Article |
Address correspondence to Steven L. Teitelbaum, Department of Pathology, Washington University School of Medicine, 216 South Kingshighway, St. Louis, MO 63110. Tel.: (314) 454-8463. Fax: (314) 454-5505. email: teitelbs{at}medicine.wustl.edu
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Abstract |
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Key Words: osteoclast; vß3 integrin; M-CSF; cytoskeleton; podosome
Abbreviations used in this paper: BMM, bone marrow macrophage; HGF, hepatocyte growth factor; LIBS, ligand-induced binding site; M-CSF, macrophage colonystimulating factor; OC, osteoclast; OPN, osteopontin; VN, vitronectin.
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Introduction |
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While serving as a matrix attachment molecule in OCs, vß3 is also a signaling receptor (Eliceiri et al., 1998; Duong et al., 2000) that induces changes in intracellular calcium (Paniccia et al., 1993; Zimolo et al., 1994), protein tyrosine phosphorylation, and cytoskeletal reorganization (Clark et al., 1998; Schlaepfer and Hunter, 1998). The signaling capacity of integrins can be regulated by extracellular matrix molecules that, interacting with the integrin external domain, stimulate "outside-in" signaling (Takagi et al., 2002). Alternatively, "inside-out" signaling is induced by trans-activated intracellular molecules, which interact with the cytoplasmic component of
vß3 and prompt conformational changes in the integrin's ligand binding site. Outside-in and inside-out signals control the affinity state of the integrin, thereby modulating its binding capabilities and, ultimately, intracellular events (Pelletier et al., 1995; Geiger et al., 2001; Butler et al., 2003). Several cytokines and growth factors enhance integrin-dependent intracellular events, via inside-out signaling. Platelet-derived growth factor induces
vß3-mediated adhesion in fibroblasts (Schneller et al., 1997), basic fibroblast growth factor augments migration in vascular endothelial cells (Kiosses et al., 2001), and macrophage colonystimulating factor (M-CSF) and hepatocyte growth factor (HGF) modulate OC function, in part by increasing activated
vß3 in the motile area of the membrane (Faccio et al., 2002).
In most cells, vß3 localizes with actin and other cytoskeletal proteins in focal adhesions (Ballestrem et al., 2001; Cukierman et al., 2001). OCs contain a related, but distinct, adhesive structure called the podosome, which consists of a core of F-actin bundles surrounded by a rosette-like structure containing
vß3, vinculin, and
-actinin (Marchisio et al., 1988). As ligand activation of
vß3 and growth factor stimulation promote podosome reorganization (Pfaff and Jurdic, 2001; Faccio et al., 2002), interest has turned to the intracellular components of the integrin and the signaling molecules linking it to cytoskeletal proteins.
c-Src is essential to OC function, as mice deleted of this tyrosine kinase develop osteopetrosis in the face of adequate numbers of dysfunctional OCs (Soriano et al., 1991). The fact that c-Src-/- OCs fail to organize a normal cytoskeleton suggests that c-Src may be a signaling molecule that associates with, and is activated by, vß3 (Duong et al., 2000; Sanjay et al., 2001). In addition, c-Cbl, a substrate of c-Src in OCs, is recruited to adhesion sites where it modulates the binding of the vitronectin (VN) receptor
vß3 (Sanjay et al., 2001). Pyk-2, a member of the FAK family of kinases, is a signaling molecule that binds c-Src and c-Cbl, and appears to be essential for bone resorption. Pyk2 is activated when OCs are plated on ligands recognized by
vß3 and is important for cytoskeletal organization during OC adhesion, migration, and sealing zone formation (Duong et al., 1998). The above compendium of events suggests that Pyk2 activation, in osteoclastic resorption, is mediated by
vß3.
Rho family GTP-ases control cytoskeletal organization and dynamics and integrin-mediated signaling, as their inhibition blocks vß3-dependent motility (Clark et al., 1998; Ridley et al., 1999; Chellaiah et al., 2000; Ory et al., 2000). Importantly, Rho and Rac regulate the OC actin ring, and their blockade blunts the resorptive activity of the cell (Razzouk et al., 1999; Ory et al., 2000).
In the present study, we establish that the ß3 cytoplasmic domain, specifically S752, is responsible for organizing the OC cytoskeleton. Furthermore, like adhesion-dependent cytoskeletal reorganization, growth factorinduced vß3 inside-out signaling activates Rho GTPases. Finally, although
vß3 is essential for activation of c-Src and c-Cbl, OCs lacking the integrin fully activate Pyk2.
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Results |
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External conformation of ß3 integrin does not depend on the cytoplasmic domain
One possible explanation for the failure of ß3-C and ß3 S752P to localize to podosomes is that the external domain of these mutants is not able to assume the activated, high-affinity conformation necessary for appropriate substrate interaction. One tool to assess the ability of ß3 integrin to assume the activated conformation of the external domain is the antiligand-induced binding site (LIBS) antibody AP5. In low calcium buffer, this Ab binds all
vß3 integrin on the cell surface, converting it to the active conformation. In high calcium buffer, AP5 binds only the integrin already in the activated state (Faccio et al., 2002). An increase in fluorescence intensity of
vß3-expressing cells when AP5 binding is assessed in low calcium, relative to high calcium buffer, indicates that the external domain of the integrin can assume the activated conformation in response to the Ab. When pre-OCs transduced with ß3-
C and ß3 S752P, as well as with ß3 WT and ß3 Y747F/Y759F, are analyzed in this manner, there is an increase in AP5 binding in low calcium, indicating that each of these ß3 constructs undergoes conformational change (Fig. 3 A). Thus, the failure of the ß3-
C and ß3 S752P mutants to properly localize in podosomes does not reflect an inability of these integrins to assume an activated conformation.
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Localization of activated vß3 integrin to lamellipodia requires the ß3 cytoplasmic domain
Upon growth factor activation, OC vß3 moves to the motile region of the cell membrane (Faccio et al., 2002). Having found that dysfunctional ß3 mutants fail to properly localize in resting OCs, and do not become activated in response to growth factors, we determined if these mutants also fail to localize properly in the newly formed lamellipodia in response to growth factor stimulation. Thus, ß3-/- OCs bearing WT ß3, ß3-
C, ß3 Y747F/Y759F, or ß3 S752P constructs grown on coverslips were exposed to HGF or M-CSF for 30 min and, after fixation, stained with AP5 (in high calcium) to detect localization of the activated form of
vß3. Unstimulated OCs carrying ß3 WT or the Y747F/Y759F mutation express the activated integrin along membrane ruffles and in lamellipodia (Fig. 4 A, CTR). Treatment with either growth factor induces AP5-positive membrane extensions (lamellipodia). This phenomenon is completely abrogated in ß3-
C and ß3 S752Pbearing cells, which are unable to spread and form lamellipodia in response to growth factors (Fig. 4 A). These observations are confirmed by counting the percentage of cells with multiple lamellipodia extensions (Table I). These data show that M-CSF and HGF induce lamellipodia in cells expressing ß3 WT and ß3 Y747F/Y759F but not in those transduced with ß3-
C or ß3 S752P.
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Growth factors promote cytoskeletal rearrangement in a ß3 integrindependent manner
The observation detailed in Fig. 4 led us to hypothesize that the ß3 integrin controls cytoskeletal changes induced by growth factors. To address this issue, we stained unstimulated and growth factortreated ß3+/+ and ß3-/- OCs with FITCphalloidin and analyzed actin organization by confocal microscopy. Once again, the absence of ß3 integrin does not alter the peripheral podosomal distribution of F-actin in untreated OCs (Fig. 5 A, CTR). However, in the presence of HGF or M-CSF, F-actin moves from the podosomes to short filamentous protrusions, consistent with lamellipodia formation, only in ß3+/+ OCs (Fig. 5 A, low and high magnification; Table I). To confirm that this observation is an integrin-dependent consequence of podosome reorganization, we examined the distribution of -actinin, a cytoskeletal protein involved in the formation and stability of podosomes, and a link between actin and integrins (Pavalko et al., 1991).
-Actinin distribution in ß3+/+ and ß3-/- OCs mirrors that of actin, both in the presence and absence of growth factors (Fig. 5 A). Consistent with this finding, immunoblot analysis shows an increase in the pool of
-actinin present in the Triton X-100 soluble fraction exclusively in ß3+/+ OCs treated with HGF and M-CSF (Fig. 5 B). In ß3-/- cells,
-actinin remains in the insoluble fraction. Similar results were obtained using OCs transduced with the different ß3 mutants. Dramatic changes in the peripheral ring of actin are seen in response to M-CSF (Fig. 5 C), and the content of
-actinin in the Triton X-100 soluble fraction increases in ß3 WT and ß3 Y747F/Y759F mutants, but not in those transduced with ß3-
C or ß3 S752P (Fig. 5 D). The failure of ß3-null cells to respond to M-CSF is not dependent on different expression levels of c-Fms (Fig. S1) or on its different localization among the various ß3 mutants (Fig. 5 E). These observations suggest that, in OCs, growth factorinduced reorganization of podosomes, leading to the formation of new membrane ruffles, is a ß3-dependent event.
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Rho family GTPase activation is impaired in ß3-/- OCs
Numerous signal transduction molecules, including Rho GTPases, associate with integrin complexes in adherent cells and regulate adhesion-dependent morphological changes (Pavalko et al., 1991; Yamada and Miyamoto, 1995; Clark et al., 1998). Furthermore, distinct aspects of adhesion and migration are controlled by different Rho family members. For example, Rho, Rac, and Cdc42, respectively, regulate the formation of stress fibers, lamellipodia, and filopodia. As ß3-/- OCs exhibit defective adhesion, spreading, and cytoskeletal rearrangement in response to growth factors, we asked if these phenomena reflect impaired activation of Rho GTPases. To this end, ß3-/- and ß3+/+ OCs were exposed to HGF or M-CSF, and Rho and Rac activation were assessed by pull-down binding assays. Mirroring the cytoskeletal defects, Rho activation in response to both growth factors is completely arrested in ß3-/- OCs, compared with the sixfold increase seen in ß3+/+ OCs (Fig. 7 A). Similarly, whereas Rac is activated in a biphasic manner in ß3+/+ OCs exposed to M-CSF, no such activation occurs in ß3-/- OCs (Fig. 7 B). To determine if integrin-mediated Rho activation is dependent on an intact ß3 cytoplasmic domain, we repeated these experiments with ß3-/- OCs transduced with the various ß3 constructs. Whereas ß3 WT and ß3 Y747F/Y759Fexpressing OCs respond to growth factor with an increase in GTP-bound Rho, no induction occurs in those expressing ß3-C or ß3 S752P (Fig. 7 C).
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Discussion |
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vß3, in OCs, exists in two conformational states, which are differentially distributed on the cell surface (Faccio et al., 2002). In its basal condition, the receptor localizes in the sealing zone and podosomes, while the activated integrin is principally associated with motile areas of the membrane. The extracellular components of
vß3 modulating its activation state are in hand (Beglova et al., 2002), and those in the cytoplasmic domain, which respond to growth factor stimulation and thus mediate inside-out signaling, have been partially identified (Takagi et al., 2002; Vinogradova et al., 2002). To further address this issue, we turned to a system of retroviral transduction, which previously permitted us to express various human ß3 integrin mutants in OCs and their precursors (Feng et al., 2001).
Our first exercise established that the ß3 cytoplasmic domain is essential for appropriate distribution of the integrin to cytoskeletal structures such as podosomes and lamellipodia. Mirroring their effect on OC spreading and matrix resorption, ß3 mutants, lacking the cytoplasmic domain or bearing S752P, distribute abnormally in OCs residing on glass and dentin, failing to colocalize with F-actin. This observation prompted us to ask if the same components of the ß3 integrin are involved in modulating the conformational state of the intact heterodimer.
Antibodies recognizing the activated conformation bind the LIBS in the NH2 terminus of integrin heterodimers (Bodeau et al., 2001). In high calcium buffer, the anti-LIBS mAb AP5 recognizes activated, high-affinity ligand-binding vß3 integrin. Importantly, in low calcium buffer, AP5 binds to all
vß3 and forces all receptors into the activated conformation. This AP5-induced conformational change of
vß3 involves a direct effect on the external domain of the integrin and does not require the ß3 cytoplasmic domain. On the other hand, the heterodimer's biological activity requires the cytoplasmic tail. Thus, ß3-/- OCs bearing ß3-
C or ß3 S752P have decreased adhesive and migratory capabilities, suggesting defective outside-in signaling. In contrast to the direct effect of AP5 in changing the external conformation of ß3, HGF and M-CSF modulate adhesion and spreading of mature OCs by activating the
vß3 integrin via the ß3 cytoplasmic domain by inside-out signaling (Insogna et al., 1997; Teti et al., 1998; Faccio et al., 2002).
Active OCs form a stable ring of actin, which delineates the ruffled membrane where the resorptive process takes place. Our previous observations, showing abnormal actin rings in ß3-null OCs generated in low dose M-CSF, indicate that ß3 contributes to the formation of this structure. We have also found that vß3 and M-CSF cooperate during osteoclastogenesis (Faccio et al., 2003). In this study, we find that ß3-/- OCs, or those expressing the human mutation S752P, exhibit normal actin distribution when generated in high dose M-CSF, indicating that M-CSF can compensate for lack of
vß3 in actin ring formation.
Podosomes, found in adherent OCs, are rosette-like structures containing vß3 around an actin core (Marchisio et al., 1988). In contrast to the relatively static actin ring, podosomes are dynamic, rapidly redistributing under the influence of extracellular stimuli such as HGF and M-CSF (Insogna et al., 1997; Teti et al., 1998; Faccio et al., 2002). Here we show that ß3 integrin is absolutely required for dynamic changes in the actin cytoskeleton in response to growth factors or cell attachment. ß3-
C or ß3 S752P mutants fail to form lamellipodia when plated on glass (Fig. 4 A) or on dentin (Fig. 4 B). In agreement with these observations,
-actinin, which links actin filaments directly to integrin receptors (Pavalko et al., 1991; Otey et al., 1993), fails to enter the Triton X-100 soluble fraction of ß3-/- pre-Ocs, or ß3-
C and ß3 S752P mutants, in response to growth factors. In other cell types, redistribution of
-actinin, from focal adhesions to the Triton X-100 soluble fraction, is associated with loss of close apposition of cell membrane to the extracellular matrix, consistent with enhanced motility (Greenwood et al., 2000).
Modulation of the OC cytoskeleton is controlled by Rho GTPases. For example, dominant negative Rho arrests podosome organization, OC motility, and bone resorption (Chellaiah et al., 2000), and Rac inhibition decreases the resorptive activity of OCs (Razzouk et al., 1999). The mechanisms of Rho and Rac activation in OCs are poorly defined. We find that the defect in migration and lamellipodia formation in ß3 S752P OCs, in response to growth factors, is associated with failure to activate Rho and Rac.
Interestingly, OCs bearing ß3 Y747F/Y759F are indistinguishable from wild-type cells in their appearance and resorptive capabilities, but adhesiveness to OPN is moderately decreased. Recently, phosphorylation levels of the ß3 Y747 and 759 have been correlated with strength of binding during adhesion (Boettiger et al., 2001). It is possible that in OCs, these tyrosines are required for stable and strong adhesion, but not for efficient migration. As motility is requisite for bone resorption, the Y747F/Y759F mutation does not compromise the physiological function of these cells.
The ability of M-CSF to induce OC spreading and actin reorganization depends on c-Src expression (Insogna et al., 1997). Sanjay et al. (2001) have shown that upon adhesion, vß3 forms a complex with Pyk2 and c-Src that, in turn, recruits c-Cbl, resulting in podosome assembly. This model holds that c-Cbl binds to Tyr 416 in the c-Src kinase domain, which down-regulates both Src kinase activity and integrin-mediated adhesion, prompting podosome detachment and subsequent disassembly. Consistent with this hypothesis, we find that activation of c-Src and c-Cbl, in response to VN adherence, is abrogated in ß3-/- OCs and in ß3-
C and ß3 S752P mutants, thereby decreasing podosome turnover and, consequently, OC adhesion and migration.
Our data, however, stand in contrast to the conclusions of Sanjay et al. (2001) and Nakamura et al. (2001), who claim that vß3 is essential for Pyk2 phosphorylation. We believe this discrepancy may reflect the fact that we directly assessed Pyk2 phosphorylation in authentic pre-OCs, deleted of the
vß3 receptor. Two possibilities present themselves as to how OCs lacking
vß3 phosphorylate Pyk-2. First, Pyk2 activation is calcium dependent (Sanjay et al., 2001). In this regard, the intracellular calcium chelator, BAPTA, blunts Pyk2 autophosphorylation in ß3+/+ and ß3-/- pre-OCs. Second, other adhesive receptors could compensate for the lack of ß3 and mediate Pyk2 phosphorylation. Pyk2 activation occurs equally in cells lacking
2ß1 integrin or CD44, another receptor for OPN, but not in ß2-/- pre-OCs. Thus, although it is formally possible that
2ß1, CD44, and ß3 compensate for each other in signaling to Pyk2, the weak Pyk2 phosphorylation detected in ß2-/- cells suggests that the ß2 integrin is dominant in this process. In support of this posture, uncommitted macrophages, which have yet to express
vß3, activate Pyk2 by ß2 integrin ligation (Duong and Rodan, 2000), and we find that the same is true in ß3-/- pre-OCs. Despite having the ß2 integrin, ß3-/- pre-OCs generated in high dose M-CSF do not retain a macrophage phenotype, as they express markers of committed OCs. As the ß2 integrin is not present in fully mature resorptive OCs (Athanasou and Quinn, 1990), in this circumstance,
vß3 may be the Pyk2 activating receptor.
Pyk2, independent of its phosphorylation status, is associated with paxillin, and this association is increased in adherent cells. Pyk2, however, fails to be recruited to the ß3 complex in adherent cells carrying ß3-C or S752P. It is possible that the failure of c-Src and c-Cbl to be activated in these mutants reflects the inability of Pyk2 to bind the integrin.
We propose, therefore, that in OCs, cytokines stimulate the formation of new membrane extensions that contain activated vß3 (Fig. 10 A). These cytoskeletal rearrangements are under the control of Rho family GTPases and require functional
vß3 (Fig. 10 B). Upon
vß3 occupancy, phosphorylated Pyk2, an event independent of the integrin, forms a complex at the ß3 cytoplasmic domain with phosphorylated c-Src and c-Cbl (Fig. 10 B). In the absence of functional
vß3, Pyk2 may be activated by other means, such as
Mß2 or increased calcium. These alternative means of activating Pyk2 permit its association with paxillin, but the Pyk2c-Srcc-Cbl adhesive complex fails to form, resulting in poorly resorptive OCs.
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Materials and methods |
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Infection of BMMs
BMMs were transduced with virus containing vectors that encode for several ß3 integrin mutants (Feng et al., 2001), in the presence of 1:10 CMG supernatant and 8 µg/ml polybrene (Sigma-Aldrich), without antibiotic selection. Cells were cultured for an additional 23 d before analysis of integrin expression or osteoclastogenesis.
Flow cytometry
Pre-OCs expressing the different mutants were lifted with Trypsin/EDTA (Sigma-Aldrich) and washed in a calcium-free buffer based on HBSS. Pretreated cells were incubated with HGF or M-CSF in -MEM supplemented with 0.5% BSA for 30 min at 37°C; control cells were incubated with medium alone. After incubation, cells were washed twice and incubated with the mAb AP5 (50 µg/ml) in high calcium buffer, which recognizes the activated ß3 integrin subunit. Binding of AP5 in HBSS calcium-free buffer served as positive control, identifying all ß3 on the cell surface. Cells were then incubated with FITC-conjugated secondary Ab, as previously described (Faccio et al., 2002).
Immunofluorescence
ß3+/+ or ß3-/- BMMs, transduced with the indicated mutants, were plated on dentin slices or glass coverslips under osteoclastogenic conditions for 4 d. For some experiments (Figs. 3 and 4), after 4 d in culture, cells were treated with HGF (50 ng/ml) or M-CSF (100 ng/ml), or media + 0.5% BSA as control, for 30 min at 37°C, and then fixed and stained as previously described (Faccio et al., 2002) and observed with a confocal microscope.
Adhesion and migration assays
Adhesion and haptotactic migration assays were performed using pre-OCs expressing the different ß3 mutants plated respectively onto 96-well plates or transwell filters, 8-µm pore size (Costar), coated with 10 µg/ml human OPN.
For both experiments, cells were preactivated with growth factors or AP5 for 30 min in suspension and then plated, and adherent cells were stained with crystal violet. For migration assay, cells that migrated to the lower side were viewed at 300x magnification and counted. Results represent the averages from 15 fields ± SEM of a representative experiment.
Western blot analysis
BMMs were cultured for 3 d in the presence of 100 ng/ml RANKL and 100 ng/ml M-CSF and starved overnight in the presence of 2% serum. Cells were lifted and replated onto the indicated matrix proteins for 30 or 60 min. In some experiments, adherent OCs were starved and restimulated with 100 ng/ml M-CSF or 50 ng/ml HGF. Cells were lysed in RIPA (Faccio et al., 2003), or in TNE (Lakkakorpi et al., 1999) for coimmunoprecipitation of c-Src and Pyk2. Precleared lysates were immunoprecipitated with 2 µg anti-Pyk2 polyclonal antibodies (Biosource International, CA), 2 µg anti-ß3 mAb (clone 7G2), or 2 µg antiphosphotyrosines (PY99) for 1 h at 4°C followed by overnight incubation with protein A/GSepharose beads at 4°C (Santa Cruz Biotechnology, Inc.) and then analysis by SDS-PAGE and immunoblotting.
Rho and Rac assay
Pre-OCs were lysed in a buffer containing 50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 500 mM NaCl, 10 mM MgCl2, 1% (vol/vol) Triton X-100, and protease inhibitors (4 µg/ml leupeptin and 30 µg/ml PMSF). Lysates were incubated with glutathioneagarose beads (Sigma-Aldrich) coupled with bacterially expressed GSTRBD fusion protein for Rho pull down or GSTPAK1 for Rac pull down (Ren et al., 1999) at 4°C for 45 min. Bound proteins were analyzed by SDS-PAGE followed by immunoblotting against RhoA (Santa Cruz Biotechnology, Inc.) or Rac1 (Upstate Biotechnology).
Online supplemental material
The supplemental material (Figs. S1 and S2) is available at http://www.jcb.org/cgi/content/full/jcb.200212082/DC1. Fig. S1 shows the expression levels of c-Fms, 2ß1, and ß2 in BMMs and pre-OCs. Fig. S2 shows the localization of viniculin, talin, and ß3 integrin in the podosomes.
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Acknowledgments |
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This work was supported by National Institutes of Health grants AR48812 and AR46852 to F.P. Ross and AR48853, AR46523, AR32788, and DK-56341 (Clinical Nutrition Research Unit) to S.L. Teitelbaum, a grant from the Italian Foundation for Cancer Research (AIRC), and a grant from the Italian Space Agency (ASI) (A. Zallone).
Submitted: 17 December 2002
Accepted: 12 June 2003
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