimi et al., 1998, Jimi et al., 1999). The selective effect of IL-1 on osteoclast activation is also supported by the recent observation that TNFα (but not IL-1) potently stimulates osteoclast differentiation of bone marrow macrophages into TRAP + osteoclasts in vitro, however, these cells are not fully active and cannot function to resorb bone unless cultured with IL-1 (Azuma et al., 2000; Kobayashi et al., 2000). Because a common component of downstream signaling utilized by both RANK and IL-1R is TRAF6 (Cao et al., 1996), we hypothesized that IL-1β would rescue the resorptive defect found in osteoclasts derived from the RANK mutant incapable of directly binding TRAF6 (RANK ). Addition of IL-1β to cells expressing RANK circumvented the block in formation of 16

17 17 larger TRAP + osteoclasts (Figure 9a) and, moreover, rescued CaPO 4 resorption both in the presence or absence of RANKL (Figures 9b and 9c). IL-1β also rescued the ability of RANK expressing cells to form authentic resorptive lacunae on dentin slices (Figure 10). The ability of IL-1β to rescue the resorptive defect of a RANK construct that cannot signal through TRAF6 suggests the obligatory role of TRAF6 in the activities of mature, differentiated osteoclasts. 17

18 18 DISCUSSION The detailed genetic program that controls each step of osteoclastogenesis has been elucidated largely through the analysis of mouse mutants having osteopetrosis. Genes essential for this program have been identified for each sequential stage in this process including the proliferation and survival of osteoclast progenitors (CSF-1), commitment and differentiation of osteoclasts (c-fos, p50 and p52 NF-κB subunits), polarization/maturation of osteoclasts (c-src, TRAF6, and αvβ3 integrins) and, finally, activation of the osteoclasts to promote resorptive function (cathepsin K, carbonic anhydrase II, H+-ATPase) (reviewed in Teitelbaum, 2000). Mice lacking the TNFR superfamily member RANK, or its cognate ligand (RANKL), are also osteopetrotic due to a cell autonomous defect in osteoclast differentiation from hematopoietic precursors (Dougall et al., 1999; Kong et al., 1999; Li et al., 2000), yet it is understood that RANKL affects multiple, diverse steps in the osteoclastogenic genetic program including osteoclast differentiation (Lacey et al., 1998; Yasuda et al., 1998), survival (Lacey et al., 2000), and stimulation of resorptive function (Burgess et al., 1999; Fuller et al., 1998). Because each of the steps may contribute to enhanced bone turnover during pathologic conditions such as osteoporosis, bone loss due to inflammatory diseases or malignancies (Teitelbaum, 2000), it is important to define the signal transduction cascades emanating from RANK that are responsible for these pleiotropic actions. Using our experimental system, we were able to demonstrate that binding of different TRAF proteins to distinct RANK cytoplasmic domains mediates divergent signals leading to osteoclast activation or osteoclast differentiation. We found that the RANK signal transduction pathway that leads to osteoclast functional resorptive activity selectively requires linkage of RANK to TRAF6. In the absence of the TRAF6/RANK protein-protein interactions, the 18

19 19 formation of F-actin rings, cytoskeletal organization of c-src and c-cbl, and resorption of CaPO 4 or genuine bone slices by osteoclasts were completely blocked. Removal of individual TRAF binding motifs from the RANK cytoplasmic domain significantly reduced the efficiency of osteoclast differentiation, but the differentiation program was not blocked unless all TRAF binding sites were removed, revealing that RANK initiated redundant differentiation paths via distinct domains that bind to distinct TRAF proteins. Mice deficient in TRAF6 have been generated and, in addition to perinatal lethality and defects in CD40, IL-1 and LPS signaling, were also shown to be osteopetrotic (Lomaga et al., 1999). The altered bone remodeling in these mice resulted not from a block in osteoclast differentiation, since these animals had normal numbers of TRAP + osteoclasts, but rather due to impaired contact of osteoclasts with bone surfaces and reduction in osteoclast ruffled borders. Ruffled borders are specialized plasma membrane structures that are necessary for resorption of bone by polarized osteoclasts. These results are consistent with the selective linkage of RANK/TRAF6 signaling to cytoskeletal organization and osteoclast resorption that we have defined in this study. A second osteopetrotic TRAF6-/- mouse has also been reported (Naito et al., 1999) and exhibits significantly reduced numbers of TRAP + OCL. The discrepancy between these two TRAF6-/- mice has not been explained. We have defined an indispensable role for the interaction of RANK and TRAF6 in osteoclast cytoskeletal formation and functional activation. The resorptive defect that we observed in osteoclasts reconstituted with the TRAF6 binding mutant of RANK cannot be explained by the absence of of carbonic anhydrase II, cathepsin K, and the OC-116kDa subunit of the H + -ATPase mrnas, however, we have not accounted for the absolute protein expression or enzymatic activity required for calcium phosphate resorption. The regulation of cytoskeletal 19

20 20 organization is a key mechanism for osteoclast activation, and proper F-actin organization into ring-like podosomal structures is crucial for the formation of the ruffled border and tight sealing zone necessary for bone resorption by osteoclasts (Lakkakorpi and Vaananen, 1996). This ordered cytoskeletal structure was absent in osteoclasts derived from RANK constructs with deletion of the TRAF6 binding site (Figure 6). Osteoclasts derived from the c-src knockout lack ruffled borders and do not form resorptive lacunae, indicating the requirement for this protein for bone resorption (Boyce et al., 1992). Interestingly, the unusual morphology and altered F-actin staining pattern seen in osteoclasts without RANK/TRAF6 signaling was similar to the morphology and unusual localization of F-actin to focal adhesion-like structures observed in src- /- osteoclasts (Sanjay et al., 2001; Schwartzberg et al., 1997), which may suggest that a TRAF6 linkage of RANK signaling to c-src activation/modification may be essential for the integrity of osteoclast cytoskeletal formation. Wong et al. (1999) have clearly defined this linkage in transfected human 293 cells by demonstrating that TRAF6 mediates RANK-dependent activation of c-src and subsequent phosphorylation of c-cbl. Considering that phosphorylation of c-cbl occurs in authentic osteoclasts in a c-src dependent manner (Tanaka et al., 1996), and c-cbl, in addition to c-src, has pivotal roles in osteoclast-mediated bone resorption (Boyce et al., 1992; Soriano et al., 1991; Tanaka et al., 1996), we analyzed c-src and c-cbl in osteoclasts reconstituted with various RANK constructs. We noted that the sub-cellular localization of these proteins was not properly organized at the cell periphery in the absence of RANK/TRAF6 signaling perhaps due to defective c-src mediated signaling. Coordinated localization of c-src and c-cbl to the cell periphery may be important for association with the F-actin cytoskeleton and/or the membrane integrins thereby affecting osteoclast function. While RANK/TRAF6 signaling has also been 20

21 21 shown to activate the antiapoptotic AKT/PKB kinase (Wong et al., 1999), we did not observe increased apoptotic nuclei in osteoclasts differentiated via the RANK/TRAF6 binding mutant (data not shown), suggesting that resorptive defects observed in these cells was probably not due to any reduction in cell survival. Alternatively, RANK/TRAF6 signaling which activates PI- 3Kinase or AKT/PKB is also c-src dependent (Wong et al., 1999) and may also influence the cytoskeletal changes and osteoclast activation observed in this study. The defects in osteoclast cytoskeletal organization that we have defined with the RANK/TRAF6 binding mutant may affect ruffled border assembly, cell migration, exocytosis or processing of bone matrix necessary for optimal resorption and osteoclast activity. In contrast to osteoclast activation signals, we found that differentiation pathways leading from each of the RANK/TRAF-binding domains are functionally redundant. Our results show that the efficiency of osteoclast differentiation was quantitatively reduced when either the membrane proximal RANK TRAF-binding motif (which interacts with TRAF6) or the C- terminal RANK TRAF-binding domain (which binds TRAFs1, 2, 3 and 5) were removed. The RANKL-independent osteoclastogenesis observed after transduction with some RANK constructs probably reflects the autoactivation of RANK as has been observed previously (Galibert et al., 1998), while the increased size of osteoclasts after RANKL treatment can be explained by the ability of this factor to rapidly increase osteoclast spreading in the absence of additional cell fusion (Fuller et al., 1998). Qualitatively, however, these cells retained many characteristics of authentic differentiated osteoclasts including expression of TRAP in multinucleated cells and expression of many other osteoclast markers, suggesting a functional overlap of these TRAF-binding domains. Of the four TRAF proteins that bind to the RANK C- terminal motif, TRAF2 or TRAF5 may contribute to early osteoclast differentiation signals. Both 21

22 22 proteins are capable of linking RANK to activation of NF-κB and AP-1 (Darnay et al., 1998; Galibert et al., 1998; Wong et al., 1998) transcription factors which are necessary for osteoclast differentiation (reviewed in Teitelbaum, 2000). RANKL treatment of osteoclast precursors results in induced levels of relevant downstream signaling intermediates including TRAF2, TRAF6, c-src, and MEKK1 (Zhang et al., 2001), suggesting that the enhanced efficiency of differentiation signals via both TRAF binding sites revealed in this study may be explained by cooperation of signaling paths that are perhaps dependent on the abundance of signaling intermediates, ultimately converging at NF-κB and AP-1. In contrast to RANK and RANKL, there is apparently no obligate role for TNF, IL-1 or their receptors TNFRp55, TNFRp75 or IL-1R in normal bone physiology, but TNF and IL-1 signals may operate during bone loss in pathological conditions such as inflammatory bone diseases, rheumatoid arthritis, prosthetic loosening or osteoporosis (Goldring and Gravallese, 2000; Lorenzo et al., 1998; Merkel et al., 1999). There are notable common features of the signaling network utilized by RANK, the TNFRs and IL-1R including the ability to activate JNK, NF-κB and MAPK pathways (reviewed in Inoue et al., 2000). It is clear that RANK provides the full spectrum of signals required for osteoclast differentiation, activation and survival. As a key component of downstream signaling utilized by both RANK and IL-1R (Cao et al., 1996; Lomaga et al., 1999), TRAF6 may then be crucial during the late activation phase of osteoclasts. On one hand, the TRAF6 knockout mice generated by Lomaga et al. (1999) support this hypothesis, while analysis of another TRAF6 knockout (Naito et al., 1999) supports a role for TRAF6 throughout osteoclast differentiation and activation. In their paper, Kobayashi et al. (2000) hypothesized that a common pathway shared by RANKL and IL-1β mediated signaling would affect activation of osteoclasts. We have demonstrated that TRAF6 is not required for 22

23 23 early RANK-mediated events, but, as discussed, these cells fail to form the appropriate cytoskeletal structures and consequently cannot resorb CaPO 4 or form resorptive lacunae on dentin slices. Our data clearly shows that IL-1β alone cannot induce osteoclastogenesis in the absence of RANK signaling (Figure 9). However, by demonstrating that IL-1R activation, which utilizes TRAF6 independently of RANK, could rescue the defect in osteoclast resorption/activation of cells transduced with the RANK/TRAF6 mutant, we have highlighted the indispensable role of RANK/TRAF6 interactions during normal RANKL-induced osteoclast activation and maturation. In summary, analysis of RANK-mediated osteoclast differentiation and bone resorbing activity indicate that while the various RANK/TRAF signaling linkages are redundant and compensatory in the pathway that leads to differentiation, only RANK/TRAF6 binding is capable of leading to bone resorbing activity. 23

24 24 Acknowledgements: We are grateful to D. Anderson, J. Peschon, K. Schooley and D. Williams for helpful discussions and critical reading of the manuscript; S. Braddy,D. Hirchstein, and C. Huang for technical assistance; G. Carlton for help with graphics; A. Aumell for editorial assistance. 24

25 25 Footnotes. The abbreviations used are: CSF, colony stimulating factor; IL, interleukin; JNK, c- jun kinase; MOI, multiplicity of infection; NF-κB, nuclear factor-κb; OPG, osteoprotegerin; RANK, receptor activator NF-κB; RANKL, receptor activator NF-κB ligand; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; TRAF, tumor necrosis factor receptorassociated factor; TRAP, tartrate-resistant acid phosphatase 25

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30 30 Kong, Y.-Y., H. Yoshida, I. Sarosi, H.-L. Tan, E. Timms, C. Capparelli, S. Morony, A. Oliveirados-Santos, G. Van, A. Itie, W. Khoo, A. Wakeham, C.R. Dunstan, D.L. Lacey, T.W. Mak, W.J. Boyle, and J.M. Penninger OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397: Lacey, D.L., H.L. Tan, J. Lu, S. Kaufman, G. Van, W. Qiu, A. Rattan, S. Scully, F. Fletcher, T. Juan, M. Kelley, T.L. Burgess, W.J. Boyle, and A.J. Polverino Osteoprotegerin ligand modulates murine osteoclast survival in vitro and in vivo. Am. J. Pathol. 157: Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, H. Hsu, J. Sullivan, N. Hawkins, E. Davy, C. Capparelli, A. Eli, Y.-X. Qian, S. Kaufman, I. Sarosi, V. Shalhoub, G. Senaldi, J. Guo, J. Delaney, and W.J. Boyle Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93: Lakkakorpi, P.T., and H.K. Vaananen Cytoskeletal changes in osteoclasts during the resorption cycle. Microsc. Res. Tech. 33: Li, J., I. Sarosi, X.Q. Yan, S. Morony, C. Capparelli, H.L. Tan, S. McCabe, R. Elliott, S. Scully, G. Van, S. Kaufman, S.C. Juan, Y. Sun, J. Tarpley, L. Martin, K. Christensen, J. McCabe, P. Kostenuik, H. Hsu, F. Fletcher, C.R. Dunstan, D.L. Lacey, and W.J. Boyle RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc. Natl. Acad. Sci. USA 97:

31 31 Lomaga, M.A., W.-C. Yeh, I. Sarosi, G.S. Duncan, C. Furlonger, A. Ho, S. Morony, C. Capparelli, G. Van, S. Kaufman, A. van der Heiden, A. Itie, A. Wakeham, W. Khoo, T. Sasaki, Z. Cao, J.M. Penninger, C.J. Paige, D.L. Lacey, C.R. Dunstan, W.J. Boyle, D.V. Goeddel, and T.W. Mak TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40 and LPS signaling. Genes Dev. 13: Lorenzo, J.A., A. Naprta, Y. Rao, C. Alander, M. Glaccum, M. Widmer, G. Gronowicz, J. Kalinowski, and C.C. Pilbeam Mice lacking the type I interleukin-1 receptor do not lose bone mass after ovariectomy. Endocrinology 139: Matsumoto, M., T. Sudo, T. Saito, H. Osada, and M. Tsujimoto Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL). J. Biol. Chem. 275: Merkel, K.D., J.M. Erdmann, K.P. McHugh, Y. Abu-Amer, F.P. Ross, and S.L. Teitelbaum Tumor necrosis factor-α mediates orthopedic implant osteolysis. Am. J. Pathol. 154: Naito, A., S. Azuma, S. Tanaka, T. Miyazaki, S. Takaki, K. Takatsu, K. Nakao, K. Nakamura, M. Katsuki, T. Yamamoto, and J. Inoue Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice. Genes Cells 4:

32 32 Riggs, B.L., R. Baron, W.J. Boyle, M. Drezner, S. Manolagas, T.J. Martin, A.F. Stewart, T. Suda, H. Yasuda, J. Aubin, and D. Goltzman Proposed standard nomenclature for new tumor necrosis factor family members involved in the regulation of bone resorption. J. Bone Miner. Res. 15: Sanjay, A., A. Houghton, L. Neff, E. DiDomenico, C. Bardelay, E. Antoine, J. Levy, J. Gailit, D. Bowtell, W.C. Horne, and R. Baron Cbl associates with Pyk2 and Src to regulate Src kinase activity, α v β 3 integrin-mediated signaling, cell adhesion, and osteoclast motility. J. Cell. Biol. 152: Schwartzberg, P.L., L. Xing, O. Hoffmann, C.A. Lowell, L. Garrett, B.F. Boyce, and H.E. Varmus Rescue of osteoclast function by transgenic expression of kinase-deficient Src in src-/- mutant mice. Genes Dev. 11: Soriano, P., C. Montgomery, R. Geske, and A. Bradley Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64: Tanaka, S., M. Amling, L. Neff, A. Peyman, E. Uhlmann, J.B. Levy, and R. Baron c-cbl is downstream of c-src in a signalling pathway necessary for bone resorption. Nature 383: Teitelbaum, S.L Bone resorption by osteoclasts. Science 289:

33 33 Wong, B.R., D. Besser, N. Kim, J.R. Arron, M. Vologodskaia, H. Hanafusa, and Y. Choi TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-src. Mol. Cell. 4: Wong, B.R., R. Josien, S.Y. Lee, M. Vologodskaia, R.M. Steinman, and Y. Choi The TRAF family of signal transducers mediates NF-κB activation by the TRANCE receptor. J. Biol. Chem. 273: Yasuda, H., N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinosaki, S. Mochizuki, A. Tomoyasu, K. Yano, M. Goto, A. Murakami, E. Tsuda, T. Morinaga, K. Higashio, N. Udagawa, N. Takahashi, and T. Suda Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl. Acad. Sci. USA 95: Yeh, W.C., A. Shahinian, D. Speiser, J. Kraunus, F. Billia, A. Wakeham, J.L. de la Pompa, D. Ferrick, B. Hum, N. Iscove, P. Ohashi, M. Rothe, D.V. Goeddel, and T.W. Mak Early lethality, functional NF-kappaB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7: Yoshida, H., S. Hayashi, T. Kunisada, M. Ogawa, S. Nishikawa, H. Okamura, T. Sudo, L.D. Shultz, and S. Nishikawa The murine mutation osteopetrosis is in the coding region of the macrophage-colony stimulating factor gene. Nature 345:

34 34 Zhang, Y.-H., A. Heulsmann, M.M. Tondravi, A. Mukherjee, and Y. Abu-Amer Tumor necrosis factor-α (TNF) stimulates RANKL-induced osteoclastogenesis via coupling of TNF type 1 receptor and RANK signaling pathways. J. Biol. Chem. 276:

35 35 Figure Legends Figure 1. Expression of a panel of RANK constructs in infected cells. (A) Surface expression of human RANK constructs expressed via retroviral vectors in NIH3T3 cells. RANK expression was determined using mab 330 (Anderson et al., 1997) and FITC-labeled secondary antibody followed by flow cytometry analysis. (B) Quantitative real-time RT-PCR analysis of RNA from cells transduced with 6 different human RANK constructs normalized to HPRT expression. RNA was isolated from RANK-/- cells after 48 h incubation with each retroviral supernatant as described in Methods and Materials. Numbers are given as relative RNA levels in percent compared with the full-length hrank (mean +/- standard deviation). (C) Splenic hematopoietic progenitors from the RANK-/- mouse were infected with each of the hrank retroviral constructs and a control retrovirus encoding lacz. Cells were then cultured for 5 d with CSF-1 in the presence of RANKL. Indirect immunofluourescence was used to reveal the human RANK protein in permeabilized (green=intracellular RANK, red=nuclei) or non-permeabilized (red=surface RANK) cells. Figure 2. Rescue of osteoclast formation in RANK-/- cells by transduction of RANK and RANK mutants. (A) Splenic hematopoietic progenitors from the RANK-/- mouse were infected with each of the hrank retroviral constructs and a control retrovirus encoding lacz. Cells were then cultured for 5 d with CSF-1 in the absence or presence of RANKL followed by staining for TRAP activity. (B) Quantitation of TRAP + osteoclast formation. Cells were cultured and stained as described in (A), and TRAP + cells containing more than three nuclei were counted. Results from triplicate wells (+/- standard deviation) are shown. 35

36 36 Figure 3. RANK transduction induces expression of calcitonin receptors. Splenic hematopoietic progenitors from the RANK-/- mouse were infected with each of the hrank retroviral constructs and a control retrovirus encoding lacz. Cells were then cultured for 5 d with CSF-1 in the absence or presence of RANKL and incubated with 125 I-salmon calcitonin. After washing cells to remove unbound calcitonin, cells were lysed in 0.5 M NaOH and total bound counts measured using a gamma counter. Results from triplicate wells (+/- standard deviation) are shown. Figure 4. Analysis of osteoclast marker mrna expression in RANK-/- cells after transduction with wild-type (WT) RANK and RANK mutants. Quantitative real-time RT-PCR analysis of MMP-9, TRAP, c-src, β3integrin (CD61), cathepsin K, and carbonic anhydrase II RNAs. RNA was isolated from RANK-/- cells cultured 5 d after infection with each retroviral supernatant in the presence of CSF-1 and RANKL. Values are normalized to HPRT expression and are expressed as relative RNA levels in percent compared to that measured in cells transduced with full-length hrank (mean +/- standard deviation). Figure 5. Expression of c-src protein in multinucleated osteoclasts after transduction with RANK constructs. Immunofluorescence showing c-src staining (green) and nuclear staining (red). RANK-/- cells were infected with each of the RANK retrovirus constructs or a retrovirus encoding lacz and differentiated in the presence of RANKL and CSF-1 for 5 d. Cells were fixed with paraformaldehyde and processed for immunofluorescence as described (Materials and 36

37 37 Methods). Note the distinctive less-spread morphology and smaller size of c-src + cells differentiated after transduction with the RANK construct. Figure 6. Actin cytoskeleton of osteoclasts after transduction with RANK constructs. RANK-/- cells were infected with each of the RANK retrovirus constructs or a retrovirus encoding lacz and differentiated in the presence of RANKL and CSF-1 for 5 d. Cells were fixed with paraformaldehyde and processed for F-actin fluorescence using ALEXA 568 conjugated phalloidin (red) and nuclei were visualized with Sytox (green). (A) Cells differentiated after transduction with the full-length/wild-type (WT) RANK, or either of the C-terminal RANK deletions (RANK or RANK ) exhibited distinct podosome assembly (F-actin rings) in the periphery of most cells. Cells differentiated after transduction with the RANK/TRAF6 binding mutant (RANK ) showed a redistribution of the actin to the ends of lamellipodia-like structures. Images were taken with a 20 X objective. (B) Higher magnification(images were taken with a 60X objective) of the F-actin fluorescence in cultures transduced with the control lacz virus, WT RANK and the RANK/TRAF6 binding mutant (RANK ). Figure 7. c-cbl and c-src localization are disrupted in cells differentiated after transduction with the RANK/TRAF6 ( ) mutation. Immunofluorescence showing c-cbl (green) and c-src (red) localization in RANK-/- cells infected with lacz, full-length RANK or RANK retroviral constructs and differentiated in the presence of RANKL and CSF-1 for 5 d. The double-ring of red c-src staining with a intervening broad band of c-cbl was observed on the leading edge of migrating, multinucleated osteoclasts transduced with the full-length/wild-type 37

38 38 (WT) hrank construct. This distinct pattern was also seen on most multinucleated osteoclasts transduced with the full-length RANK (shown) or the RANK C-terminal deletions (RANK or RANK ) (data not shown). Note that the organization of c-src and c-cbl is disrupted in osteoclasts transduced with the RANK construct. Figure 8. Resorption of CaPO 4 by cells differentiated after transduction with different RANK constructs. Splenic hematopoietic progenitors from the RANK-/- mouse were infected with each of the hrank retroviral constructs and a control retrovirus encoding lacz. Cells were then cultured on a synthetic matrix of CaPO 4 for 5 d with CSF-1 in the absence (left panel) or presence (right panel) of 200 ng/ml RANKL. Following this cell culture, cells were removed and the remaining CaPO 4 matrix was revealed by staining with the calcium-specific dye, alizarin red. Areas of resorption by osteoclasts are visible as clear areas on the red background of remaining CaPO 4. Note that osteoclastic resorption was only observed after RANKL addition in cells transduced with the full-length (WT) human RANK or C-terminal deletion constructs (RANK or RANK ) lacking the binding sites for TRAFs1,2,3 and 5. Cells differentiated after transduction with the RANK/TRAF6 binding mutant (RANK ) were completely defective in CaPO 4 resorption even in the absence of exogenously added RANKL. Quantitation of the resorbed area was performed by measuring the surface area of cleared CaPO4 areas after staining the matrix with alizarin red as described in the Methods section. Results shown are representative of more than 5 replicate experiments. Values are expressed as relative resorbed area in percent compared to that measured in cells transduced with full-length hrank and treated with RANKL and were measured from wells in triplicate for each experiment and expressed as mean +/- standard deviation. 38

39 39 Figure 9. IL-1β rescues the defect in osteoclast resorption/activation of cells differentiated after transduction with the RANK/TRAF6 binding mutant (RANK ). Splenic hematopoietic progenitors from the RANK-/- mouse were transduced with the wild-type (WT) hrank, the RANK/TRAF6 binding mutant (RANK ), or the RANK construct and incubated with either RANKL (200 ng/ml), IL-1β (20ng/ml) or a combination of RANKL and IL-1β for a period of 5 d. (A) Cells were monitored for osteoclast differentiation by TRAP staining in situ. (B) Cells were assayed for their ability to resorb CaPO 4 illustrating that the block in resorption seen with cells transduced with RANK could be circumvented by the addition of IL-1β or IL-1β plus RANKL. Neither IL-1β nor RANKL promoted the osteoclast differentiation or the CaPO 4 resorption of cells transduced with the RANK construct which cannot bind any TRAFs and lacks all RANK-dependent signaling activities. (C) Quantitative analysis of the resorbed area. Figure 10. Dentin resorbing activity of OCL formed in culture with RANKL and/or IL-1β. Splenic hematopoietic progenitors from the RANK-/- mouse were transduced with the indicated constructs and cultured as described in figure 9. Transduced cells were assayed for the formation of resorption lacunae on dentin slices after culture for 7 days with the indicated cytokines. The C-terminal RANK deletions (RANK or RANK ) formed resorptive lacunae on dentin after addition of RANKL in a similar manner to that shown for the full-length RANK. Note that RANK expressing cells can only form authentic resorptive lacunae on dentin 39

40 40 slices when cultured with IL-1β plus RANKL. Similar results were obtained in three independent experiments. 40

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