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Departments of Pathology and Immunology, Washington University School of Medicine (X.F., S.T., N.N., S.W., S.L.T., F.P.R.), St. Louis, Missouri 63110; and Department of Pediatrics, Okayama University Graduate School of Medicine and Dentistry (N.N.), Okayama 700-8558, Japan
Address all correspondence and requests for reprints to: F. Patrick Ross, Ph.D., Washington University School of Medicine, Department of Pathology, Barnes-Jewish Hospital North, Mailstop 90-31-649, 216 South Kingshighway, St. Louis, Missouri 63110. E-mail: rossf{at}medicine.wustl.edu.
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Abstract |
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B (RANK) and c-Fms, receptors for RANK ligand (RANKL) and M-CSF, respectively. Activated c-Fms autophosphorylates cytoplasmic tail tyrosine residues, which, by recruiting adaptor molecules, initiate specific signaling pathways. To identify which tyrosine residues are involved in c-Fms signaling in primary cells, we retrovirally transduced M-CSF-dependent bone marrow macrophages with a chimera comprising the external domain of the erythropoietin (Epo) receptor linked to the transmembrane and cytoplasmic domains of c-Fms. Transduced cells differentiate into bone-resorbing osteoclasts when treated with RANKL and either M-CSF or Epo, confirming that both endogenous and chimeric receptors transmit osteoclastogenic signals. Cells expressing chimeric receptors with Y697F, Y706F, Y721F, and Y921F single point mutations generate normal numbers of bone-resorbing OCs, with normal bone-resorbing activity when treated with RANKL and Epo. In contrast, those expressing Y559F generate fewer OCs, whereas theY807F mutant is incapable of osteoclastogenesis. Finally, although mature OCs expressing Y559F exhibit impaired bone resorption, those bearing Y807F do not. Thus, we have identified specific tyrosine residues in the cytoplasmic tail of c-Fms that are critical for transmitting M-CSF-initiated signals individually required for OC formation or function, respectively. |
Introduction |
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B (RANK) ligand (RANKL) (1, 2, 3, 4). The key osteoclastogenic role of M-CSF is established by the fact that op/op mice, which secrete no functional M-CSF, are devoid of OCs (1, 2), a phenotype that can be rescued by recombinant M-CSF (5). M-CSF initiates intracellular signaling by binding to its receptor, c-Fms, which, like other members of the receptor tyrosine kinase family, comprises an extracellular ligand-binding region, a single transmembrane sequence, and a cytoplasmic tail containing a tyrosine kinase domain. Upon ligand binding, the receptor dimerizes, stimulating its tyrosine kinase activity and thus autophosphorylating six tyrosine residues within the cytoplasmic tail, namely 559, 697, 706, 721, 807, and 921 (numbers based on the murine c-Fms sequence) (6). Experiments based on tyrosine point mutational analysis indicate that Y559, Y706, and Y721 are binding sites for c-Src, signal transducer and activator of transcription 1, and phosphoinositol 3-kinase, respectively, whereas Y697 and Y921 recognize Grb2 (6, 7). On the other hand, no protein has been shown to interact directly with Y807, the most heavily phosphorylated residue (7). All prior experiments relating to tyrosine residue function involved expression of c-Fms mutants in transformed cell lines and are thus of questionable physiological relevance. To address this issue, we turned to M-CSF-dependent bone marrow macrophages (MDBMs), cells of the myeloid lineage and precursors of fully differentiated OCs, which are M-CSF targets. To obviate the impact of endogenous c-Fms in these cells, we transduced them with a chimeric receptor comprising the external domain of the erythropoietin (Epo) receptor linked to the transmembrane and intracellular domains of c-Fms. The chimeric receptor-bearing MDBMs were then exposed to the key osteoclastogenic cytokine RANKL and recombinant Epo. The latter protein, on binding the chimeric receptor, activates a signaling cascade that mimics that generated by ligation of endogenous c-Fms by its ligand M-CSF. We also transduced MDBMs with forms of the chimeric receptor in which individual cytoplasmic tail tyrosine residues were replaced with phenylalanine, again followed by RANKL and Epo treatment. We show that although tyrosines 697, 706, 721, and 921 are not critical for the formation and/or function of osteoclasts, Y559 and Y807 have essential and distinct roles in these processes.
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Materials and Methods |
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Plat-E packaging cells were cultured in DMEM with 10% heat-inactivated FBS supplemented with puromycin and blasticidin as previously described (9). The plasmids were transiently transfected into Plat-E cells using Lipofectamine Plus reagent (Life Technologies, Inc., Gaithersburg, MD). Virus was collected at 48, 72, and 96 h after transfection.
Infection, selection, and expansion of bone marrow macrophages
M-CSF-dependent bone marrow macrophages (MDBMs) were cultured in the presence of 0.1 vol of culture supernatant of M-CSF-producing cells from bone marrow cells of 6- to 9-wk-old mice for 2 d as previously described (10). Then, cells were infected with virus for 24 h in the presence of 0.1 vol of culture supernatant of M-CSF-producing cells and 4 µg/ml polybrene (Sigma, St. Louis, MO). Cells were further cultured in the presence of M-CSF and 2 µg/ml puromycin for selection and expansion of transduced cells. Transduced cells were used for the studies described below.
Generation and use of osteoclasts
Retrovirally transduced precursors were cultured in the presence of 20 U/ml recombinant human Epo (or 20 ng/ml M-CSF for control) and 100 ng/ml RANKL in 96-well culture plates or 48-well culture plates with or without dentin slices. Osteoclasts began to form on d 3 and were stained for tartrate-resistant acid phosphatase (TRAP) on d 5 using a commercial kit (Sigma, 387-A). TRAP activity was measured by TRAP solution assay (11). For resorption studies osteoclasts were generated on whale dentin slices from infected or uninfected marrow macrophages as described above. Dentin was harvested on d 7, cells were removed with 0.25 M ammonium hydroxide and mechanical agitation, and the matrix was subjected to scanning electron microscopy (12). For analysis of bone resorption by cells expressing chimeric receptors bearing cytoplasmic tail point mutations, we prepared TRAP-positive mononuclear preosteoclasts (pOCs) by culturing infected MDBMs for 2 d with RANKL and M-CSF in the presence of puromycin (2 µg/ml). Cells were lifted and replated on dentin slices in the presence of RANKL and either Epo or, as a positive control, M-CSF. Dentin was harvested on d 2, and resorption pits were stained with Coomassie brilliant blue (10).
Western analysis
MDBMs infected with various point mutations or control virus were washed twice with ice-cold PBS. Western blot analysis was performed on 10-µg protein aliquots as described previously (13). In brief, electrophoresis in 6% SDS-PAGE was followed by transfer to nitrocellulose, with blocking using 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20. Membranes were exposed overnight at 4 C to rabbit polyclonal antibodies, washed, and incubated with secondary goat antirabbit IgG horseradish peroxidase-conjugated antibody for 1 h. The polyclonal antibodies used were c-Fms (C-terminal, C-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), phospho-c-Fms (Y721; Cell Signaling Technology, Inc., Beverly, MA), and ß-actin (Sigma). Membranes were washed extensively, and an enhanced chemiluminescence detection assay (Pierce Chemical Co., Rockford, IL) was performed following the manufacturer’s directions.
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Results |
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). Epo exposure may stimulate a chimeric receptor to initiate signals from either its wild-type or mutated cytoplasmic domain. Alternatively, the same cells will activate endogenous c-Fms when exposed to M-CSF (Fig. 1), again resulting in signals resulting in OC formation and function. Thus, Epo or M-CSF treatment of transduced MDBMs permits delineation of those cytoplasmic tail tyrosine residues mediating specific cellular events in physiologically relevant cells. As our studies were aimed at defining tyrosine residues important for osteoclast differentiation and function, all subsequent experiments were performed in the presence of optimal amounts of RANKL.
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). In addition, MDBMs transduced with either chimera exhibited bands of the expected size, namely 90 kDa, with the levels of both chimeric receptors being almost identical and approximating those of mature, endogenous c-Fms. Next, as a marker of signal transduction, we examined phosphorylation of the EpoR/c-Fms chimera in cells stimulated with Epo. As shown in Fig. 2b, Epo exposure of EpoR2/ c-Fms-expressing cells leads to the transient phosphorylation of residue Y721, whereas EpoR1/c-Fms does not (data not shown).
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). These correspond to the previous signaling data, as shown in Fig. 2b
. Establishing that EpoR2/c-Fms signals like endogenous c-Fms, cells bearing this chimera also survive and proliferate after Epo exposure. In contrast, Epo fails to support the viability of EpoR1/c-Fms-expressing cells. Consistent with the essential survival signals generated by c-Fms, all cells exposed to RANKL alone die. On the other hand, transduced cells treated with M-CSF plus RANKL differentiate into osteoclasts in all circumstances. Finally, after treatment with RANKL and Epo, cells expressing EpoR2/c-Fms give rise to osteoclasts whose number and appearance are indistinguishable from those generated from the same cells with M-CSF and RANKL. In contrast and consistent with its failure to promote macrophage survival or autophosphorylation in response to Epo, the EpoR1/c-Fms chimera fails to transmit osteoclastogenic signals.
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), findings that parallel the capacity of precursors carrying the same chimera to generate TRAP-positive osteoclasts. Thus, Epo treatment of cells carrying a chimeric receptor containing the EpoR external domain coupled to the transmembrane and cytoplasmic tail portions of c-Fms can functionally replace the signals created by ligation of endogenous c-Fms by M-CSF, its natural ligand.
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). As expected, treatment of cells bearing the different mutants with RANKL and M-CSF led to the formation of many osteoclasts (Fig. 5b, upper panels). Furthermore, treatment with Epo and RANKL of OC precursors containing any of the single Y to F mutations at position 697, 706, 721, or 921 gave rise to OCs in numbers equivalent to those bearing the nonmutated chimera or exposed to M-CSF and RANKL. In contrast, substantially fewer OCs were derived by Epo/RANKL treatment of cells bearing either Y559F or Y807F mutation (Fig. 5b, lower panels). In this single experiment the numbers of OCs in the presence of M-CSF vs. Epo were 51/0, 55/52, 54/5, 54/48, 55/45, 46/42, 52/17, and 55/45 in vector alone (vehicle), wild-type EpoR/c-Fms, Y549F, Y697F, Y706F, Y721F, Y807F, and Y921F mutants, respectively. Furthermore, to quantify the effects of each individual Y to F mutation for osteoclastogenesis, a TRAP solution assay was performed. Figure 5c clearly shows that Y559 and Y807 are critical for osteoclastogenesis, confirming the results in Fig. 5b.
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, upper panels). On the other hand, Epo and RANKL treatment generated numerous OCs using as targets pOCs expressing wild-type chimera (control), Y697F, Y706F, Y721, Y807F, and Y921F mutants. Although vector alone (vehicle) generated no osteoclasts, as expected, the number of TRAP-positive multinucleated cells was slightly lower with Y807F and was almost zero in the case of Y559F. These data demonstrate that pOCs expressing all mutant receptors except Y559F can fully differentiate into mature OCs from pOCs within 24 h. TRAP staining of cells kept in culture for 2 d revealed that, once again, all mutants except Y559F generated numerous OCs (Fig. 6b, top panels). To evaluate the resorptive capacity of these cells, they were removed from the dentin, which was then stained by Coomassie brilliant blue, a procedure identifying the presence of resorption pits. Once again, cells bearing a Y to F mutation at positions 697, 706, 721, or 921 in the cytoplasmic tail all generated numerous pits, whereas those bearing Y599F were functionally inactive (Fig. 6b, lower panels). pOCs expressing wild-type chimera receptor could not generate any pits in the absence of RANKL (data not shown). Of note, and in contrast to the role of Y807 in osteoclast formation, mutation of this residue to phenylalanine failed to suppress the capacity of cells to resorb bone. pOCs bearing the Y559F mutant generated equivalent numbers of OCs and pits compared with the wild-type chimera receptor when cultured in the presence of M-CSF and RANKL (data not shown). These data show that the expression of this chimeric receptor does not inhibit signaling through the endogenous c-Fms receptor.
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Discussion |
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Previous studies of the role of individual c-Fms tyrosine residues in cell function and signaling were performed by transfecting wild-type or mutated c-Fms into either fibroblasts or IL-3-dependent myeloid cells, neither of which normally expresses the receptor. The presence of wild-type c-Fms renders such cells responsive to treatment with M-CSF, manifest either as a change in the proliferation rate or, in the case of the myeloid cells, a limited degree of differentiation. The data obtained from these and related studies are conflicting and/or incomplete (6). For example, expression of the Y807F point mutant in NIH-3T3 cells leads to almost complete loss of proliferation in response to M-CSF. In contrast, the same mutant, when expressed on Rat-1 fibroblasts, minimally alters the capacity of the cells to divide in response to M-CSF. Finally, in the IL-3-dependent FDC-P1 line, activation of c-Fms Y807F again enhances proliferation (16). Importantly, fibroblasts and M-CSF-independent myeloid cells cannot become osteoclasts, a major consequence of M-CSF action, and thus it is not possible to gain meaningful insights into the role of c-Fms in differentiation into bone-resorptive polykaryons when using such cells. In summary, although M-CSF is clearly essential for differentiation of MDBMs, the role of individual tyrosine residues in the cytoplasmic tail of its receptor c-Fms in signal transduction is unclear. To address this issue we turned to transduction of appropriate chimeric receptors into these cells, which represent authentic OC precursors.
We have previously expressed the ß3 integrin subunit in MDBMs by retroviral transduction (17). We used an analogous approach to investigate the role of individual cytoplasmic tyrosine residues of c-Fms in OC differentiation and function. The fact that the c-Fms null mouse, if available, would have few or no bone marrow macrophages as targets for transduction, required that we use wild-type MDBMs, which express endogenous c-Fms. To overcome this problem we developed a chimeric receptor strategy. We believed that this approach might be successful, as chimeric transmembrane receptors have, in other circumstances, provided important information on the role of individual tyrosine residues in intracellular signaling (18, 19, 20, 21, 22, 23). Of greater relevance, similar methods have been applied to analysis of the role of specific cytoplasmic tail tyrosine residues in c-Fms signaling, albeit not in cells endogenously expressing the receptor (24, 25).
We first engineered two chimeric proteins, each containing the external domain of the EpoR and the cytoplasmic tail of c-Fms. We chose to use the external domain of the EpoR because it, like c-Fms, signals as a dimer. Given this fact and reports in which the external domain of EpoR has been used to initiate signaling from the cytoplasmic tail of receptor tyrosine kinases, we reasoned that one or more of these chimeras would activate c-Fms-induced intracellular signals. In addition, as neither osteoclasts nor their precursors express EpoR, Epo would stimulate exclusively the chimeric receptor. Our data indicate that only the chimeric protein comprising the extracellular domain of EpoR linked to the transmembrane and intracellular domains of c-Fms is capable of mimicking the endogenous c-Fms in mediating osteoclast differentiation. These findings initially suggest that the transmembrane domain of c-Fms is required for signaling by the chimera. However, an alternative explanation is provided by recent reports that the transmembrane domain of the EpoR plays an unexpected role in transducing signals (26). This region of the molecule has dimerizing capacity in the absence of receptor liganding and also mediates changes in the conformation of the intracellular tail after binding of Epo (27). Thus, it is possible that the Epo transmembrane domain acts as a dominant negative regulator of c-Fms activation.
This chimeric receptor approach allowed us to identify Y559 and Y807 as two important tyrosine residues in the c-Fms cytoplasmic tail involved in osteoclast differentiation and/or function. Previous studies have suggested that Y559 in the c-Fms cytoplasmic tail associates with c-Src (28). Given that osteoclastogenesis is not impaired in c-Src knockout mice (29), it is unlikely that c-Src plays a direct role in c-Fms-mediated osteoclast differentiation. Thus, the mechanism by which Y559 in the cytoplasmic tail of c-Fms contributes to OC differentiation and function is unknown.
Consistent with a previous report demonstrating that Y807 plays a role in M-CSF-induced FDC-P1 cell differentiation (16), we found that this residue also involved in osteoclast differentiation. In contrast, signals emanating from this residue are not required for the resorptive capacity of mature osteoclasts. As studies have failed to identify the signaling molecule(s) binding to Y807, the downstream signaling pathway derived from this tyrosine residue is also unknown.
In summary, the current report represents the first study of c-Fms signaling in which identification of functional tyrosine residues was performed in a physiologically relevant cellular background. This approach enables us to establish that Y559 and Y807 in the c-Fms cytoplasmic tail are two critical residues mediating osteoclast differentiation and that the former residue is also important for the resorptive capacity of the mature bone-resorbing polykaryon. These data serve as a starting point to elucidate c-Fms signaling pathways in osteoclast differentiation and function.
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Acknowledgments |
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Footnotes |
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1 X.F. and S.T. equally contributed to this study. 
Abbreviations: Epo, Erythropoietin; EpoR, erythropoietin receptor M-CSF, macrophage colony-stimulating factor; MDBM, macrophage colony-stimulating factor-dependent bone marrow macrophages; OC, osteoclast; pOC, preosteoclast; RANK, receptor activator of nuclear factor B; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase.
Received May 1, 2002.
Accepted for publication August 6, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand. J Clin Invest 106:1481–1488[Medline]
B in osteoclast precursors. Endocrinology 142:1290–1295This article has been cited by other articles:
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) linked to the cytoplasmic domains of c-Fms (
), with transmembrane domains derived from either c-Fms or EpoR. When transduced MDBMs are treated with M-CSF and RANKL, they will differentiate into osteoclasts, using endogenous c-Fms (left panel). The same cells, when treated with Epo and RANKL, may differentiate into osteoclasts using either and/or both chimeric receptors (middle and right panels).
