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Vol. 18, Issue 12, 4899-4910, December 2007

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CD44 and β3 Integrin Organize Two Functionally Distinct Actin-based Domains in Osteoclasts

Anne Chabadel^*, Inmaculada Bañon-Rodríguez, David Cluet, Brian B. Rudkin, Bernhard Wehrle-Haller, Elisabeth Genot^||, Pierre Jurdic^*, Ines M. Anton, and Frédéric Saltel^*,||

*Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Institut Fédératif Biosciences Gerland Lyon Sud, Université Lyon 1, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, Ecole Normale Supérieure de Lyon, 69364 Lyon, France; Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Cientificas-Universidad Autonoma de Madrid, 28049 Madrid, Spain; Laboratoire de Biologie Moléculaire de la Cellule, Unite Mixte de Recherche 5239 Centre National de la Recherche Scientifique/Ecole Normale Supérieure Lyon, Université Lyon I, Institut Fédératif de Recherche "BioSciences Lyon-Gerland," Ecole Normale Superieure de Lyon, 69364 Lyon Cedex 07, France; Department of Cellular Physiology and Metabolism, Centre Médical Universitaire, 1211 Geneva 4, Switzerland; and ^||European Institute of Chemistry and Biology, Unité Institut National de la Santé et de la Recherche Médicale 889, Université Victor Segalen Bordeaux 2, L'Institut Fédératif de Recherche 66, 33 600 Pessac, France

Submitted April 26, 2007; Revised September 4, 2007; Accepted September 14, 2007
Monitoring Editor: David Drubin

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The actin cytoskeleton of mature osteoclasts (OCs) adhering to nonmineralized substrates is organized in a belt of podosomes reminiscent of the sealing zone (SZ) found in bone resorbing OCs. In this study, we demonstrate that the belt is composed of two functionally different actin-based domains: podosome cores linked with CD44, which are involved in cell adhesion, and a diffuse cloud associated with β3 integrin, which is involved in cell adhesion and contraction. Wiskott Aldrich Syndrome Protein (WASp) Interacting Protein (WIP)–/– OCs were devoid of podosomes, but they still exhibited actin clouds. Indeed, WIP–/– OCs show diminished expression of WASp, which is required for podosome formation. CD44 is a novel marker of OC podosome cores and the first nonintegrin receptor detected in these structures. The importance of CD44 is revealed by showing that its clustering restores podosome cores and WASp expression in WIP–/– OCs. However, although CD44 signals are sufficient to form a SZ, the presence of WIP is indispensable for the formation of a fully functional SZ.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Podosomes are highly dynamic adhesion structures found in monocyte-derived cells and in v-src–transformed fibroblasts, and they can be induced by cytokines or phorbol esters in vascular cells. They are thought to carry out two major functions: adhesion and extracellular matrix (ECM) degradation (Tarone et al., 1985; Correia et al., 1999; Linder et al., 1999, 2007a; Burns et al., 2001; Moreau et al., 2003; Varon et al., 2006). Recently, podosomes found in leukocytes have been shown to initiate transcellular diapedesis (Carman et al., 2007). Podosomes are formed by an F-actin core surrounded by several focal adhesion proteins such as integrins, vinculin, paxillin, and talin (Marchisio et al., 1988; Zambonin-Zallone et al., 1989; Pfaff and Jurdic, 2001; Linder and Aepfelbacher, 2003; Linder, 2007a). However, podosomes clearly differ from focal adhesions by their shorter lifetime and by the architectural organization of these proteins. Actin-associated proteins such as cortactin, Arp2/3. and Wiskott Aldrich Syndrome Protein (WASp) are associated with the F-actin core. WASp is associated with WASp Interacting Protein (WIP), which exerts its functions by regulating the spatial organization of actin polymerization in all cell types studied so far (Anton and Jones, 2006). WIP can also interact with other proteins implicated in podosome formation such as cortactin or actin itself (Ramesh et al., 1997; Martinez-Quiles et al., 2001; Kinley et al., 2003; de la Fuente et al., 2007). WIP has indeed been detected in the podosomes of endothelial cells (Moreau et al., 2003) and more recently in dendritic cells (Chou et al., 2006) where it was found to regulate podosome formation. In these cells, knock down of WIP resulted in a decreased number of podosomes (Chou et al., 2006). However, the role of WIP in osteoclasts (OCs) has not yet been investigated.

OCs are giant multinucleated bone-resorbing cells derived from the fusion of monocytic precursors stimulated by the receptor activator of nuclear factor B-ligand (RANK-L) and macrophage colony-stimulating factor (M-CSF) (Lacey et al., 1998; Boyle et al., 2003). OCs can adhere to several substrates on which they form distinct F-actin structures. During the in vitro differentiation of OCs, cells exhibit numerous podosomes that self-organize along the differentiation process. Podosome clusters are found in early OCs, which evolve into dynamic and transient rings at intermediate stages and end up forming peripheral podosome belts in mature cells (Destaing et al., 2003). Based on in vitro observations, it has been proposed that podosomes are essential for ECM degradation, invasion, and migration in vivo (Mizutani et al., 2002; Calle et al., 2006; Redondo-Munoz et al., 2006; Tatin et al., 2006; Linder, 2007b). In vivo, bone resorbing OCs exhibit a sealing zone (SZ), made of a large circular band of F-actin with an inner and outer lining of vinculin (Lakkakorpi et al., 1991; Saltel et al., 2004; Jurdic et al., 2006; Luxenburg et al., 2007. The SZ delineates the area of enzyme secretion and the transcytosis of degraded materials occurs from basal-to-apical membranes (Nesbitt and Horton, 1997; Salo et al., 1997). OCs adhering to glass spread and form podosome belts, whereas they become polarized and exhibit SZ when seeded onto apatite (Saltel et al., 2004). The spatiotemporal relationship between these two structures formed in cultured and bone attached osteoclasts is still open to conjecture. Whereas no individual podosomes could be detected at the SZ formed in OCs seeded on a mineralized substrate, by means of time-lapse video confocal microscopy (Saltel et al., 2004), a novel approach based on high-resolution electron microscopy revealed that the structure is made of a highly dense network of podosomes interconnected with radial bundles of actin (Luxenburg et al., 2007).

OC adhesion is critical for its function of bone resorption and OCs express several matrix receptors, including vβ1, 2β1, and vβ3 integrins (Clover et al., 1992; Nesbitt et al., 1993). Although vβ3 integrins were described to be the most implicated in OC biology (Faccio et al., 2003a,b; Zou et al., 2007), another major receptor is represented by CD44 (Chellaiah et al., 2003a,b). CD44 is a cell surface, single-pass transmembrane proteoglycan expressed in most cell types. The roles played by CD44 are diverse, but great interest in CD44 results from its function as a receptor for hyaluronic acid (HA) (Ponta et al., 2003). Although HA is the principal ligand for CD44, it also interacts with osteopontin (OPN), collagen, and laminin (Goodison et al., 1999).

In the present study, we analyzed the molecular composition and F-actin organization of the OC podosome belts. We first confirmed that the belt is composed of two distinct entities, the podosome core visualized as an F-actin dot that we described herein associated with CD44 and an actin cloud surrounding the core and that matched the β3 integrin pattern. This finding demonstrates for the first time the presence of CD44 in podosomes, and it describes the first nonintegrin receptor in the podosome core. Interestingly, podosome core formation, but not the actin cloud, was abrogated in OCs obtained from WIP–/– mice, demonstrating a structural independence between these different F-actin structures. The absence of podosomes could be explained by decreased WASp expression levels in WIP–/– OCs. In addition, by providing CD44 signals, we were able to reinduce podosomes in WIP–/– OCs, and the reappearance of podosomes was correlated with WASp recovery, indicating that CD44 activation could regulate WASp and promote podosome formation in the absence of WIP. We further show that podosome cores and actin clouds are involved in distinct functions in OC physiology where podosome cores play a major role in adhesion whereas the actin cloud controls contractility. We conclude that the clustering and activation of CD44 is sufficient to form a SZ. However, because WIP–/– OCs displayed reduced resorbing activities, we demonstrate that the presence of WIP is necessary for the formation of a fully functional SZ.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mice
The generation of WIP–/– mice has been described previously (Anton et al., 2002). Mice are from a mixed 129/SvJ x C57Bl/6 background. They were kept under specific pathogen-free conditions at the Centro de Biología Molecular "Severo Ochoa" (Madrid, Spain). Comparisons are made between cells obtained from age and sex-matched WIP–/– and wild-type (WT) mice.

Cell Cultures
Spleen and bone marrow cells of WT and WIP–/– mice were cultured for 8 d in six-well or 12-well plates in differentiation medium: -minimal essential medium (MEM) containing 10% fetal bovine serum, M-CSF, and RANK-L (Destaing et al., 2003). For Western blot analysis, OCs were rapidly washed in ice-cold phosphate-buffered saline (PBS), and proteins were extracted with lysis buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 1% Triton X-100, 10% glycerol, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, and 1 µg/ml each of leupeptin, pepstatin, and aprotinin).

Reagents
Recombinant human RANK-L and human M-CSF were produced as described previously (Destaing et al., 2003). Culture media were from Invitrogen (Invitrogen). Anti-WIP antiserum was raised by Proteintools (Madrid, Spain) by immunizing rabbits with a 19-amino acid C-terminal peptide of WIP (KLARNESRSGSNRRERGGP) (Lanzardo IJBCB 39 2007). Anti-paxillin (clone 349) was from BD Transduction Laboratories (Lexington, KY). Anti-phospho-paxillin was from Invitrogen (Carlsbad, CA). Anti-vinculin (clone Vin 11-5), anti--actinin (clone BM-75.2), and anti-myosin (cloneMY-32) were from Sigma-Aldrich (St. Louis, MO). Anti-CD44 (H-CAM [H-300], sc-7946; H-CAM [F-4] sc-9960) and anti-WASp (sc-13139) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Cortactin (p80/85; clone 4F11) and anti-phospho-tyrosin (clone 4G10) were from Upstate Biotechnology (Charlottesville, VA). Anti-CD61 (integrin β3 subunit) and anti-CD29 (integrin β1 chain; clone 9EG7 and clone Ha2/5) were from BD Biosciences PharMingen (San Diego, CA). Anti-Arp3 was a generous gift from E. Znameroski (Welch laboratory, University of California, Berkeley, CA). Alexa Fluor-555-Phalloidin was from Molecular Probes (Eugene, OR). The myosin inhibitors blebbistatin (Sigma-Aldrich) and ML-7 (Calbiochem, San Diego, CA) were used for 20 min at 50 and 0.5 µM respectively. Vitronectin (VN), collagen, and laminin were obtained from Sigma-Aldrich, and they were incubated on glass slides at 10 µg/ml for 1 h. Osteopontin and hyaluronic acid (Sigma-Aldrich) were incubated on glass slides at 5 µg/ml for 1 h. After rinsing, coated slides were blocked for 1 h in bovine serum albumin 0.5% in PBS (Sigma-Aldrich). To study the blocking of CD44 or β3 integrin subunit signaling, OCs were incubated with 10 µg/ml CD44 blocking antibodies for 20 min (sc-7946; Santa Cruz Biotechnology), or 10 µM cyclic Arg-Gly-Asp (cRGD) peptides (Bachem, Bubendorf, Switzerland; Legler et al., 2001) for 30 min, respectively. For the activation of CD44 signaling, OCs were incubated on glass coverslips coated first with 10 µg/ml poly-L-lysine (Sigma-Aldrich) for 2 h and then overnight with a CD44-activating antibody (clone A3D8; Sigma C7923). Cell cultures were fixed 4 h after being seeded on these matrices.

Immunofluorescence
Cells were fixed in 3.7% paraformaldehyde, pH 7.2, for 10 min, permeabilized with 0.2% Triton X-100 for 7 min, and incubated with various antibodies. F-actin distribution was revealed by Alexa Fluor-555-Phalloidin. Cells were imaged with a confocal LSM 510 (Carl Zeiss Microimaging, Jena, Germany) by using a 63x (numerical aperture [NA] 1.4) Plan Neofluar objective. To prevent contamination between fluorochromes, each channel was imaged sequentially using the multitrack recording module before merging. Z-cut pictures were obtained using LSM 510 software. Deconvoluted images were obtained from Z-cut pictures (X = 40 nm, Y = 40 nm, Z = 131 nm), by using Huygens essential software (BioVision Technologies, Exton, PA).

Interference Reflection Microscopy (IRM)
The contact areas of WT or WIP–/– OCs were assessed by IRM. Adherent cells were imaged with an Antiflex Plan-Neofluor Ph3, 63x NA 1.25 oil immersion objective, equipped with a /4 plate and custom-made polarization and analysis filter, mounted on an Axiovert 100M (both from Carl Zeiss Microimaging). Images were captured with a 12-bit digital charge-coupled device camera (model ORCA 4742-95; Hamamatsu Photonics, Massy, France), controlled by Openlab software (Improvision, Coventry, United Kingdom). Image analysis was performed using the MetaMorph software package (Molecular Devices, Sunnyvale, CA).

Measurement of OC Surface Areas
OCs were stained for F-actin and imaged with a 20x (NA 1.0) Plan-Achromat objective (Carl Zeiss Microimaging). OC surface area was measured using MetaMorph software. Results are plotted as the mean number of pixels included in the OC surface area, ± SD, out of three wells.

Measurement of Resorption Efficiency
At the end of the differentiation process on plastic dishes, mature OCs were removed with four washes with PBS– 0.25 mM EDTA for 30 min each and then seeded on various substrates. OCs were seeded on dentin slices (a generous gift from Dr N. Takahashi, Matsumoto Dental University, Nagano, Japan) or on apatite collagen complex (ACC) slides in differentiation medium for 2 d. ACC was prepared using the method described previously (Shibutani et al., 2000; Saltel et al., 2004). Remaining cells were removed by immersion in water, and ACC slides were stained with silver nitrate to detect resorption pits under a light microscope. Resorbed areas were assessed using a stereomicroscope (MZ12; Leica, Wetzlar, Germany) and using MetaMorph and Photoshop software (Adobe Systems, Mountain View, CA). Results were plotted as the mean number of pixels ± SD out of three wells, and they were representative of three independent experiments.

Measurement of F-Actin Intensities
WT and WIP–/– cells were fixed and stained for F-actin. Thirty pictures of the actin cytoskeleton were acquired at high magnification with identical excitation and exposure settings by using the confocal microscope described above. The intensity of every single pixel was measured with MetaMorph software, as described by Cluzel et al. (2005). After smoothing (3 x 3 kernel), background substraction, and manual selection of the cell surface using MetaMorph software, data were exported to Excel (Microsoft, Redmond, WA) for further analysis. Cumulative fluorescence intensity histograms were obtained by multiplying the number of pixels with their respective gray value. This measurement was repeated on 30 different podosome belts, and an average graph was drawn. The intensity limit between actin cloud and podosome cores was fixed at an intensity value of 150, corresponding to the separation between the curve and its tangent.

OC Microinjections
Mouse spleen cell-derived OCs, differentiated in vitro on Eppendorf CELLocate coverslips for 7 d in differentiation medium, were transferred to medium buffered with 20 mM HEPES, pH 7.4. Intranuclear microinjections of cDNA (3.5 mg/ml in 0.05 M Tris-HCl, pH 7.4) were carried out at room temperature using an Eclipse TE 200 inverted microscope (Nikon, Tokyo, Japan) with an InjectMan micromanipulator and an Eppendorf 5246 microinjector. After injection, cells were further maintained at 37°C and 5% CO₂ for 6 h in differentiation medium, before imaging.

OCs were transfected with either control plasmid (pcDNA), or with plasmid containing the human WIP coding sequence (pcDNA-WIP), as described previously (Anton et al., 2002). These plasmids were kindly provided by N. Ramesh (Children's Hospital, Boston, MA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CD44 and β3 Integrin Receptors: Mediators of Two Distinct F-Actin–based Domains of the Podosome Belt
Podosomes are considered as the structural unit of the OC actin cytoskeleton in vitro. During osteoclastogenesis, the pattern of podosome organization evolves from clusters in the early stages to expanding dynamic rings at intermediate stages and finally to belts localized at the cell periphery in mature OCs (Destaing et al., 2003). To get more insight into the organization of F-actin in this continuously evolving structure, phalloidin labeling was performed and analyzed using confocal microscopy. Data presented in Figure 1 revealed that the podosome belt (Figure 1A) was, in fact, composed of two distinct F-actin entities (Figure 1B). Strong fluorescence intensity matching dense dot-like fibrillar actin (Figure 1B, black star), delimited the podosome cores, which were found connected with a more diffuse, low-intensity F-actin staining (Figure 1B, white star), which we defined as the actin cloud. To examine the contribution of these two distinct structures to the podosome belt, the pixel/intensity distribution from >30 cells was quantified, averaged, and expressed in the form of a histogram, reflecting the surface and relative fluorescence intensity of these two domains (Figure 1C). A Z-section of a podosome belt showed podosome cores as intense columns of fibrillar actin, whereas the actin cloud manifested itself as a thin continuous network connecting podosomes with each other (Figure 1D, top). Deconvolution analysis of this Z-section confirmed that podosomes and clouds were interconnected (Figure 1D, bottom). These data unambiguously established that podosome belts of mature OCs, presented two distinct structural arrangements of F-actin consisting of the podosome core embedded in a cloud of polymerized actin. Because OC adhesion plays a crucial role in the bone resorption process, we next examined the localization of receptors for components of the extracellular bone matrix expressed in this cell type, namely, vβ1, 2β1, and vβ3 integrins and CD44 (Clover et al., 1992; Nesbitt et al., 1993; Chellaiah et al., 2003b). We were unable to detect β1 subunits with two antibodies (9G7 and Ha2/5), which easily reacted with murine fibroblasts (data not shown). vβ3 integrins and CD44 receptors showed specific and nonoverlapping distribution profiles within the cells. Interestingly, CD44 selectively localized to podosome cores (Figure 1E). This finding identifies CD44 as the first nonintegrin receptor at the podosome core. In contrast, β3 subunit staining matched perfectly that of the actin cloud, excluding actin dots corresponding to podosome cores (Figure 1F). Because actin dots and clouds colocalized with distinct receptors, we hypothesized that the formation of the two structures may emanate from the activation of distinct signaling pathways.

View larger version (44K): [in this window] [in a new window]   Figure 1. OC podosome belt is composed of two F-actin domains specifically associated with CD44 and the β3 integrin subunit. (A) Mature WT OCs plated on glass coverslips were observed by confocal microscopy after F-actin staining with phalloidin, and they exhibited podosome belts. Bar, 20 µm. (B) Magnified deconvoluted view of the area within the white inset in A shows that podosome belts are formed by two F-actin–containing structures: the cloud (white star) and dots (black star), referred to as podosome cores, embedded in the cloud. Bar, 1.5 µm. (C) Cumulative fluorescence intensity histograms were obtained by multiplying the number of pixels by the respective gray value of each pixel obtained from 30 images. This analysis illustrates the difference between podosome clouds and podosome cores (n = 30). (D) Z-cut section of a magnified view of the F-actin cytoskeleton (dotted white line in B) before (top) and after (bottom) deconvolution, confirmed the existence of the actin cloud, which interconnects with the actin dots of podosome core fluorescence. Bar, 0.5 µm. (E and F) Differentiated wild-type OCs were observed after actin staining by phalloidin and immunolabeling for CD44 and the β3 integrin subunit. CD44 receptor (E) colocalized with actin dots, whereas the β3 integrin subunit (F) was specific to the actin cloud.

View larger version (59K): [in this window] [in a new window]   Figure 2. WIP is required for podosome core formation. (A) Differentiated WIP–/– OCs plated on glass coverslips were observed by confocal microscopy after F-actin staining by phalloidin and exhibited a continuous actin belt. Bar, 20 µm. (B) Magnified deconvoluted view of the area within the white inset in A showed that the actin belt of the WIP–/– OCs consisted of the cloud domain only (white star), with the complete absence of podosome cores. Bar, 3 µm. (C) Comparison between cumulative intensity histograms (n = 30) of WT versus WIP –/– OC actin cytoskeleton confirmed the absence of the podosome core fluorescence domain, and the exaggerated fluorescence of the cloud domain. (D) Z-cut sections of a magnified view of the F-actin cytoskeleton before (top) and after (bottom) deconvolution confirmed that the actin cloud is homogeneous and devoid of any actin dots. Bar, 2 µm. (E). Immature WIP–/– OCs were microinjected with a WIP full-length cDNA and observed after F-actin and WIP staining: actin dots were reinduced in microinjected cells. Bar, 5 µm. (F) Magnified view of the area within the white inset in (E), showing podosome cores. Bar, 1.5 µm. (G–I) Differentiated WIP–/– OCs were observed after actin staining by phalloidin and immunolabeling for WIP, CD44, and β3 integrin subunits. The expression level of these proteins was also revealed by Western blots. As expected, WIP (G), which colocalized with actin dots in WT OCs, was absent. CD44 receptor (H) was diffusely expressed at the cell membrane, whereas the β3 integrin subunit (I) remained colocalized with the actin cloud.

CD44 Activation Restores Podosome Assembly in WIP–/– OCs
The localization of CD44 at the base of podosome cores in WT OCs suggested to us that this receptor may play a role in their formation. To analyze the contribution of CD44 in this process WT or WIP–/– mature OCs were seeded on various matrices displaying distinct CD44 binding capacities. WT OCs exhibited podosomes on glass (Figure 3A) and on all other matrices tested (data not shown). In contrast, WIP–/– OCs were unable to assemble podosome cores on uncoated glass or VN, an vβ3 integrin ligand (Figure 3, B and C). However, despite the absence of WIP, podosome core assembly was restored when WIP–/– OCs were seeded on collagen I, OPN, or HA (Figure 3, D–F), all ligands for CD44. Consistent with this, CD44 colocalized with podosome cores in WIP–/– OCs seeded on HA (Figure 3F, inset). Furthermore, when plated on coverslips coated with an activating anti-CD44 antibody (clone A3D8), podosome formation was equally restored in WIP deficient OCs (Figure 3G). These results show that triggering CD44 signaling reinduced podosome cores in WIP–/– OCs and confirm the direct implication of CD44 in podosome core formation in OCs. To investigate the mechanisms by which CD44 restores podosome core formation, WIP–/– OCs were seeded either on coverslips where WT OCs had been cultured for 2 d, or with WT OC conditioned medium. Podosome cores were induced only in the first situation (Figures 3, H and I). This indicates that OCs provided CD44 activating signals, possibly by secreting insoluble matrix components necessary for CD44-mediated podosome core formation. In a converse experiment, WT OCs were treated with CD44 antibodies endowed with blocking properties (sc-7946). As expected, a dramatic decrease in podosome core numbers was observed (Figure 3, J and K) and confirmed by actin fluorescence quantitation (Figure 3L). Together, these data provide evidence that CD44 clustering promotes podosome core formation in WIP–/– OCs, thereby bypassing the need for WIP during podosome formation.

View larger version (91K): [in this window] [in a new window]   Figure 3. CD44 plays a key role in the induction of actin dots. Mature WT OCs formed actin dots when seeded on different substrates such as glass (A) but also, vitronectin, collagen type I, OPN, and HA (data not shown). Mature WIP–/– OCs formed an homogenous actin cloud but no podosome cores on glass (B) or vitronectin (C). In contrast, podosome cores were reinduced when WIP –/– OCs were seeded on different ligands of CD44: collagen type I (D), OPN (E), or HA (F). Bar, 4 µm. The CD44 receptor colocalized with these podosome cores (inset). Bar, 8 µm. Podosome cores were also reinduced when mature WIP–/– OCs were plated on coverslips, either coated with CD44-activating antibodies (A3D8) (G) or coated for 3 d with mature WT OCs, which were then removed (H); mature WIP–/– OCs seeded on glass first coated with mature OC-derived conditioned medium, did not form podosome cores (I). Bar, 16 µm. Insets show a magnified view of the actin belts under each condition. Bar, 8 µm. (J) In contrast, incubation of WT OCs plated on glass with CD44 blocking antibodies (sc-7946) induced actin dot dissolution. Bar, 16 µm. (K) Magnified view of the area within the white inset in J. Bar, 4 µm. (L) Cumulative intensity histograms (n = 30) confirmed this disappearance of the podosome core fluorescence domain.

View larger version (80K): [in this window] [in a new window]   Figure 4. CD44 activation compensates WIP deletion by WASp stabilization. Localization and expression levels of three podosome cores markers, Arp3 (A–C), cortactin (D–F), and WASp (G–I) were analyzed in WT and WIP –/– OCs seeded on glass slides by confocal microscopy and Western blot, respectively. In WIP–/– OCs, Arp3 was expressed but diffusely distributed at the cell membrane; cortactin was confined to the actin cloud, and WASp was absent. In WIP–/– OCs seeded on HA-coated slides, WASp (J) and Arp3 (K) were present in podosome cores. Western blot analysis confirmed the restoration of WASp levels in these OCs. Bar, 3 µm.

View larger version (43K): [in this window] [in a new window]   Figure 5. Podosome cores and actin clouds play different roles in OCs. Mature WT and WIP–/– OCs plated on glass were observed by confocal microscopy after F-actin staining (A and C) or by IRM (B and D). Bar, 6 µm. Both cell types showed a large dark band at their periphery, corresponding to a tight attachment to the substratum, which perfectly colocalized with podosome belts. (E) Quantification of the adhesion strength of WT and WIP–/– mature osteoclast on glass was obtained after incubation of these cells with PBS/EDTA to induce their detachment. Remaining adherent osteoclasts were counted 0, 7, or 15 min after treatment. (F) To quantify adhesion strength, WT and WIP–/– mature OCs seeded on glass or on HA were incubated with CD44 blocking antibody (sc-7946) or β3 blocking peptides (RGD). Remaining OCs were recorded 0, 20, or 30 min after treatment. Only adhesion of WT and WIP–/– OCs seeded on HA, which contained podosomes cores, decreased with sc-7946; in contrast, cell adhesion in the three cultures decreased with RGD peptides, demonstrating that both podosome cores and the actin cloud, and consequently the CD44 receptor and β3 integrin subunits, participate in OC adhesion. Mature WT and WIP–/– OCs were plated on glass coverslips and observed by confocal microscopy after actin staining by phalloidin and immunolabeling for myosin II motor protein. Myosin II is localized in actin cloud WT (G) and WIP–/– (H) OCs. Bar, 4 µm. (I–M) WT and WIP–/– OCs were plated on glass and treated with blebbistatin, an inhibitor of myosin II. Confocal microscopy after F-actin staining showed that the drug induced spreading of WT (I and J, dotted lines) and WIP–/– OCs (K and L, dotted lines), as indicated by pixel number. Bar, 60 µm. (M) Quantification of cell spreading was achieved by measuring WT and WIP–/– mature OC surface areas, before and after the addition of 50 µM blebbistatin (Bb), on glass or ACC slides. WIP–/– OCs have the same state and ability of contraction as WT OCs, demonstrating that the sole presence of the actin cloud allows OC contractility.

CD44/WASp Is a Major Pathway for SZ Formation and Bone Resorption
Adhesion and contractility are crucial for the OC resorption process. Bone resorbing OCs exhibit a SZ, a large band of F-actin that does not result from the simple fusion of podosomes (Saltel et al., 2004). Results presented so far suggested to us that the SZ could be derived from 1)the actin cloud only, 2) the reorganization of podosome cores only, or 3) podosome cores fusing with the actin cloud. To explore these possibilities, we first investigated how podosomal markers distributed in OCs seeded on ACC, a bone matrix mineralized in vitro containing apatite, the mineral part of bone. CD44 localized at the SZ in WT OCs but also in WIP–/– OCs (Figure 6, A and C). These data show that the podosome core moiety is integrated in the SZ. Likewise, other specific components of podosome cores, such as cortactin and Arp3, were found in WIP–/– SZ (data not shown). Confirming the results obtained on HA or OPN, WIP–/– OCs seeded on ACC showed a classical SZ. WASp was detected at the SZ, and it appeared as in WT OCs (Figure 6, B and D). WASp expression under these conditions was also detected by Western blot (data not shown). We conclude that CD44 activation maintains WASp expression and localization at the SZ. Together, these data demonstrate the complexity of the SZ, which corresponds to the integration and reorganization of the two actin structures: podosome cores and actin clouds.

View larger version (49K): [in this window] [in a new window]   Figure 6. CD44/WASp is a major pathway for SZ formation and bone resorption. Mature WT and WIP–/– OCs were seeded on ACC coverslips. Confocal microscopy, after actin staining by phalloidin (insets) and immunolabeling for CD44 and WASp, showed that these proteins were present in the sealing zone of WT (A and B) and WIP–/– OCs (C and D). Bar, 10 µm. After cell lysis and staining, resorbed surfaces areas by WT (E) and WIP–/– (F) OCs were observed under a stereomicroscope (MZ12; Leica). Bar, 200 µm. Images were quantified (G) using MetaMorph and Photoshop software (see Materials and Methods). WIP–/– OC were still able to resorb mineralized matrix, but with a 30% lower efficiency than WT OCs (t test; p < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fully differentiated multinucleated OCs exhibit two different actin structures, podosome belts and SZ, when adherent on artificial or apatite-mineralized substrates, respectively. In this study, we have extended our previous observation (Destaing et al., 2003), recently confirmed by high-resolution scanning electron microscopy (Luxenburg et al., 2007), that podosome belts are composed of two F-actin–containing domains, namely, a diffuse actin cloud surrounding actin dots referred to as podosome cores. The use of WIP-deficient OCs enabled us to define the molecular architectures of the two F-actin domains and to show for the first time that they fulfill different roles. The actin cloud is composed of proteins such as vinculin, paxillin, and signaling phosphoproteins, together with the vβ3 integrin linking this domain to the ECM. In contrast, podosome cores are made of a dense F-actin network associated with proteins such as cortactin, Arp2/3, WASp, and the transmembrane receptor CD44 (Figure 7 and Table 1). As expected, WIP, which can interact directly with actin, WASp, and cortactin (Ramesh et al., 1997; Martinez-Quiles et al., 2001; Kinley et al., 2003), and regulates podosome formation in dendritic cells (Chou et al., 2006), colocalized with podosome cores. The potential role played by this protein in podosome formation was further emphasized by the analysis of WIP-deficient mature OCs; the actin cloud domain remained, but no podosome cores could be observed by deconvolution confocal microscopy. In addition, WASp and Arp2/3 could not be detected, whereas cortactin redistributed within the cloud domain.

View larger version (30K): [in this window] [in a new window]   Figure 7. Proposed schematic molecular organization of the two F-actin domains in OCs. Top left, detail of the cloud domain. A transversal F-actin network is nucleated via the vβ3 integrin and the multiprotein complex consisting of vinculin and paxillin. This complex is linked via talin and vinculin to a radial F-actin network. Only a few markers are represented for simplification purposes. Top right, detail of podosome core domain. A vertical F-actin network is nucleated at the membrane via the CD44 receptor and the actin polymerization machinery comprising WIP, WASp, cortactin, and Arp2/3. Bottom, schematic drawing of the F-actin belt in OC with an optical cut perpendicular to the substratum. The plasma membrane is delimited by an orange line. Adapted from Linder and Aepfelbacher, 2003 and Luxenburg et al., 2007.

Podosome belts exhibit two transmembrane receptors with specific patterns, integrin vβ3 underneath the actin cloud and CD44 associated with podosome cores, whereas β1 integrin could not be detected at the belt. Colocalization of CD44 with podosome cores is the first observation of a receptor distinct from integrins, directly associated with the actin core of podosomes. Podosome cores were reinduced in WIP–/– OCs seeded on HA, bringing new insight to understanding the role of HA in bone resorption (Spessotto et al., 2002). Moreover, the use of blocking antibodies against CD44 induced podosome disassembly in WT OCs. Both vβ3 integrin and CD44 are transmembrane receptors for several extracellular matrix elements, such as vitronectin for integrin vβ3 and HA for CD44. One interesting point is that they share a common ligand, OPN, which is largely expressed and secreted by OCs (Chellaiah et al., 2003b; Chellaiah and Hruska, 2003). These two receptors were presumed to play important functions in OC adhesion, migration, and resorption. Surprisingly, β3 or CD44 null mice show only mild defects in bone resorption and only a weak osteopetrotic phenotype (McHugh et al., 2000; de Vries et al., 2005). Our results suggest that suppression of only one of these two receptors is not sufficient to block OC resorption. In contrast, OPN gene knockout induces a decrease in CD44 expression in OCs, which are hypomotile and less efficient in resorbing bone matrix (Chellaiah et al., 2003b). Together these data suggest cooperativity between these two receptors in OCs. WIP–/– OCs seeded on coverslips previously coated by WT OCs exhibit podosome cores, suggesting that OCs secrete their own matrix to induce podosome core formation and subsequently bone resorption. Moreover, it can be anticipated that during the resorption cycle, the complex bone matrix underneath the OC will be altered as long as resorption progresses. Thus, a possible scenario could be the following: in an early phase of the process, OCs meet apatite, the mineral component of bone that is able to bind and concentrate matrix components such as OPN or HA (Reinholt et al., 1990; Rees et al., 2002). This high concentration of OPN or HA concentrates and activates CD44 to organize a SZ, a prerequisite for bone resorption. In the resorption lacuna delimited by the SZ, acidic dissolution of apatite resulting from H⁺ secretion and proteolysis of the different bone matrix proteins, profoundly alters the bone matrix which can then promote the dissociation of the SZ and stimulate OC migration. This cycle could then be repeated.

Our present work has confirmed the essential role of WIP in podosome core formation. This molecule seems to act as a nucleator of actin-regulating proteins, because in its absence, OC podosome core components are completely dissociated, with delocalization of cortactin and Arp2/3. WIP has been shown to protect WASp from degradation via the proteasome or from proteolytic cleavage by calpain, in lymphocytes and dendritic cells (Chou et al., 2006; de la Fuente et al., 2007). In WIP–/– OCs, no specific staining for WASp was detected in the actin cloud, nor for Arp2/3 or phospho-tyrosine proteins (Figure 7 and Table 1). However in the absence of WIP, the activation of CD44 by specific ligands such as HA, OPN, or laminin was sufficient to stabilize WASp and to reinduce podosome cores. Now, it will be interesting to determine precisely how CD44 restores WASp level, probably by protecting it from degradation. A direct CD44/WASp interaction could mask a site used by calpain, but CD44 could also inhibit ubiquitinylation of WASp and its degradation by the proteasome. This identification of CD44 as the first matrix receptor associated directly with the podosome core is consistent with its ability to interact with key elements involved in podosome core formation such as N-WASp, c-Src, and cortactin (Bourguignon et al., 2001, 2007; Hill et al., 2006).

Finally, the resorption capacity of WIP–/– OCs was found to be slightly but significantly reduced compared with control OCs, even though the appearance of the SZ was normal. Thus, the clustering and activation of CD44 is sufficient to form a SZ, but the presence of WIP is necessary for the formation of a fully functional SZ. We can speculate that apatite allows WIP–/– OC to bypass WIP deletion to some extent. This could be explained by the high-affinity of apatite for secreted matrix elements such as OPN or HA, which concentrate CD44 receptors and thereby trigger CD44 signaling pathways. How WIP contributes to the functionality of the SZ and its exact role in bone physiology are presently under investigation.

	ACKNOWLEDGMENTS

 
We acknowledge stimulating discussions with E. Plannus and I. Maridonneau-Parini. This work was supported by grants from Fondation pour la Recherche Médicale (to P.J.), Association Recherche contre le Cancer (to P.J.), Swiss National foundation (3100AO-103805), Fundación Ramón Areces (to I.M.A.). and the Spanish Ministry of Education and Science BFU2004-00128 (to I.M.A.). A.C. is the recipient of an "allocation couplée" grant from ENS Lyon. F.S. was the recipient of a European Molecular Biology Organization postdoctoral fellowship and is presently supported through an Institut National de la Santé et de la Recherche Médicale postdoctoral contract. I.B. is supported by a predoctoral contract from Consejería de Educación de la Comunidad de Madrid and Fondo Social Europeo. I.M.A. is recipient of a "Ramón y Cajal" contract from the Spanish Ministry of Education and Science.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-04-0378) on September 26, 2007.

Address correspondence to: Pierre Jurdic (pjurdic{at}ens-lyon.fr).

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