Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, Montpellier, FranceUniversité de Montpellier, FranceInstitut Albert Bonniot Centre de Recherche INSERM-UJF U823, CNRS ERL 5284, Grenoble, FranceUniversité de Grenoble, France
a r t i c l e i n f o
rticle history:eceived 3 December 2012eceived in revised form 28 February 2013ccepted 11 March 2013
eywords:steoclastodosomectin
a b s t r a c t
Podosomes are adhesion structures characteristic of the myeloid cell lineage, encompassing osteoclasts,dendritic cells and macrophages. Podosomes are actin-based structures that are dynamic and capableof self-organization. In particular in the osteoclast, podosomes densely pack into a thick ring called thesealing zone. This adhesion structure is typical of osteoclasts and necessary for the resorption of the bonematrix. We thought to explore in more details the role of podosomes during osteoclast differentiation andmigration. To this end, we made from soft to stiff substrates that had not been functionalized with extra-cellular matrix proteins. Such substrates did not support podosome formation in osteoclasts. With suchdevices, we could show that integrin activation was sufficient to drive podosome assembly, in a substrate
ofilinntegrindhesionanganeseyeloidMP14
stiffness independent fashion. We additionally report here that osteoclast differentiation is a podosome-independent process. Finally, we show that osteoclasts devoid of podosomes can migrate efficiently. Ourstudy further illustrates the great capacity of myeloid cells to adapt to the different environments theyencounter during their life cycle.
Osteoclasts are giant multinucleated cells of the mono-yte/macrophage lineage specialized for bone resorption. Duringifferentiation, osteoclast precursors rapidly exhibit podosomes,dhesion structures typical of myeloid cells. Osteoclast podosomesave the characteristics to organize into superstructures thatvolve during differentiation from podosome clusters to podosomeings that finally fuse into a peripheral podosome belt (Destaingt al., 2003). This belt prefigures the sealing zone, a thick ringf densely packed podosomes, which is essential for the boneesorbing activity of osteoclasts and only assembles on mineralizedatrices (Luxenburg et al., 2007).
Receptor Activator of NF-�B Ligand (RANKL) is the key
ytokine that commits hematopoietic precursors into osteoclasts.ANKL elicits a complex transcriptional program involving the
∗ Corresponding author at: CRBM CNRS UMR 5237, 1919 route de Mende, F-34293ontpellier Cedex 5, France. Tel.: +33 43435 9505; fax: +33 43435 9410.
E-mail address: [email protected] (A. Blangy).1 These authors contributed equally to this work.2 Present address: Institut de Génétique Humaine, CNRS UPR 1142, Montpellier,
fundamental transcription factor NFATc1 that will drive theexpression of a series of osteoclast specific genes (Takayanagiet al., 2002). Osteoclastogenesis involves the expression of genesessential for precursor fusion, such as DC-STAMP and Atp6V0d2(Kim et al., 2008; Yagi et al., 2007) as well as the expression ofgenes essential for bone matrix degradation such as CtsK encodingthe matrix protease Cathepsin K (Gowen et al., 1999) or Acp5 thatencodes the Tartrate Resistant Acid Phosphatase (TRAP) (Kirsteinet al., 2006). It is also characterized by the expression of manygenes controlling cell adhesion and more specifically podosomesuperstructure organization in osteoclasts including Src (Destainget al., 2008), Integrin ˇ3 (McHugh et al., 2000), the Rho-familyGTPase Wrch1 (Brazier et al., 2009; Ory et al., 2007) and the Racactivator Dock5 (Vives et al., 2011).
The in vitro culture of monocyte/macrophage hematopoieticlineage precursor cells allowed detailed characterization of themolecular mechanisms downstream of RANKL that control thedifferentiation of osteoclasts. Conversely, the actual origin ofosteoclasts in vivo still remains a matter of debate. In particular, itis not known if osteoclasts derive from circulating or bone resident
precursors, or from both. It was shown that osteoclast precursorscould migrate to and from the bone marrow vasculature (Ishiiet al., 2010). More over, osteoclasts precursors can also be recruitedalso from systemic circulation. The recruitment of the circulating
onocytes into the bone marrow cavity is induced by RANKLKotani et al., 2013), suggesting that osteoclast precursor commit-
ent can occur outside the bone marrow. It was also reported thatendritic cells could differentiate into osteoclasts in vivo (Wakkacht al., 2008). Regarding differentiated osteoclasts, they are usuallyound in contact with the bone in physiological conditions butsteoclasts were also found in the tumor stroma of breast cancerone metastases (Saltel et al., 2006) and in the pannus of rheuma-oid joints (Okamoto and Takayanagi, 2011). Therefore, osteoclastsnd their precursors can accommodate to various environment,s it is the case for the other myeloid cells: macrophages andendritic cells, which are found in multiple locations in the body.
The dynamics of osteoclast podosomes has also been extensivelytudied in culture cells. As the elementary components of the seal-ng zone, podosomes are clearly essential for the bone resorbingunction of osteoclasts. But their importance is less clear duringther aspects of osteoclast biology. For instance, the suppressionf Dock5, Src and Integrin �3 leads to impaired organization ofodosomes into a sealing zone but the RANKL-induced transcrip-ional program and osteoclast precursor fusion remain unaffectedDestaing et al., 2008; McHugh et al., 2000; Vives et al., 2011). Onhe other hand, interfering with the activity of Wrch1 affects osteo-last fusion and integrin-mediated adhesion signaling, still withoutmpacting on osteoclastic gene expression (Brazier et al., 2006,009). We reported recently that podosomes could drive a salta-ory mode of migration in osteoclasts (Hu et al., 2011). But M-CSF,hich induces podosome dissolution, is a potent activator of osteo-
last migration (Novack and Faccio, 2009). Moreover, as osteoclastsesorb the bone, they alternate between resorbing phases, duringhich podosomes are present, and a migrating phase, during whichodosomes disassemble (Saltel et al., 2004). Thus, the importancef podosomes for osteoclast differentiation and migration is notrmly established.
In this context, we sought to address further the importance ofodosomes during these aspects of osteoclast biology. To this end,e worked on the establishment of artificial cell culture substrates
f defined stiffness that did not support podosome formation insteoclasts. Our results indicate first that podosome assembly insteoclast is very permissive regarding substrate stiffness as soons integrins are activated. In a context where podosomes could not
ig. 1. Podosome assembly in osteoclasts plated on soft substrates. Osteoclasts differentitronectin (VN) functionalized polyacrylamide (PAA) gels or polydimethylsiloxane (PDMSnd vinculin (green). Panels at the bottom are magnified areas of the merged top panels.
of Cell Biology 92 (2013) 139– 149
assemble, we further found that the differentiation of osteoclastsis normal and that they can migrate efficiently.
Results
Podosome assembly can occur in osteoclasts plated on softmatrices
As podosomes are mechanosensors (Albiges-Rizo et al., 2009;Collin et al., 2008), we first sought to interfere with podosomeassembly in osteoclasts by lowering substrate stiffness. To this end,we generated cell culture substrates using polydimethylsiloxaneto obtain substrates with various shear moduli ranging approxi-mately from 50 to 1800 kPa depending on the ratio of cross linkeragent to base (Brown et al., 2005) and also produced polyacryl-amide hydrogels (Pelham and Wang, 1997) with a stiffness of28.2 ± 3.7 kPa, as measured by atomic force microscopy. Thesesubstrates were functionalized with collagen or vitronectin. Thestiffness of these substrates was several orders of magnitude belowthat of bone (10–25 GPa), polystyrene dishes (3 GPa) or glass cover-slips (50 GPa), the substrates classically used to study podosomesin osteoclasts.
To analyze the formation of podosomes, we differentiated osteo-clasts from bone marrow-derived macrophages and plated themon polyacrylamide gels or polydimethylsiloxane gels functional-ized with vitronectin or collagen or on glass coverslips. Osteoclastpodosomes are made of a central column of densely packed F-actinfibers running perpendicular to the substrate and containing cor-tactin: the podosome core. The core is surrounded by an array of lessdense F-actin filaments, running parallel to the substrate and asso-ciated with vinculin: the podosome cloud (Chabadel et al., 2007).As expected, analysis by confocal microscopy revealed the pres-ence of actin-containing podosomes in osteoclasts plated on glasscoverslips (Fig. 1, Glass). In these cells, vinculin was concentratedaround the podosome cores in ring-shaped structures (Fig. 1, Glass).
Similarly, we observed podosomes assembly in osteoclasts platedonto polyacrylamide gels or polydimethylsiloxane gels coated withvitronectin (Fig. 1, VN functionalized-PAA and -PDMS) or with col-lagen (not shown), even with a low stiffness of 30 kPa
iated on glass coverslips were harvested and seeded on glass coverslips (Glass) or) gels of the indicated stiffness. After 5 h, cells were fixed and stained for actin (red)
Scale bars: 30 �m.
H. Touaitahuata et al. / European Journal of Cell Biology 92 (2013) 139– 149 141
Fig. 2. Podosome assembly is impaired on gels non-functionalized with extracellular matrix proteins. (A–F) Osteoclasts plated on glass coverslips (Glass) (A–C) or non-functionalized 30 kPa polyacrylamide (NF-PAA) gels (D–F) for 5 h were stained for actin (red) and the podosome cloud marker vinculin (green) (A, D) or the podosome coremarker cortactin (green) (B, E) or the matrix metalloproteinase 14 (MMP14) (green) (C, F). Lower panels are magnification of the boxed area in the upper panels. Note themutually exclusive localization of actin and vinculin on glass coverslips whereas these proteins are co-localized on polyacrylamide gels. Actin and cortactin, as actin andMMP14, are co-localized in all conditions. Scale bars: 30 �m. (G) Osteoclasts plated on non-functionalized polydimethylsiloxane (NF-PDMS) gels of the indicated stiffnessw ow ab
ct
Ao
to
ere stained for actin (red) and vinculin (green). For each condition, left images shoxed areas. Scale bars: 30 �m.
These observations suggest that podosome assembly in osteo-lasts is highly permissive regarding the mechanical constraints ofhe environment.
ctivation of integrins is a prerequisite for podosome assembly insteoclasts
In order to interfere with podosome formation, we decidedo hinder integrin activity. To this end, osteoclasts were platedn the same polyacrylamide gels or polydimethylsiloxane gels,
n overlay of actin and vinculin staining and right images are magnifications of the
but which had not been functionalized with extracellular matrixproteins. Indeed, contrarily to glass or polystyrene, cell-secretedextracellular proteins are not able to adhere on polyacrylamidegels or polydimethylsiloxane gels (Damljanovic et al., 2005). Incontrast with glass coverslips (Fig. 2A–C), osteoclasts were ableto adhere and spread but were unable to form podosomes when
plated onto non-functionalized polyacrylamide gels (Fig. 2D–F).Instead, they displayed a diffuse distribution of vinculin in thecytoplasm demonstrating an absence of podosome cloud formation(Fig. 2D). Staining of cortactin and of metalloprotease 14 (MMP14)
142 H. Touaitahuata et al. / European Journal of Cell Biology 92 (2013) 139– 149
Fig. 3. Impaired cofilin localization and activation in osteoclasts on non-functionalized gels. (A-B) Osteoclasts differentiated on glass coverslips (Glass) (A) or non-functionalized 30 kPa polyacrylamide (NF-PAA) gels (B) were stained for actin (red) and cofilin (green). Lower panels are magnification of the boxed area in the upperpanels. Note the co-localization of actin and cofilin in the podosome belt on glass coverslips. On non-functionalized polyacrylamide gels, cofilin does not co-localize withactin. Scale bars: 30 �m. (C) Bone marrow-derived macrophages were cultured for 3 days in the presence of M-CSF alone (−) or of M-CSF and RANKL (R+) on glass coverslips(Glass) or non-functionalized polyacrylamide (NF-PAA) gels. Cells were lysed and equal amounts of proteins were immunoblotted for phospho-cofilin (P-COF) and totalc n levet st).
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ofilin (COF). (D) Densitometric analysis of the ratio of phospho-cofilin to total cofilihree independent experiments (NS, non significant, ***P < 0.001, Mann–Whitney te
lso revealed a diffuse distribution throughout the cytoplasmevealing the lack of podosome core organization (Fig. 2E and F).urthermore, cortactin, MMP14 and vinculin, co-localized withctin, in structures morphologically unrelated to podosomes. Inter-stingly, we found that osteoclasts plated onto non-functionalizedolydimethylsiloxane gels displayed a similar disorganization ofheir cytoskeleton, whatever their stiffness (Fig. 2G).
We recently described the activation of the actin-severing pro-ein cofilin during the formation of osteoclast podosome belt,nd its accumulation there (Blangy et al., 2012). Confocal analy-is of cofilin subcellular distribution confirmed the co-localizationetween cofilin and actin in podosome belts of osteoclasts seedednto glass coverslips (Fig. 3A). By contrast, cofilin was excludedrom actin-rich structures when osteoclasts were plated on non-unctionalized polyacrylamide gels (Fig. 3B). The absence ofodosomes correlated with a high level of cofilin phosphorylationFig. 3C and D), thus revealing that cofilin activation did not occurn these conditions.
We finally verified that the absence of podosome formation insteoclasts plated onto non-functionalized substrates (Fig. 4B and) was indeed a consequence of impaired integrin activation. Tohis end, we forced integrin �1 and �3 activation in osteoclastslated on non-functionalized polyacrylamide gels (Fig. 4E) or poly-imethylsiloxane gels (Fig. 4F) by adding 0.5 mM manganese inhe culture medium (Orr et al., 2006), which is known to acti-ate integrins �1, �2 and �3 (Chen et al., 2003; Evans et al., 2010;ould et al., 2002), the major integrins in osteoclasts (Schmidt
t al., 2011). After 1 h of manganese treatment and 2 h of washut, we observed that around 70% of the osteoclasts had organized
odosomes (Fig. 4E–G).
Taken together, these results suggest that podosome formationn osteoclasts can occur in a context of low stiffness as soon asntegrins are activated.
ls in cells prepared as described in (C). Bar graph shows average and SD ratios from
Osteoclast differentiation does not require podosomes but requiresadhesion
Having set up experimental conditions in which osteoclastscould not make podosomes, we further addressed the question oftheir function during osteoclast differentiation. To obtain osteo-clasts, bone marrow-derived macrophages were directly seededonto glass coverslips or onto non-functionalized polyacrylamidegels and grown in the presence of M-CSF and RANKL to induceosteoclastic differentiation.
After 3–5 days of culture in the presence of M-CSF andRANKL, TRAP-positive multinucleated cells were obtained inboth conditions. Nevertheless, cells exhibited dramatic morpho-logical differences. On glass coverslips, bone marrow-derivedmacrophages formed TRAP-positive well-spread cells (Fig. 5A,Glass) whereas on non-functionalized polyacrylamide gels thesecells exhibited a very contracted morphology and elongated cyto-plasmic extensions (Fig. 5A, NF-PAA). Surprisingly, we found thatthe overall distribution of nuclei per osteoclasts was similarbetween the two conditions, but with more small osteoclasts (lessthat 10 nuclei) and less big ones (over than 30 nuclei) on the non-functionalized polyacrylamide gels as compared to glass coverslips(Fig. 5B). These data suggest that the overall process of precursorcell fusion was not impaired in the absence of podosomes.
We next analyzed the RANKL-induced expression of osteoclasticmarker genes by real-time Q-PCR. Out of the 8 genes analyzed, 6were induced by RANKL at the same level on non-functionalizedpolyacrylamide gels as compared to glass coverslips (Fig. 5C). Inthe case of DC-STAMP and ATP6v0d2, we observed a lower level of
expression on polyacrylamide gels. As these genes are involved inosteoclast precursors fusion (Kim et al., 2008; Yagi et al., 2007),this result might be related to the absence of osteoclasts displayinga very large number of nuclei on this substrate (Fig. 5B). Finally,
H. Touaitahuata et al. / European Journal of Cell Biology 92 (2013) 139– 149 143
Fig. 4. Integrin activation is sufficient to induce podosome assembly on non-functionalized soft gels. (A-F) Osteoclasts seeded on glass coverslips (Glass) (A, D) or non-functionalized 30 kPa polyacrylamide (NF-PAA) gels (B, E) or 48 kPa polydimethylsiloxane (NF-PDMS) gels (C, F) were treated for 1 h in the absence (A–C) or in presenceo After
m ficatioa
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f 0.5 mM manganese (MnCl2) followed by a 2-h recovery without MnCl2 (D–F).
agnification of the boxed area in the upper panels. Scale bars: 30 �m. (G) Quantind SD proportions from three independent experiments.
oth types of osteoclasts assembled sealing zones when plated onalcium-phosphate-coated coverslips (Fig. 5D).
In order to establish the importance of cellular adhesion duringsteoclastogenesis, bone marrow-derived macrophages were
ifferentiated on soft agar, a non-adhesive substrate (Fig. 5E).
n theses conditions, we observed no induction of Src, Acp5 andtgb3 expression after 4 days of culture in the presence of RANKL,
hereas these markers were induced in bone marrow-derived
fixation, cells were stained for actin (red) and vinculin (green). Lower panels aren of the proportion of osteoclasts (OC) with podosomes. Bar graph shows average
macrophages differentiated on glass coverslips (Fig. 5F). Whenthe cells on soft agar were plated for 1 h onto glass coverslips,cells adhered on the substrate but more than 98% were mononu-cleated and negatives for the TRAP staining (Fig. 5G). 5 h after
plating, cells remained mononucleated, round and the stainingof actin and vinculin revealed the presence of podosome rosettes(Fig. 5H), which characterize macrophages (Guiet et al., 2012).These observations are consistent with a recent report showing
144 H. Touaitahuata et al. / European Journal of Cell Biology 92 (2013) 139– 149
Fig. 5. Osteoclasts can differentiate in the absence of podosomes but not without adhesion. (A) Osteoclasts differentiated on glass coverslips (Glass) or non-functionalizedpolyacrylamide (NF-PAA) gels were stained for TRAP activity. Scale bars: 50 �m. (B) Distribution of the number of nuclei in osteoclasts (OC) prepared as described in A.Results are representative of 3 independent experiments. (C) Total RNAs were prepared from bone marrow-derived macrophages grown for 3 days in the presence of M-CSFalone (−) or of M-CSF and RANKL (R+) on glass coverslips (Glass) (black bars) or non-functionalized polyacrylamide (NF-PAA) gels (gray bars). The mRNAs levels of osteoclastcharacteristic genes relative to Gapdh were determined by RT-PCR. (D) Osteoclasts differentiated on glass coverslips (Glass) or on non-functionalized polyacrylamide(NF-PAA) gels were seeded on calcium phosphate coated coverslips for 24 h and stained for actin (red) and vinculin (green) to reveal the sealing zones. Lower panels are mag-nification of the boxed area in the upper panels. Scale bars: 30 �m. (E) Morphology of non-adherent bone marrow-derived macrophages grown for 4 days in the presence ofM-CSF and RANKL on soft agar (Agar). Scale bar: 30 �m. (F) Total RNAs were prepared from bone marrow-derived macrophages grown for 4 days in the presence of M-CSF alone
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hat non-adhesive substrate methyl-cellulose does not supportsteoclast differentiation in response to RANKL (Mochizuki et al.,012). Taken together, these results suggest that adhesion, but not
ntegrin activation, is required for osteoclast differentiation.We further verified that in osteoclasts differentiated on
on-functionalized polyacrylamide gels, the RANKL-induced trans-riptional program was no affected for osteoclasts to organizeodosomes when exposed to permissive conditions. To this end,steoclasts were lifted from the polyacrylamide gels and platednto glass coverslips in the absence or in the presence of acti-omycin D, in order to block transcription. In the absence ofctinomycin D, individual podosomes were visible 4 h after platingnd displayed the characteristic structure of actin dots surroundedy vinculin (Fig. 6A). After 5 h, podosomes began to arrange intoeripheral belts (Fig. 6B). In the presence of actinomycin D, theame structures were observed with the same kinetics (Fig. 6C and). The efficiency of the actinomycin D treatment was ascertainedy Q-PCR (Fig. 6E). These data demonstrate that osteoclasts differ-ntiated on non-functionalized polyacrylamide gels express all theRNA components required to form and organize podosomes.Overall, these results show that the establishment of the
ANKL-specific transcriptional program is not impaired on non-unctionalized polyacrylamide gels but is inhibited on agar,uggesting that podosomes formation is not essential for osteoclastifferentiation while adhesion is crucial for this process.
odosome are dispensable for osteoclast migration
We reported recently that podosomes could drive osteoclastigration on polyacrylamide gels functionalized with vitronectin
Hu et al., 2011). Nevertheless, the osteoclast transmigration pro-ess does not rely on podosomes (Saltel et al., 2006) and leukocytesan migrate in 3-dimensional collagen matrices in an integrin-ndependent fashion (Lammermann et al., 2008). In addition, theissolution of podosomes in activated dendritic cells is associatedith high-speed migration (van Helden et al., 2006) and M-SF-stimulated osteoclast migration is associated with podosomeissolution (Novack and Faccio, 2009). Therefore the link betweenodosomes and the migration of myeloid cells is far from beingrmly established.
Hence, we followed the behavior of live podosome-free osteo-lasts differentiated on non-functionalized polyacrylamide gels byideo microscopy. Consistent with previous studies (Saltel et al.,004), we found that the well-spread, podosome belt-containingsteoclasts generated on glass coverslips were mostly static (Fig. 7And B, Glass and Movie 1). Movements were essentially due todditional fusion of mononucleated precursors and hence to thextension of osteoclast surface. By contrast, osteoclasts plated onon-functionalized polyacrylamide gels were highly motile (Fig. 7And B, NF-PAA and Movie 2). On this surface, the multinucleatedells showed extension and retraction of numerous membrane pro-rusions. This was consistent with the membrane protrusions webserved on fixed cells (Fig. 2D–F). These data indicate that osteo-lasts do not require podosomes for random migration.
iscussion
In this study we explored the importance of podosomes forsteoclast differentiation and migration. We found that osteo-lasts can differentiate and migrate in conditions where they arenable to form podosomes. We further found that the formation of
−) or of M-CSF and RANKL (R+) on glass coverslips (Glass) (black bars) or soft agar (Agar)ere determined by RT-PCR. (G–H) Bone marrow-derived macrophages grown for 4 days
or 1 h and stained for TRAP activity (G, arrows indicate non-adherent cells) or for 5 h andnd vinculin staining and arrows indicate podosome rosettes structure. Scale bars: 30 �m
of Cell Biology 92 (2013) 139– 149 145
podosomes can occur in a wide range of substrate stiffness, as longas integrins are activated.
Podosomes are essential for osteoclasts to fulfill their primaryfunction, which is to resorb the bone matrix. When plated onto non-mineralized substrates, podosomes organize into a peripheral belt.In the presence of minerals, this belt gets thicker and podosomescompact to form the osteoclast specific resorption apparatus calledthe sealing zone (Luxenburg et al., 2007; Saltel et al., 2004). Theformation of these structures and the efficient resorbing activ-ity of osteoclasts require integrins �1, �2, �3 and �v as well asthe integrin activating protein Kindlin-3 (Schmidt et al., 2011). Inagreement, we observed the absence of podosomes in osteoclastsplated on substrates not compatible with extracellular matrix pro-tein immobilization. The activation of integrins with manganeseor by functionalizing the substrates with vitronectin or collagenwas sufficient to induce podosome formation, even on substrateswith a low stiffness of 30 kPa That is several orders of magnitudebelow bone, polystyrene or glass, the common natural and artifi-cial substrates on which osteoclasts are usually studied. Podosomescan sense the physical properties of the substrate and substratestiffness influences their capacity to transmit mechanical stressesinside-out and outside-in the cell (Collin et al., 2008). But, ourresults suggest that substrate stiffness does not control the for-mation of podosomes itself, at least in osteoclasts, which does notexclude a potential effect on their dynamics, as shown for instancein the case of podosome rosettes in NIH3T3 cells (Collin et al., 2006).
Previous studies reported that the suppression of each individ-ual core or cloud component of podosomes did not affect osteoclastdifferentiation. On one hand, Kindlin-3 was recently shown to bedispensable for the completion of the transcriptional program char-acteristic of osteoclast differentiation (Schmidt et al., 2011). Thelack of integrins or of kindlin-3, which is necessary for the acti-vation of integrins, prevents the formation of the peripheral cloudregion of podosomes (Schmidt et al., 2011). On the other hand, theCD44 receptor is necessary for the formation of the podosome corebut not of the podosome cloud (Chabadel et al., 2007). Similar tokindlin 3 or integrins, the lack of CD44 did not preclude the dif-ferentiation process of osteoclasts (de Vries et al., 2005). In ourexperimental device, both the cloud and core regions of podosomeswere absent. Instead, actin appeared as a disorganized meshwork,colocalized with cortactin, matrix metalloprotease 14, but also withvinculin, which is normally a component of the podosome cloudexcluded from the core. Moreover, we recently reported that theactin-severing protein cofilin, a regulator of podosomes organi-zation, is activated during osteoclastogenesis induced by RANKLand localized in podosome cores of mature osteoclasts (Blangyet al., 2012). In osteoclasts differentiated on non-functionalizedgels, cofilin was excluded from actin-rich structures and remainedinactive. In such conditions, multinuclear TRAP-positive cells couldarise and the RANKL-induced osteoclastic transcriptional programwas unaffected, confirming that podosomes are not essentials forosteoclastogenesis. Still, we observed a reduced tendency to formvery large polykaryons similar to what was reported for Kindlin-3 deficient osteoclasts (Schmidt et al., 2011). This may resultfrom the lower expression level of ATP6v0d2 and DC-STAMP, twogenes essential for osteoclast precursor fusion, that we observedon non-functionalized gels. The observation that podosomes are
dispensable for osteoclast differentiation suggests that integrin-mediated extracellular matrix adhesion signaling is not essentialduring the osteoclast differentiation process. In line with this,we recently found that the GTPase Wrch1, which associates with
(white bars). The mRNAs levels of osteoclast characteristic genes relative to Gapdh in the presence of M-CSF and RANKL on soft agar were seeded on glass coverslips
stained for actin (red) and vinculine (green) (H). Images show an overlay of actin.
146 H. Touaitahuata et al. / European Journal of Cell Biology 92 (2013) 139– 149
Fig. 6. Osteoclasts differentiated on polyacrylamide gels are competent for podosome formation. (A–D) Osteoclasts differentiated on non-functionalized polyacrylamide gelswere lifted and plated onto glass coverslips in the absence (Without ActinoD) (A and B) or in the presence of 5 �M actinomycin D (Actinomycin D) (C-D). Cells were fixed4 h and 5 h after plating and stained for actin (red) and vinculin (green). Lower panels are magnification of the boxed area in the upper panels. Scale bar: 20 �m. (E) TotalRNAs were prepared from osteoclasts differentiated on glass coverslips (Glass) or on non-functionalized polyacrylamide (NF-PAA) gels treated for 5 h in absence (WithoutActinoD) or in presence of 5 �M actinomycin D (ActinoD). The mRNAs levels of Src relative to 28S RNA were determined by RT-PCR.
Fig. 7. Osteoclasts can migrate in the absence of podosomes. (A) Osteoclast migration tracks on glass coverslips (Glass) or on non-functionalized polyacrylamide gels (NF-PAA)were obtained from time-lapse video microscopy (Videos S1 and S2). Digital images were taken every 7 min for a total of 15 h. Each wind rose plot shows centroid tracks from10 representative cells per condition, with the initial position of each track superimposed at a common origin. Scale bars: 150 �m. (B) Osteoclast migration speed deducedfrom time-lapse video microscopy (***P < 0.001, Mann–Whitney test, n = 10 per condition).
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steoclast podosomes (Ory et al., 2007), is an inhibitor of integ-in signaling in osteoclasts (Brazier et al., 2009). Wrch1 reducessteoclast precursor adhesion to the substrate and favors cell-celldhesions, similar to the effect of the cytokine RANKL. This mech-nism involving the inhibition of integrin signaling by Wrch1, isssential for osteoclast precursor cell fusion (Brazier et al., 2009).
Matrix elasticity can regulate lineage commitment of mes-nchymal stem cells (Engler et al., 2006; Fu et al., 2010) andodulate differentiation processes such as striation of myotubes
Engler et al., 2004) or mineral deposition by osteoblasts (Khatiwalat al., 2006). Whereas their ultimate function is to resorb bone,steoclasts differentiate from hematopoietic precursors of theonocyte–macrophage lineage, present in the circulating blood,
he bone marrow or other hematopoietic tissues such as spleen oretal liver (Roodman, 1999). Elastic moduli of vascular and epithe-ial layers are in the order of several kPa, whereas bone elastic
odulus is several orders of magnitude higher, reaching 10–25 GPa.ere we performed osteoclasts differentiation on various artificial
ubstrates with elastic moduli ranging between 30 kPa in the casef polyacrylamide gels and several GPa in the case of glass cover-lips. In all conditions, we found TRAP-positive cells and efficientctivation of the RANKL-induced osteoclastogenic differentiationrogram. This suggests that osteoclast differentiation is permissiveo a wide range of physical constrains, maybe reflecting the mul-iple origin of their precursors. However, an adhesive context isequired for the differentiation of precursor cells into osteoclasts.n the non-adhesive soft agar, we found that the expression ofsteoclastic marker genes was not up-regulated in bone marrow-erived macrophages in response to RANKL, in agreement with aecent report showing that methylcellulose did not support osteo-last differentiation (Mochizuki et al., 2012).
We observed that on non-functionalized polyacrylamide gels,steoclasts are highly motile cells, even though they are devoidf podosomes. On the other hand, we previously reported thatodosomes could drive saltatory osteoclast migration (Hu et al.,011). These observations suggest that osteoclasts can developarious migratory behaviors depending on their extracellular envi-onment. The motile phenotype we observed for osteoclasts onon-functionalized polyacrylamide gels is in agreement with the
act that M-CSF induces the dissolution of podosomes and theigration of osteoclasts (Novack and Faccio, 2009). Moreover,igration of colorectal cancer cells line SW480 and squamous
ell carcinoma-type cells is increased when cofilin is phospho-ylated (Jang et al., 2012; Zhou et al., 2013), consistent with theigh motility of osteoclasts on non-functionalized polyacrylamideels where phospho-cofilin levels are high. Similar to osteoclasts,he other myeloid cell types appear highly versatile regardingheir need for podosomes and integrins to be able to migrate. Fornstance, dendritic cells can exhibit integrin-independent migra-ion (Lammermann et al., 2008) and macrophages can adopt anmoeboid-type of migration that does not require podosomesnd strong adhesive interactions (Friedl and Weigelin, 2008;an Goethem et al., 2010). This indicates that specific integrin-ependent adhesive interaction with the extracellular matrix isot a prerequisite for the migration of cells of myeloid origin. Thisay be a way for these cells, which encounter different environ-ents during their life cycle, to retain the ability to migrate without
equiring the modification of their integrin repertoire.
aterials and methods
thics statement
Harvesting of murine bone marrow from sacrificed mice waspproved by the regional ethic committee of Languedoc-Roussillon
of Cell Biology 92 (2013) 139– 149 147
(France). Approval ID number: CEEA-LR-1054. Mice were main-tained at the animal facilities of the CNRS in Montpellier.
Isolation, culture and osteoclastic differentiation of mouse bonemarrow-derived macrophages
Bone marrow cells were isolated from long bones of 4-to 8-week-old C57BL/6 mice sacrificed by cervical dislocation,as described (Brazier et al., 2009). Non-adherent bone marrowcells were cultured in �MEM containing 10% heat-inactivatedfetal calf serum (Hyclone) and 2 mM glutamine, supplementedwith 30 ng/ml M-CSF (Peprotech) for 48 h and used as bonemarrow-derived macrophages. For osteoclast differentiation, bonemarrow-derived macrophages were harvested, seeded in 6-well plates onto glass coverslips or polyacrylamide gels orpolydimethylsiloxane gels or agar substrate at the density of1.5 × 105 cells/well and cultured in the presence of 100 ng/mlRANKL and 30 ng/ml M-CSF (Peprotech). Media was changed andcytokines were replenished every 2 days. Osteoclasts generallyappeared after 3 to 5 days. Alternatively, bone marrow-derivedmacrophages were maintained in M-CSF only as a control for undif-ferentiated cells.
To switch osteoclasts between substrates, osteoclasts culturedon polyacrylamide gels or polydimethylsiloxane gels or agar sub-strate were recovered by flushing the cells in fresh medium.Alternatively, osteoclasts were scraped from plastic culture dishesafter a wash in PBS containing 0.025 mM EDTA (Vives et al., 2011).
Preparation of polyacrylamide gels
Polyacrylamide gels exhibiting a rigidity of 30 kPa were obtainedusing the ratio 8% acrylamide/0.2% bis-acrylamide and preparedon glass coverslips (Pelham and Wang, 1997). Briefly, a 2.5 mlsolution was obtained by mixing 500 �l acrylamide 40%, 250 �lbis-acrylamide 2%, 25 �l HEPES (1 M, pH 8.5) and water. Then12.5 �l ammonium persulfate and 1.25 �l tetramethylethylene-diamine were added to allow polymerization. The final solution(50 �l) was dropped on a Sigmacote-treated glass surface, whichwas then covered by a Bind-Silane-treated glass coverslip. After20 min of polymerization, the gel was recovered together with theglass coverslip.
For functionalization, the gel surface was activated withvitronectin (BD Biosciences) or collagen (Sigma) as described(Damljanovic et al., 2005). Briefly, pure hydrazine hydrate (Sigma)was added to the gels for 2 h The gels were then washed firstwith 5% glacial acetic acid for 1 h and then with distilled waterfor 1 h. The extracellular matrix protein solution (10 �g/ml) wasdiluted in 50 mM sodium acetate buffer at pH 4. The oxidation ofthe proteins was achieved by adding 3.6 mg/ml sodium periodatecrystals (Sigma) and incubating the gels at room temperature for30 min Oxidized proteins (150 �l) were added on the hydrazinehydrate-treated gel and incubated at room temperature for 1 hbefore washing with PBS.
Preparation of polydimethylsiloxane gels
Polydimethylsiloxane gels with increasing elastic moduli ofapproximately 48, 259 and to 1783 kPa were produced from sili-cone elastomer base (Sylgard 184, Dow Corning) using respective50:1, 30:1 and 10:1 ratio of base to cross-linked according toa classical procedure (Brown et al., 2005). Briefly, the silicone
elastomer base and the cross-linked were mixed thoroughly andtransferred (50 �l) onto glass coverslips. After degassing under vac-uum for 30 min, the polydimethylsiloxane substrates were curedovernight (18–20 h) at 60 ◦C. For functionalization, the gels were
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Spatiotemporal dynamics of actin-rich adhesion microdomains: influence ofsubstrate flexibility. J. Cell Sci. 119, 1914–1925.
Damljanovic, V., Lagerholm, B.C., Jacobson, K., 2005. Bulk and micropatterned
48 H. Touaitahuata et al. / European Jo
oated by hydrophobic absorption with 10 �g/ml vitronectin (BDiosciences) or collagen (Sigma) by incubation for 1 h at 37 ◦C.
reparation of soft agar substrates
3.3% agar substrate obtained by dissolving agarose (Sigma)n ultrapure water was mixed to the differentiation medium at:5 ratio then transferred on 6-well plates (2 ml/well). Soft agarubstrates were used for osteoclast culture after 1 h at room tem-erature.
ntibodies and reagents
Monoclonal antibodies to vinculin, bisbenzimide Hoechst dye,ctinomycin D, MnCl2 and TRITC-labeled Phalloidin were fromigma. Other antibodies were as follow: monoclonal anti-cortactinMillipore), polyclonal anti-cofilin (Cytoskeleton) and polyclonalnti-phospho-cofilin (Cell Signaling Technologies), monoclonalnti-MMP14 (Abcam). Alexa Fluor 488- or 546-conjugated sec-ndary antibodies and Alexa Fluor 350-conjugated Phalloidin wererom Invitrogen. Osteologic Biocoat mineralized substrates wererom BD Biosciences.
mmunofluorescence and microscopy
Indirect immunofluorescence was performed after fixation ofhe cells with 3.7% formalin in PBS for 10 min, permeabilizationith 0.1% Triton X-100 in PBS for 5 min, and saturation with
% BSA in PBS, followed by incubation with primary antibod-es diluted with 2% BSA in PBS for 45 min After three washes inBS, primary antibodies were revealed with Alexa Fluor® 488- or46-conjugated goat anti-mouse or anti-rabbit antibodies (Invitro-en). Where indicated, TRITC- or Alexa350-conjugated phalloidinas added to reveal F-actin. DNA was stained using bisbenz-
mide Hoechst dye (Sigma). Cells were mounted in Mowiol® 40-88Sigma) and observed under an Axioplan2/LSM 510 META confocal
icroscope (Zeiss) using Zeiss 40× IR ACHROPLAN 0.8 W or Zeiss3× C-Apochromat 1.2 W Korr U-V-I objectives or under an SP5onfocal microscope (Leica) using Leica 40× 1.3 or 63× 1.4 oil HCXlan Apochromat objectives. To ensure that only one fluorochromeas detected at a time, each channel was imaged sequentially using
he multitrack recording module before merging.To assess osteoclast differentiation, cells were fixed and stained
or TRAP activity as described (Vives et al., 2011). Cells werebserved under an upright Zeiss AxioImager microscope using a0× or a 20× PLAN-APOCHROMAT 0.8 (Zeiss) objective. Imagesere acquired with MetaMorph 7.0 software (Molecular Devices).
andom cell migration assay
After cells were plated, tissue culture dishes were transferred to time-lapse microscope with a 37 ◦C, 5% CO2 incubation chamberor 1 h to allow cells to settle and for the plastic ware to equilibrate.mages were taken using an inverted microscope system (Zeiss)
ith a 20× objective and bright-field channels at 7-min intervalsver up to 15 h. Analysis of experimental data was performed usingmageJ. Individual cells were manually tracked to calculate the cov-red distance and speed.
eal-time PCR analyses
Real time PCR analyses were performed as described earlier
Brazier et al., 2006). Briefly, RNA extractions were carried outsing high Pure RNA Isolation kit (Roche) according to the manu-acturer’s instructions. RNA was quantified by spectrophotometrynd cDNA synthesized from 1 �g of total RNA with oligo(dN)20
of Cell Biology 92 (2013) 139– 149
by using the Superscript II First-Strand Synthesis System for RT-PCR (Invitrogen). Real-time PCR was then performed using 1 �l ofcDNA per 25 �l total volume. The primers used have been describedpreviously (Brazier et al., 2006; Vives et al., 2011). Real-time PCRmeasures to quantify cDNAs were carried out in triplicate and the95% confidence limits of the ratios to Gapdh or 28S RNA were deter-mined by Student’s t test, as described (Brazier et al., 2006; Viveset al., 2011).
Western blots
Whole cell extracts were prepared in Laemmli sample buffer,resolved on SDS-PAGE and electrotransferred on nitrocellulosemembranes. Immunoblotting was performed and signals wererevealed using the ECL Western Lightning Plus detection system(Perkin Elmer) with horseradish peroxidase-conjugated secondaryantibodies (GE Healthcare), according to manufacturer’s instruc-tions.
Acknowledgments
We wish to thank G. Massiera (LCVN, UMR CNRS/UM2 5587,Montpellier, France) for technical advices with polydimethyl-siloxane substrates. This work was supported by research grantsfrom the Fondation pour la Recherche Medicale (FRM grant #DVO20081013473 to AB), the Institut National du Cancer (grant# INCa-4361 to AB), the Agence Nationale de la Recherche (ANRgrant # ANR-2011-BLAN-006 to AB) and the Ligue contre le Cancer(CAR Equipe Labellisée Ligue 2010). We thank the Montpellier RIOImaging Facility (http://www.mri.cnrs.fr/) for imaging advice andfacilities.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ejcb.2013.03.001.
References
Albiges-Rizo, C., Destaing, O., Fourcade, B., Planus, E., Block, M.R., 2009. Actinmachinery and mechanosensitivity in invadopodia, podosomes and focal adhe-sions. J. Cell Sci. 122, 3037–3049.
Blangy, A., Touaitahuata, H., Cres, G., Pawlak, G., 2012. Cofilin activation duringpodosome belt formation in osteoclasts. PLoS ONE 7, e45909.
Brazier, H., Pawlak, G., Vives, V., Blangy, A., 2009. The Rho GTPase Wrch1 regu-lates osteoclast precursor adhesion and migration. Int. J. Biochem. Cell Biol. 41,1391–1401.
Brazier, H., Stephens, S., Ory, S., Fort, P., Morrison, N., Blangy, A., 2006. Expressionprofile of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogen-esis: identification of essential genes in osteoclasts. J. Bone Miner. Res. 21,1387–1398.
Brown, X.Q., Ookawa, K., Wong, J.Y., 2005. Evaluation of polydimethylsiloxanescaffolds with physiologically-relevant elastic moduli: interplay of substratemechanics and surface chemistry effects on vascular smooth muscle cellresponse. Biomaterials 26, 3123–3129.
Chabadel, A., Banon-Rodriguez, I., Cluet, D., Rudkin, B.B., Wehrle-Haller, B., Genot,E., Jurdic, P., Anton, I.M., Saltel, F., 2007. CD44 and beta3 integrin organizetwo functionally distinct actin-based domains in osteoclasts. Mol. Biol. Cell 18,4899–4910.
Chen, J., Salas, A., Springer, T.A., 2003. Bistable regulation of integrin adhesivenessby a bipolar metal ion cluster. Nat. Struct. Biol. 10, 995–1001.
Collin, O., Tracqui, P., Stephanou, A., Usson, Y., Clement-Lacroix, J., Planus, E., 2006.
conjugation of extracellular matrix proteins to characterized polyacrylamidesubstrates for cell mechanotransduction assays. Biotechniques 39, 847–851.
de Vries, T.J., Schoenmaker, T., Beertsen, W., van der Neut, R., Everts, V., 2005. Effectof CD44 deficiency on in vitro and in vivo osteoclast formation. J. Cell. Biochem.94, 954–966.
estaing, O., Sanjay, A., Itzstein, C., Horne, W.C., Toomre, D., De Camilli, P., Baron,R., 2008. The tyrosine kinase activity of c-Src regulates actin dynamics andorganization of podosomes in osteoclasts. Mol. Biol. Cell 19, 394–404.
ngler, A.J., Griffin, M.A., Sen, S., Bonnemann, C.G., Sweeney, H.L., Discher, D.E., 2004.Myotubes differentiate optimally on substrates with tissue-like stiffness: patho-logical implications for soft or stiff microenvironments. J. Cell Biol. 166, 877–887.
vans, E., Kinoshita, K., Simon, S., Leung, A., 2010. Long-lived, high-strength statesof ICAM-1 bonds to beta2 integrin, I: Lifetimes of bonds to recombinant alphaL-beta2 under force. Biophys. J. 98, 1458–1466.
u, Q., Rahaman, M.N., Bal, B.S., Brown, R.F., 2010. Preparation and in vitro evaluationof bioactive glass (13–93) scaffolds with oriented microstructures for repair andregeneration of load-bearing bones. J. Biomed. Mater. Res. A 93, 1380–1390.
owen, M., Lazner, F., Dodds, R., Kapadia, R., Feild, J., Tavaria, M., Bertoncello, I.,Drake, F., Zavarselk, S., Tellis, I., Hertzog, P., Debouck, C., Kola, I., 1999. CathepsinK knockout mice develop osteopetrosis due to a deficit in matrix degradationbut not demineralization. J. Bone Miner. Res. 14, 1654–1663.
uiet, R., Verollet, C., Lamsoul, I., Cougoule, C., Poincloux, R., Labrousse, A., Calder-wood, D.A., Glogauer, M., Lutz, P.G., Maridonneau-Parini, I., 2012. Macrophagemesenchymal migration requires podosome stabilization by filamin A. J. Biol.Chem. 287, 13051–13062.
u, S., Planus, E., Georgess, D., Place, C., Wang, X., Albiges-Rizo, C., Jurdic, P., Gemi-nard, J.C., 2011. Podosome rings generate forces that drive saltatory osteoclastmigration. Mol. Biol. Cell 22, 3120–3126.
shii, M., Kikuta, J., Shimazu, Y., Meier-Schellersheim, M., Germain, R.N., 2010.Chemorepulsion by blood S1P regulates osteoclast precursor mobilization andbone remodeling in vivo. J. Exp. Med. 207, 2793–2798.
ang, I., Jeon, B.T., Jeong, E.A., Kim, E.J., Kang, D., Lee, J.S., Jeong, B.G., Kim, J.H., Choi,B.H., Lee, J.E., Kim, J.W., Choi, J.Y., Roh, G.S., 2012. Pak1/LIMK1/cofilin path-way contributes to tumor migration and invasion in human non-small cell lungcarcinomas and cell lines. Korean J. Physiol. Pharmacol. 16, 159–165.
hatiwala, C.B., Peyton, S.R., Putnam, A.J., 2006. Intrinsic mechanical properties ofthe extracellular matrix affect the behavior of pre-osteoblastic MC3T3-E1 cells.Am. J. Physiol. Cell Physiol. 290, C1640–C1650.
im, K., Lee, S.H., Ha Kim, J., Choi, Y., Kim, N., 2008. NFATc1 induces osteoclast fusionvia up-regulation of Atp6v0d2 and the dendritic cell-specific transmembraneprotein (DC-STAMP). Mol. Endocrinol. (Baltimore, MD) 22, 176–185.
irstein, B., Chambers, T.J., Fuller, K., 2006. Secretion of tartrate-resistant acid phos-phatase by osteoclasts correlates with resorptive behavior. J. Cell. Biochem. 98,1085–1094.
otani, M., Kikuta, J., Klauschen, F., Chino, T., Kobayashi, Y., Yasuda, H., Tamai, K.,Miyawaki, A., Kanagawa, O., Tomura, M., Ishii, M., 2013. Systemic circulation andbone recruitment of osteoclast precursors tracked by using fluorescent imagingtechniques. J. Immunol. 190, 605–612.
ammermann, T., Bader, B.L., Monkley, S.J., Worbs, T., Wedlich-Soldner, R., Hirsch,K., Keller, M., Forster, R., Critchley, D.R., Fassler, R., Sixt, M., 2008. Rapid leuko-
cyte migration by integrin-independent flowing and squeezing. Nature 453,51–55.
uxenburg, C., Geblinger, D., Klein, E., Anderson, K., Hanein, D., Geiger, B., Addadi, L.,2007. The architecture of the adhesive apparatus of cultured osteoclasts: frompodosome formation to sealing zone assembly. PLoS ONE 2, e179.
of Cell Biology 92 (2013) 139– 149 149
McHugh, K.P., Hodivala-Dilke, K., Zheng, M.H., Namba, N., Lam, J., Novack, D., Feng,X., Ross, F.P., Hynes, R.O., Teitelbaum, S.L., 2000. Mice lacking beta3 integrins areosteosclerotic because of dysfunctional osteoclasts. J. Clin. Invest. 105, 433–440.
Mochizuki, A., Takami, M., Miyamoto, Y., Nakamaki, T., Tomoyasu, S., Kadono, Y.,Tanaka, S., Inoue, T., Kamijo, R., 2012. Cell adhesion signaling regulates RANKexpression in osteoclast precursors. PLoS ONE 7, e48795.
Mould, A.P., Askari, J.A., Barton, S., Kline, A.D., McEwan, P.A., Craig, S.E., Humphries,M.J., 2002. Integrin activation involves a conformational change in the alpha 1helix of the beta subunit A-domain. J. Biol. Chem. 277, 19800–19805.
Novack, D.V., Faccio, R., 2009. Osteoclast motility: putting the brakes on bone resorp-tion. Ageing Res. Rev. 10, 54–61.
Okamoto, K., Takayanagi, H., 2011. Osteoclasts in arthritis and Th17 cell develop-ment. Int. Immunopharmacol. 11, 543–548.
Ory, S., Brazier, H., Blangy, A., 2007. Identification of a bipartite focal adhesion local-ization signal in RhoU/Wrch-1, a Rho family GTPase that regulates cell adhesionand migration. Biol. Cell. 99, 701–716.
Pelham Jr., R.J., Wang, Y., 1997. Cell locomotion and focal adhesions are regulatedby substrate flexibility. Proc. Natl. Acad. Sci. U.S.A. 94, 13661–13665.
Roodman, G.D., 1999. Cell biology of the osteoclast. Exp. Hematol. 27, 1229–1241.Saltel, F., Chabadel, A., Zhao, Y., Lafage-Proust, M.H., Clezardin, P., Jurdic, P., Bonnelye,
E., 2006. Transmigration: a new property of mature multinucleated osteoclasts.J. Bone Miner. Res. 21, 1913–1923.
Schmidt, S., Nakchbandi, I., Ruppert, R., Kawelke, N., Hess, M.W., Pfaller, K., Jurdic, P.,Fassler, R., Moser, M., 2011. Kindlin-3-mediated signaling from multiple integrinclasses is required for osteoclast-mediated bone resorption. J. Cell Biol. 192,883–897.
Takayanagi, H., Kim, S., Koga, T., Nishina, H., Isshiki, M., Yoshida, H., Saiura, A.,Isobe, M., Yokochi, T., Inoue, J., Wagner, E.F., Mak, T.W., Kodama, T., Taniguchi,T., 2002. Induction and activation of the transcription factor NFATc1 (NFAT2)integrate RANKL signaling in terminal differentiation of osteoclasts. Dev. Cell 3,889–901.
Van Goethem, E., Poincloux, R., Gauffre, F., Maridonneau-Parini, I., Le Cabec, V., 2010.Matrix architecture dictates three-dimensional migration modes of human
macrophages: differential involvement of proteases and podosome-like struc-tures. J. Immunol. 184, 1049–1061.
van Helden, S.F., Krooshoop, D.J., Broers, K.C., Raymakers, R.A., Figdor, C.G., vanLeeuwen, F.N., 2006. A critical role for prostaglandin E2 in podosome disso-lution and induction of high-speed migration during dendritic cell maturation.J. Immunol. 177, 1567–1574.
Vives, V., Laurin, M., Cres, G., Larrousse, P., Morichaud, Z., Noel, D., Cote, J.F., Blangy,A., 2011. The Rac1 exchange factor Dock5 is essential for bone resorption byosteoclasts. J. Bone Miner. Res. 26, 1099–1110.
Wakkach, A., Mansour, A., Dacquin, R., Coste, E., Jurdic, P., Carle, G.F., Blin-Wakkach,C., 2008. Bone marrow microenvironment controls the in vivo differentiationof murine dendritic cells into osteoclasts. Blood 112, 5074–5083.
Yagi, M., Ninomiya, K., Fujita, N., Suzuki, T., Iwasaki, R., Morita, K., Hosogane, N.,Matsuo, K., Toyama, Y., Suda, T., Miyamoto, T., 2007. Induction of DC-STAMP
by alternative activation and downstream signaling mechanisms. J. Bone Miner.Res. 22, 992–1001.
Zhou, Y., Su, J., Shi, L., Liao, Q., Su, Q., 2013. DADS downregulates the Rac1-ROCK1/PAK1-LIMK1-ADF/cofilin signaling pathway, inhibiting cell migrationand invasion. Oncol. Rep. 29, 605–612.
