Ecole Normale Supérieure de Lyon, 46
allée d'Italie, 69364 Lyon Cedex 07,
France
*
Present address: Institut Albert-Bonniot, LEDAC (UMR5538),
Faculté de Medecine, F 38706 La Tronche Cedex,
France
Author for correspondence (e-mail:
martin.pfaff{at}ujf-grenoble.fr
)
Accepted May 1, 2001
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SUMMARY |
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Key words: Osteoclast, Podosome, Integrin, Cytoskeleton, Cell adhesion
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INTRODUCTION |
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It has been recognized that podosomes share major structural components
with focal adhesions, e.g. -actinin, vinculin and talin (David-Pfeuty
and Singer, 1980
; Marchisio et
al., 1984
; Marchisio et al.,
1987
). Furthermore, both
structures are sites of increased protein tyrosine phosphorylation (Tarone et
al., 1985
; Burridge and
Chrzanowska-Wodnicka, 1996
). On
the other hand, proteins, like gelsolin (Yin,
1987
) and fimbrin (Tplastin;
Bretscher, 1981
), which
regulate actin filament organization, are enriched only in podosomes (Wang et
al., 1984
; Carley et al.,
1986
; Marchisio et al.,
1984
; Marchisio et al.,
1987
). Recent genetic evidence
underlines the particular importance of two such proteins for podosome
function in vivo, of gelsolin (Chellaiah et al.,
2000
) and WASP (Linder et al.,
1999
). These data suggest that
the mechanisms linking cell adhesive contacts to the F-actin cytoskeleton are
similar in focal adhesions and podosomes, but that both structures differ in
the mechanisms that regulate the microarchitecture of actin filaments.
Using time-lapse video and photobleaching techniques in RSV-transformed rat
kidney cells microinjected with fluorescent -actinin, Stickel and Wang
(Stickel and Wang, 1987
)
observed that podosomes underwent rapid formation, movement and breakdown.
Fluorescing
-actinin molecules were replaced in podosomes with a
halftime of fluorescence recovery of 4 seconds, more than 10 times faster than
in focal adhesions of non-transformed cells. Thus, podosomes are highly
dynamic structures, that rapidly move and change shape and size. Chen and
co-workers have noted that podosomes in RSV-transformed cells, but not focal
adhesions in the non-transformed cells, colocalize with proteolytic activities
that degrade extracellular matrix proteins (Chen et al.,
1984
; Chen,
1989
). In transmission
electron microscopic images, they associated these proteolytic activities with
membrane protrusions, called invadopodia, formed adjacent to the rosette
contact sites. Two other studies (Nitsch et al.,
1989
; Ochoa et al.,
2000
) have described
invaginations of the plasma membrane reaching into the center of podosomes.
These are associated with membrane transport phenomena, as indicated by the
presence of the GTPase dynamin 2 (Ochoa et al.,
2000
), and might represent
sites of local protease release. Moreover, podosomes co-distribute with matrix
metalloproteinases in osteoclasts (Blavier and
Delaissé,
1995
; Sato et al.,
1997
). Therefore, podosomes
appear to be structures that uniquely combine adhesive functions with
proteolytic degradation of the extracellular matrix, characteristics that are
especially important for highly invasive cells.
The adhesive mechanisms establishing the podosome contact with the
extracellular matrix are not well understood. Experimental evidence that
demonstrates a direct implication of integrins in podosome function is scarce
compared with their well-established role in the formation of focal adhesion
plaques (Burridge and Chrzanowska-Wodnicka,
1996). Yet integrins have been
localized to podosomes that co-distribute either with F-actin (Marchisio et
al., 1988
; Nakamura et al.,
1999
) or with the plasma
membrane region directly adjacent to it (Marchisio et al.,
1988
; Helfrich et al.,
1996
; Zambonin-Zallone et al.,
1989
). One study (Johansson et
al., 1994
) has localized a
subpopulation of ß1 integrin subunits that contain a phosphorylated
tyrosine in its cytoplasmic domain in podosomes of RSV-transformed cells. In
osteoclasts, podosomes are putative precursors of the sealing zone, a tight
adhesion structure that seals the cells onto bone during resorptive activities
(Väänänen
and Horton, 1995
). Osteoclasts
express integrin
Vß3 as major integrin adhesion receptor, which is
required for the formation of the sealing zone ex vivo and for bone resorbing
activity in vivo (McHugh et al.,
2000
). However, it is highly
questioned whether this integrin provides the adhesive link between the
sealing zone and bone, because it could not be localized in this adhesive
structure (Lakkakorpi et al.,
1991
; Helfrich et al.,
1996
;
Väänänen
et al., 2000
). By contrast,
several reports demonstrate an association of this integrin with podosomes in
non-resorbing osteoclasts or osteoclast-like cells (Nakamura et al.,
1999
; Helfrich et al.,
1996
; Zambonin-Zallone et al.,
1989
).
We have decided to study podosomes in cells of the monocytic lineage, which
represent the sole untransformed cell types forming podosomes. We observed the
organization and the molecular architecture of these structures during the
differentiation of human and chicken monocyte-derived precursor cells to
bone-resorbing osteoclasts. This differentiation was initiated by the cytokine
RANKL-ODF (ligand of receptor activator of NFB-osteoclast
differentiation factor; Yasuda et al.,
1998
; Anderson et al.,
1997
), which induced high
expression levels of integrin
Vß3, reinforced the distribution of
podosomes at the cell periphery, and initiated their partial fusion to larger
F-actin-containing structures. We also observed changes in the distribution of
tyrosine-phosphorylated proteins in the podosome zone, together with the
terminal steps of differentiation. Moreover, we demonstrate the colocalization
of integrin
Vß3 in the juxtamembrane region adjacent to podosomes
with the non-receptor tyrosine kinase Pyk2 and the cytoskeletal adapter
protein paxillin. Both of these latter proteins, especially paxillin,
incorporated high amounts of phosphate into tyrosine residues during cell
adhesion. Finally, we provide evidence from in vitro binding experiments
pointing to a novel, direct interaction of both paxillin and Pyk2 with the
C-terminal region of the integrin ß3 tail. This interaction might be a
crucial element in podosome function in osteoclast-like cells.
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MATERIALS AND METHODS |
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Cell culture
Chicken monocytes were isolated from Ficoll-separated peripheral blood
cells and cultured as described previously (Solari et al.,
1995; Boissy et al.,
1998
). After 2 days of initial
culture, adherent macrophages were trypsinized and replated onto coverslips or
plastic dishes. For RANKL-ODF treatment, cells were cultured in
MEM
containing 10% fetal calf serum (HyClone Laboratories) and 30 ng/ml RANKL-ODF
for 2 to 5 days. Adherent human peripheral blood macrophages, similarly
obtained from Ficoll-separated peripheral blood leukocytes, were cultured in
MEM, 10% fetal calf serum (HyClone Laboratories) and treated for 6-9
days with 50 ng/ml RANKL-ODF and 20 ng/ml M-CSF. Murine RAW 264.7 cells were
obtained from ATCC (Rockville, Maryland) and cultured in DMEM, 10% fetal calf
serum (HyClone Laboratories).
Immunofluorescence
Cells grown on glass coverslips were for a short period immersed in
phosphate-buffered saline (PBS) and immediately fixed in 2.5%
paraformaldehyde, 5% sucrose in PBS for 10 minutes at room temperature. Cells
were permeabilized in 0.1% Triton X-100, 1 mM sodium orthovanadate in PBS for
5 minutes. After washing, coverslips were blocked with 1% bovine serum
albumin, 1 mM sodium orthovanadate in PBS and incubated with first and
secondary antibodies and rhodamine phalloidin in this same buffer. PBS-washed
samples were mounted in FluorsaveTM (Calbiochem) containing 0.05%
p-phenylenediamine and observed the next day with an epifluorescence
microscope (Axioplan 2, Zeiss) equipped for confocal immunofluorescence
analysis (LSM510, Zeiss).
Peptide and protein synthesis
Peptides were synthesized as described (Boissy et al.,
1998). They consisted of
N-terminally biotinylated penetratin peptide (RQIKIWFQNRRMKWKK), which was
directly followed by integrin ß cytoplasmic domain sequences (represented
in Fig. 6A). Peptide purity was
controlled by HPLC and their correct mass confirmed by electrospray mass
spectrometry. Recombinant structural mimics of human integrin ß1 and
ß3 tails were provided by David Calderwood and Mark Ginsberg (La Jolla,
CA). These proteins consist of the full-length integrin cytoplasmic domains
(Fig. 8) linked at their N
terminus to a heptad-repeat and a His-Tag sequence, as described (Calderwood
et al., 1999
; Pfaff et al.,
1998
).
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A pGEX-2TKTM vector for bacterial expression of
glutathione-S-transferase-(GST)-linked human paxillin was kindly provided by
Dr Mark Ginsberg (La Jolla, CA) with kind permission of Dr Ravi Salgia
(Boston, MA). Recombinant GST-paxillin was produced in Escherichia
coli strain DH5 (GIBCO Life Technologies) and purified on
glutathione crosslinked agarose (Sigma) from bacterial lysates. Lysis and
purification were performed in 20 mM Tris/HCl pH 8.0, 0.1% Triton X-100, 100
mM sodium chloride, 1mM EDTA, 1 mM DTT. Bound paxillin was eluted from
glutathione beads with 20 mM reduced glutathione, 0.75 M Tris/HCl pH 9.6 and
added to 1/2 volume of 1.5 M Tris/HCl pH 6.8.
A pEGB mammalian GST-fusion expression vector to produce GST-linked human
Pyk2 N-terminal domain (amino acids 1-407) (GST-Pyk2-NT) was provided by David
Schlaepfer (La Jolla, CA; Sieg et al.,
2000). GST-Pyk2-NT was
purified on glutathione agarose from lysates of transiently transfected 293T
cells.
Immunoprecipitation and western blotting
After two short washes with PBS, cells were lysed in buffer A (50 mM
Tris/HCl pH 7.4, 75 mM sodium chloride, 50 mM sodium fluoride, 40 mM sodium
pyrophosphate, 1 mM sodium orthovanadate, 1 mM EDTA and protease inhibitors
(CømpleteTM, Boehringer Mannheim)) containing 1% Triton
X-100, 0.5% sodium deoxycholate and 0.1% SDS. After 30 minutes on ice, lysates
were cleared by centrifugation and agitated with Protein G Sepharose 4B
(Sigma) for 1 hour at 4°C. Precleared lysates were incubated overnight
with antibodies and Protein G Sepharose at 4°C. Immunoprecipitates were
washed five times with buffer A containing 1% Triton X-100, 0.5% sodium
deoxycholate and 0.1% SDS, and processed for SDS polyacrylamide
electrophoresis followed by electrotransfer onto nitrocellulose (Amersham).
Transfer efficiency was verified by Ponceau S staining. Nitrocellulose
membranes were blocked overnight with 100 mM Tris/HCl pH 7.4, 150 mM sodium
chloride containing either 5% fat-free milk powder
(Régilait, France) or 3% bovine serum albumin,
0.1% Tween 20 (for incubations with antiphosphotyrosine mAbs). After
incubations with first and peroxydasecoupled secondary antibodies, bound
antibodies were detected by chemiluminescence (ECL, Amersham).
Affinity precipitation with peptides
For peptide binding experiments, cell lysates were prepared in buffer A
containing 1% Triton X-100, 0.2% sodium deoxycholate. Cleared lysates were
diluted with one volume buffer A and incubated with biotinylated peptides
prebound to crosslinked streptavidin agarose (Sigma) overnight at 4°C.
After five to six washes with buffer A, containing 0.5% Triton X-100 and 0.1%
sodium deoxycholate, bound proteins were eluted by boiling in reducing sample
buffer for SDS gel electrophoresis. The presence of equal amounts of the
different peptides in each experiment was verified on Coomassie-stained 15%
SDS polyacrylamide gels and bound proteins were identified by specific
antibodies on 8-10% SDS polyacrylamide gels transferred to nitrocellulose.
Binding to recombinant GST, GST-paxillin and GST-Pyk2-NT was performed under
identical conditions, except that 0.5 mM DTT and 2 mg/ml bovine serum albumin
were added to the binding buffer. Recombinant structural mimics of integrin
ß tails were used prebound to a Ni2+-resin (Novagen) as
described (Pfaff et al.,
1998). To maintain their
interaction with the resin, EDTA in buffer A had to be replaced with 1 mM
CaCl2 and 2 mM MgCl2 throughout the experiment.
Digital image processing
Confocal images, scanned immunofluorescence photographs, as well as
autoradiograms of western blots and scans of Coomassie Blue stained SDS-PAGE
gels were digitally processed using the program Adobe Photoshop for the layout
of the figures.
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RESULTS |
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Podosome-associated tyrosine phosphorylation
To gain insight into the mechanisms that regulate podosome architecture and
function, we have analyzed the association of tyrosine-phosphorylated proteins
with these cytoskeletal structures during in vitro osteoclast differentiation.
The major structures labeled by monoclonal anti-phosphotyrosine antibodies at
all stages of differentiation were indeed podosomes
(Fig. 3). Podosome-associated
phosphotyrosine predominantly localized to the extracellular
substrate-oriented podosome tips in early macrophages and in
non-RANKL-ODF-treated cells (Fig.
3). This was especially evident in confocal images scanned in the
z-axis, that sectioned individual podosomes in their centers
(Fig. 3A,C, insets). However,
the particularly F-actin-rich podosomes in RANKL-ODF treated osteoclast-like
cells showed a reduced phospho-tyrosine staining
(Fig. 3B,C, arrows in
right-hand panels). Only the podosomes located at the side of the podosome
ring facing the cell-centers were strongly stained at the tips
(Fig. 3B,C, arrowheads in
righthand panels). Reduced phospho-tyrosine staining of particularly large,
F-actin-rich podosomes was also occasionally observed in non-RANKL-ODF-treated
cells (see arrow in Fig. 3A,
right-hand panel). These variations in the association of podosomes with
tyrosine-phosphorylated proteins indicate a functional specialization of the
podosome ring during RANKL-ODF-induced differentiation, which remains to be
further elucidated.
Adhesion-dependent tyrosine phosphorylation in cellular lysates
To identify podosome proteins phosphorylated on tyrosine in a cell
adhesion-dependent manner, we biochemically analyzed cellular lysates in
western blotting with anti-phosphotyrosine antibodies. A distinct profile of
tyrosine-phosphorylated proteins with major bands at 60, 70 and 85, and
between 110 to 130 kDa was detected in lysates of adherent osteoclast
precursors (Fig. 4). This
profile was essentially unaffected during RANKL-ODF-induced differentiation
(Fig. 4A). Tyrosine
phosphorylation was strongly reduced in lysates of cells kept in suspension
for 2 hours, but reappeared during subsequent cell adhesion in a time course
reflecting the extent of cell spreading
(Fig. 4B). The major tyrosine
phosphorylated protein migrating at 70 kDa in SDS-PAGE gels was identified as
paxillin (Fig. 4C). Because
immunoprecipitation with antiphosphotyrosine antibodies did not markedly
reduce paxillin levels in the lysates, we conclude that only a small portion
of paxillin was actually phosphorylated on tyrosine
(Fig. 4D). In similar
experiments, we detected adhesion-dependent tyrosine phosphorylation of the
115 kDa protein kinase Pyk2 (Fig.
4E) and of p 130cas (data not shown). We also noted that the band
at 60 kDa contained tyrosine-phosphorylated pp60c-src (data not shown), which
remained markedly tyrosine phosphorylated in the suspended cells
(Fig. 4B,E). So far, we were
unable to identify the prominent tyrosine-phosphorylated protein migrating at
85 kDa. In conclusion, cell adhesion and spreading of osteoclasts and
osteoclast precursors result in high-level tyrosine phosphorylation of a
characteristic set of proteins, most notably, however, of a minor
subpopulation of paxillin molecules.
|
Colocalization of integrin Vß3, paxillin and Pyk2 in the
podosome zone
We next analyzed the precise cellular location of podosome proteins by
immunofluorescence (Fig. 5).
Both paxillin and Pyk2 accumulated in the zone containing podosomes, but they
were essentially confined to regions between the individual F-actin-containing
core structures of podosomes (Fig.
5). Confocal images scanned in the z-axis narrowed down
their location as being close to the extracellular substrate-oriented plasma
membrane between the podosomes (Fig.
5, insets in left panels). A broader analysis showed that most of
the proteins known to associate with focal adhesion plaques in other cells,
like talin, vinculin, -actinin, zyxin, tensin, p130cas and the integrin
Vß3, share a very similar subcellular location with paxillin and
Pyk2 in the podosome zone (Fig.
5, and data not shown). A different distribution was observed for
the src-substrate cortactin, which colocalized with the F-actin core of
podosomes in horizontal confocal sections (resulting in yellow podosomes in
images with merged F-actin and cortactin channels,
Fig. 5, bottom panels).
Vertical confocal sectioning (Fig.
5, inset in bottom left panel) revealed its preferential location
in the basal tips of podosomes. A similar distribution was observed for the
protein WASP (data not shown). Thus, the subcellular location of the proteins
previously identified as major substrates of adhesion-dependent tyrosine
phosphorylation (Fig. 4) was to
some extent different from the subcellular structures stained during
immunofluorescence with anti-phosphotyrosine antibodies
(Fig. 3). Most notably, the
prominent phosphotyrosine signal detected on the basal tips of podosomes
(Fig. 3) corresponded best to
the distribution of cortactin, but much less to that observed for paxillin,
Pyk2 and integrin
Vß3 (Fig.
5). However, we could not detect adhesion-dependent
phosphorylation on tyrosine in immunoprecipitates of cortactin. Moreover,
although cortactin migrates as a 85 kDa band in SDS-PAGE gels, its
immunodepletion from the cell lysates did not remove the major
tyrosine-phosphorylated 85 kDa band (see
Fig. 4; data not shown). Hence,
an as yet unidentified protein might be responsible for the phosphotyrosine
signal detected in situ on the podosome tips or, alternatively, the small
subpopulation of paxillin molecules, which is actually phosphorylated on
tyrosine (Fig. 4D), might be
located close to the basal podosome tips.
Pyk2 and paxillin interact with the integrin ß3 cytoplasmic
tail
To gain insight into molecular interactions that link adhesion receptors to
the particular actin cytoskeleton in macrophages and osteoclast-like cells, we
performed affinity precipitation experiments with peptides containing the
C-terminal third of integrin ß cytoplasmic domains
(Fig. 6A). In a previous study,
we have shown that such a peptide containing the 17 C-terminal amino acids of
the chicken integrin ß3 tail, which is rendered membrane permeable by its
coupling to penetratin (Derossi et al.,
1994), blocked the spreading
of cultured macrophages expressing high levels of
Vß3 (Boissy et
al., 1998
). When incubated with
a lysate of macrophages or osteoclast-like cells, this peptide precipitated
high amounts of two tyrosine-phosphorylated proteins, which were identified as
paxillin and Pyk2 (Fig. 6B, D). Two other cytoskeletal proteins, talin and vinculin, did not bind to this
peptide (Fig. 6B and data not
shown). Peptides containing single amino-acid substitutions (S-752-P;
Y-759-A), previously reported to compromise ß3 integrin function (Chen et
al., 1992
; O'Toole et al.,
1995
;
Ylänne et al.,
1995
; Schaffner-Reckinger et
al., 1998
) or a peptide
containing the homologous region of the integrin ß1 tail bound these
proteins only very weakly or not at all
(Fig. 6B). Experiments with
lysates of suspended and attached cells indicated that the interactions with
both paxillin and Pyk2 did not correlate with the extent of adhesion-triggered
tyrosine phosphorylation (Fig.
6C). Moreover, Pyk2 binding to the ß3 cytoplasmic tail was
unaffected by immunodepleting cell lysates for paxillin
(Fig. 6D). This indicates that
it was independent of the known interaction between Pyk2 and paxillin
(Schaller and Sasaki, 1997
).
Finally, binding of both latter proteins to the ß3 tail was observed in
additional experiments using the chicken ß3-tail peptide (shown in
Fig. 6A) with lysates of a
murine monocyte-derived cell line RAW 264.7 and of human 293T cells
transfected with human Pyk2 (data not shown).
Purified glutathione-S-transferase fusion proteins of full-length human paxillin and of the N-terminal part of Pyk2 (amino acids 1-407) also strongly bound to the unmodified integrin ß3 tail peptide, whereas glutathion-S-transferase alone showed negligible binding (Fig. 7). Therefore, both interactions appear to be direct.
|
To further confirm these results, we used recombinant structural mimics of
integrin ß tails containing full-length human cytoplasmic domains in
similar experiments (Fig. 8;
Pfaff et al., 1998). These
studies revealed again significant binding of both paxillin and Pyk2 in
lysates of chicken osteoclast precursors
(Fig. 8) and in mammalian cell
lines (data not shown) to the integrin ß3 cytoplasmic domain. We also
noted somewhat reduced, but still significant binding of Pyk2 and paxillin to
the full-length ß1 tail (Fig.
8). As expected from other studies, both full-length ß1 and
ß3 tails bound to talin, and talin binding was disrupted by
tyrosine-alanine mutations in their membrane-proximal NPXY motifs (Pfaff et
al., 1998
; Calderwood et al.,
1999
;
Fig. 8). Interestingly, this
same mutation also abolished paxillin and Pyk2 binding to the integrin ß3
tail, but not to the integrin ß1 tail
(Fig. 8), suggesting differing
binding requirements for ß1 and ß3 tails. Therefore, these data
provide evidence for novel and direct interactions of the integrin ß3
tail C-terminal region with paxillin and Pyk2.
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DISCUSSION |
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Our results suggest key roles of these three proteins in the regulation of cell adhesion-triggered cytoskeletal organization and signal transduction in podosome-containing cells derived from the monocytic lineage.
During RANKL-ODF-induced osteoclast differentiation of peripheral blood
monocytes, podosomes formed early in macrophage-like precursor cells. At this
stage, podosomes were found throughout the basal cell body
(Fig. 1). During subsequent
culture, the cells increasingly spread and began to fuse; concomitantly, their
podosomes redistributed towards the cell periphery. This change in podosome
distribution might reflect a change in the mechanism of podosome-mediated cell
movement: we have observed that mononuclear osteoclast precursors migrate as
`whole cells', whereas the movement of large multinucleated osteoclast
precursors rather consists in extensions and retractions of peripheral cell
parts (S. Ory and P. J., unpublished). In these osteoclast precursors, the
podosome tips facing the extracellular substrate are strongly enriched in
tyrosine-phosphorylated proteins. RANKL-ODF-treatment reinforced the
peripheral podosome distribution and led to more tightly packed podosomes,
which occasionally fused to larger F-actin containing aggregates. This
possibly reflects initial differentiation of the podosome ring to a functional
sealing zone, which normally forms only in bone-resorbing osteoclasts
(Väänänen
and Horton, 1995). The densely
packed podosomes in RANKL-ODF-treated cells contained reduced amounts of
phosphotyrosine, which did not appear to be topologically associated with the
prominent F-actin containing structures. Hence, the osteoclastogenic factor
RANKL-ODF induced discrete alterations in the microarchitecture of podosomes
and in their association with tyrosine-phosphorylated proteins.
However, a very similar set of proteins in cell lysates was phosphorylated
on tyrosine in a cell-adhesion-dependent manner in both RANKL-ODF-treated and
untreated cells (Fig. 4A).
Thus, changes in the cellular distribution
(Fig. 3) rather than in the
tyrosine phosphorylation of these proteins predominate during osteoclast
differentiation. Most of the phosphotyrosine generated in adherent and spread
macrophages or osteoclasts was detected in the LIM protein paxillin (Turner,
1994a), a scaffolding protein with binding sites for many proteins, including
the cytoplasmic protein tyrosine kinases FAK, Pyk2 and c-src (Schaller and
Sasaki, 1997; Weng et al.,
1993
; Sabe et al.,
1994
), the protein-tyrosine
phosphatase PTP-Pest (Shen et al.,
1998
), and the cytoskeletal
protein vinculin (Turner and Miller,
1994
). Paxillin also provides
a link between regulators of p21 GTPases and adhesion complexes by its binding
to a protein complex containing the p21 GTPase-activated kinase (PAK) and the
guanine nucleotide exchange factor PIX (Turner et al.,
1999
). Thus, paxillin is in a
key position to regulate adhesion-dependent cytoskeletal organization and
signal transduction. So far, its role in osteoclast adhesion and in podosome
function has not been thoroughly addressed, although several recent studies
noticed its colocalization with F-actin, vinculin, Pyk2 and p130cas in the
sealing zone of mouse osteoclasts (Lakkakorpi et al.,
1999
; Duong et al.,
1998
). In addition, a 70 kDa
protein, probably identical to paxillin, has been identified as predominant
tyrosine-phosphorylated adhesion substrate in human monocytes (Lin et al.,
1994
). We now identify
paxillin as the major tyrosine-phosphorylated protein in differentiating
osteoclasts. It co-distributes with the integrin
Vß3, the
non-receptor tyrosine kinase Pyk2 and with many other proteins, which are
typically found in focal adhesion complexes, in the membrane-proximal region
immediately adjacent to podosomes (Fig.
5; M. P. and P. J., unpublished; see also Lakkakorpi et al.,
1999
; Duong et al.,
1998
; Nakamura et al.,
1999
; Helfrich et al.,
1996
; Zambonin-Zallone et al.,
1989
; David-Pfeuty and Singer,
1980
; Marchisio et al.,
1984
; Marchisio et al.,
1987
).
Cell adhesion phenomena associated with podosomes are not very well
characterized. It remains to be demonstrated in molecular detail, how
podosomes contact the extracellular substrate. IRM techniques have revealed
that podosomes contact the substrate within a small ring (a rosette)
co-distributing with the protein vinculin around the central actin core
(David-Pfeuty and Singer,
1980; Marchisio et al.,
1984
). This topology of the
contact zone of a podosome is consistent with the subcellular location of
paxillin, Pyk2 and the major osteoclast integrin,
Vß3, revealed in
this study. It implies that strong integrin-dependent adhesion and
cytoskeletal linkage occur predominantly in this delimited region, where
adhesion receptors most closely approach the central actin-rich podosome core.
Moreover, these locally restricted cell adhesive contacts could also trigger
the strong protein tyrosine phosphorylation that we observed at the podosome
tips. This phosphotyrosine staining observed in situ could correspond to the
predominant tyrosine phosphorylation of paxillin detected in lysates of
adherent osteoclast precursors, assuming that only a small subpopulation of
paxillin molecules located near the podosome contact zone is involved.
We report for the first time that the 17 C-terminal residues of the
integrin ß3 chain strongly and directly bind to both paxillin and Pyk2
(Figs
6,7,8).
An interaction between paxillin and integrin ß cytoplasmic domains has
been suggested in earlier studies (Schaller et al.,
1995; Tanaka et al.,
1996
). Schaller et al.
reported paxillin binding to the membrane-proximal regions of integrin
ß1, ß2 and ß3 tails, but they did not provide evidence that the
interaction was direct (Schaller et al.,
1995
). Tanaka et al.
demonstrated direct paxillin binding to the integrin ß1 cytoplasmic
domain (Tanaka et al., 1996
).
Consistent with the former studies, we detected paxillin binding to
full-length ß1 tails, which was not abolished by a point mutation
adjacent to and not overlapping the proposed binding motif (Schaller et al.,
1995
;
Fig. 8). In contrast, our
results show that paxillin interacts in a clearly different way with the
integrin ß3 tail. A peptide consisting of its 17 C-terminal amino acids
alone contains strong paxillin binding activity and mutations at three
critical positions in its C-terminal region abrogate this interaction. The
serine(752)-proline mutation has been identified in a patient with Glanzmann's
thrombasthenia and resulted, like the tyrosine-alanine mutations at positions
747 and 759, in compromised capacities of the integrin to become competent for
ligand binding, to localize to focal adhesion plaques and to promote cell
spreading (Chen et al., 1992
;
O'Toole et al., 1995
;
Ylänne et al.,
1995
; Schaffner-Reckinger et
al., 1998
). Hence, our results
provide a potential molecular explanation for at least some of the defects
observed with these mutants, notably those that interfere with cell spreading
and focal adhesion localization.
We also observed strong binding of the protein kinase Pyk2 to the integrin
ß3 tail. Although the binding requirements in the ß3 cytoplasmic
domain were very similar for Pyk2 and for paxillin, we excluded the
possibility that Pyk2 binds via paxillin to the ß3 tail
(Fig. 6D). By using GST-fusion
proteins, we obtained additional evidence indicating that both interactions
are indeed independent and direct (Fig.
7). However, the requirements for ß3 tail binding to paxillin
and Pyk2 differed strongly from its binding to talin. This was expected, as
talin binding requires more membrane-proximal regions of integrin ß
tails, which are absent in our synthetic peptides (Tapley et al.,
1989; Patil et al.,
1999
).
We further show that neither paxillin nor Pyk2 binding to peptides
containing the C-terminal 17 amino acids of the integrin ß3 cytoplasmic
domain are altered by their cell adhesion-triggered tyrosine phosphorylation
(Fig. 6C). Therefore, tyrosine
phosphorylation of paxillin and Pyk2 is unlikely to provide a direct
regulatory cue for their binding to the ß3 integrin cytoplasmic domain.
This binding has then to be regulated by other means, for example by the
availability of free ß3 tails. Previous studies indicate indeed that the
availability of integrin ß tails for cytoskeletal interactions is
constrained in unoccupied integrins. This constraint involves integrin
tails and it is released during binding of the extracellular ligand (LaFlamme
et al., 1992
; Briesewitz et
al., 1993
;
Ylänne et al.,
1993
). Pyk2- and
paxillin-binding to the integrin ß3 tail could thus trigger their
recruitment to ß3 integrin-dependent cell contacts. A major current issue
will now be to study the role of this novel interaction observed primarily in
a cell-free system in the context of intact cells.
On the basis of these observations, podosomes emerge as dynamic
cytoskeletal structures with many molecular and functional homologies to focal
adhesions. Both adhesion structures use a similar set of adhesion receptors
and of adaptor proteins that link the extracellular contact to the actin
cytoskeleton as well as to signal transduction pathways. The tyrosine kinase
Pyk2, which is normally absent in focal adhesions (Schaller and Sasaki,
1997), could functionally
replace in podosomes the focal adhesion kinase FAK, which is only weakly
expressed in osteoclasts and in cells of the monocytic lineage (Duong et al.,
1998
; Lin et al.,
1994
; Li et al.,
1998
). The integrin
Vß3, which is highly expressed during later stages of osteoclast
differentiation, might be important to adapt podosomes to their function in
osteoclast migration on bone and in the formation of the sealing zone during
bone resorption (McHugh et al.,
2000
). Finally, the molecular
complex that contains paxillin and Pyk2 bound to the C terminus of the
integrin ß3 chain could represent a molecular core structure that governs
the distribution of regulatory cues linking integrin-dependent cell adhesion
to podosome functions in osteoclasts.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Anderson, D., Maraskovsky, E., Billinglsey, W. L., Dougall, W. C., Tometsko, M. E., Roux, E. R., Teepe, M. C., DuBose, R. F., Cosman, D. and Galibert, L. (1997). A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic cell function. Nature 390,175 -179.[Medline]
Blavier, L. and Delaissé, J.
M. (1995). Matrix metalloproteinases are obligatory for the
migration of preosteoclasts to the developing marrow cavity of primitive long
bones. J. Cell Sci. 108,3649
-3659.
Boissy, P., Machuca, I., Pfaff, M., Ficheux, D. and Jurdic,
P. (1998). Aggregation of mononucleated precursors triggers
cell surface expression of Vß3 integrin, essential to formation of
osteoclast-like multinucleated cells. J Cell Sci.
111,2563
-2574.
Bretscher, A. (1981). Fimbrin is a cytoskeletal protein that crosslinks F-actin in vitro. Proc. Natl. Acad. Sci. USA 78,6849 -6853.[Abstract]
Briesewitz, R., Kern, A. and Marcantonio, E. E.
(1993). Ligand-dependent and -independent integrin focal contact
localization: the role of the chain cytoplasmic domain.
Mol. Biol. Cell 4,593
-604.[Abstract]
Burridge, K. and Chrzanowska-Wodnicka, M. (1996). Focal adhesions, contractility, and signaling. Annu. Rev. Cell Dev. Biol. 12,463 -518.[Medline]
Calderwood, D., Zent, R., Grant, R,. Rees, D.J.G., Hynes, R. O.
and Ginsberg, M. H. (1999). The talin head domain binds to
integrin ß subunit cytoplasmic tails and regulates integrin activation.
J. Biol. Chem. 274,28071
-28074.
Carley, W., Bretscher A. and Webb, W. W.
(1986). F-actin aggregates in transformed cells contain
-actinin and fimbrin but apparently lack tropomyosin. Eur.
J. Cell Biol. 39,313
-320.[Medline]
Chellaiah, M., Kizer, N., Silva, M., Alvarez, U., Kwiatkowski,
D. and Hruska, K. A. (2000). Gelsolin deficiency blocks
podosome assembly and produces increased bone mass and strength. J.
Cell Biol. 148,665
-678.
Chen, W.-T. (1989). Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells. J. Exp. Zool. 251,167 -185.[Medline]
Chen, W.-T., Olden, K., Bernard, B. A. and Chu, F.-F. (1984). Expression of transformation-associated protease(s) that degrade fibronectin at cell contact sites. J. Cell Biol. 98,1546 -1555.[Abstract]
Chen, Y.-P., Djaffar, D., Pidard, D., Steiner, B., Cieutat, A.-M., Caen, J. P. and Rosa, J.-P. (1992). Ser752-Pro mutation in the cytoplasmic domain of integrin ß3 subunit and defective activation of platelet integrin aIIbß3 (glycoprotein IIbIIIa) in a variant of Glanzmann thrombasthenia. Proc. Natl. Acad. Sci. USA 89,10169 -10173.[Abstract]
David-Pfeuty, T. and Singer, S. J. (1980).
Altered distributions of the cytoskeletal proteins vinculin and
-actinin in cultured fibroblasts transformed by Rous sarcoma virus.
Proc. Natl. Acad. Sci. USA
77,6687
-6691.[Abstract]
Derossi, D., Joliot, A. H., Chassaing, G. and Prochiantz, A.
(1994). The third helix of the Antennapedia homeodomain
translocates through biological membranes. J. Biol.
Chem. 269,10444
-10450.
Duong, L., Lakkakorpi, P. T., Nakamura, I., Nagy, R. M. and
Rodan, G. A. (1998). Pyk2 in osteoclasts is an adhesion
kinase, localized in the sealing zone, activated by ligation of
Vß3 integrin, and phosphorylated by src kinase. J.
Clin. Invest. 102,881
-892.
Helfrich, M. H., Nesbitt, S. A., Lakkakorpi, P. T., Barnes, M. J., Bodary, S. C., Shankar, G., Mason, W. T., Mendrick, D. L., Väänänen, H. K. and Horton, M. A. (1996). ß1 integrins and osteoclast function: involvement in collagen recognition and bone resorption. Bone 19,317 -328.[Medline]
Hynes, R. O. (1992). Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25.[Medline]
Jockusch, B. M., Bubeck, P., Giehl, K., Kroemker, M., Moschner, J., Rothkegel, M., Rüdiger, M., Schlüter, K., Stanke, G. and Winkler, J. (1995). The molecular architecture of focal adhesions. Annu. Rev. Cell Dev. Biol. 11,379 -416.[Medline]
Johansson, M. W., Larsson, E., Lüning, B., Pasquale, E.B. and Ruoslahti, E. (1994). Altered localization and cytoplasmic domain-binding properties of tyrosine-phosphorylated ß1 integrin. J. Cell Biol. 126,1299 -1309.[Abstract]
LaFlamme, S., Akiyama, S. K. and Yamada, K. M. (1992). Regulation of fibronectin receptor distribution. J. Cell Biol. 117,437 -447.[Abstract]
Lakkakorpi, P., Horton, M. A., Helfrich, M. H., Karhukorpi, E. K. and Väänänen, H. K. (1991). Vitronectin receptor has a role in bone resorption, but does not mediate tight sealing zone attachment of osteoclasts to the bone surface. J. Cell Biol. 115,1179 -1186.[Abstract]
Lakkakorpi, P., Nakamura, I., Nagy, R. M., Parsons, J. T.,
Rodan, G. A. and Duong, L. T. (1999). Stable association of
PYK2 and p130cas in osteoclasts and their co-localization in the sealing zone.
J. Biol. Chem. 274,4900
-4907.
Li, X., Hunter, D., Morris, J., Haskill, J. S. and Earp, H.
S. (1998). A calcium-dependent tyrosine kinase splice variant
in human monocytes. J. Biol. Chem.
273,9361
-9364.
Lin, T.H., Yurochko, A., Kornberg, L., Morris, J., Walker, J. J., Haskill, S. and Juliano, R. L. (1994). The role of protein tyrosine phosphorylation in integrin-mediated gene induction in monocytes. J. Cell Biol. 126,1585 -1593.[Abstract]
Linder, S., Nelson, D., Weiss, M. and Aepfelbacher, M.
(1999). Wiskott-Aldrich syndrome protein regulates podosomes in
primary human macrophages. Proc. Natl. Acad. Sci. USA
96,9648
-9653.
Marchisio, P., Cirillo, D., Naldini, L., Primavera, M. V., Teti, A. and Zambonin-Zallone, A. (1984). Cell substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures. J. Cell Biol. 99,1696 -1705.[Abstract]
Marchisio, P., Cirillo, D., Teti, A., Zambonin-Zallone,A. and Tarone, G. (1987). Rous Sarcoma virus-transformed fibroblasts and cells of monocytic origin display a peculiar dot-like organization of cytoskeletal proteins involved in microfilament-membrane interaction. Exp. Cell Res. 169,202 -214.[Medline]
Marchisio, P., Bergui, L., Corbascio, G.C., Cremona, O., D'Urso, N., Schena, M., Tesio, L. and Caligaris-Cappio, F. (1988). Vinculin, talin, and integrins are localized at specific adhesion sites of malignant B-lymphocytes. Blood 72,830 -833.[Abstract]
McHugh, K., Hodivala-Dilke, K., Zheng, M.-H., Namba, N., Lam,
J., Novack, D., Feng X., Ross, F. P., Hynes, R. O. and Teitelbaum, S. L.
(2000). Mice lacking ß3 integrins are osteosclerotic because
of dysfunctional osteoclasts. J. Clin. Invest.
105,433
-440.
Nakamura, I., Pilkington, M. F., Lakkakorpi, P. T., Lipfert, L.,
Sims, S.M., Dixon, S. J., Rodan, G. A. and Duong, L.T.
(1999). Role of a Vß3 integrin in osteoclast migration and
formation of the sealing zone. J. Cell Sci.
112,3985
-3993.
Nitsch, L., Gionti, E., Cancedda, R. and Marchisio, P. C. (1989). The podosomes of Rous Sarcoma virus transformed chondrocytes show peculiar ultrastructural organization. Cell Biol. Int. Rep. 13,919 -926.[Medline]
Ochoa, G.-C., Sepnev, V. I., Neff, L., Ringstad, N., Takei, K.,
Daniel, L., Kim, W., Cao, H., McNiven, M., Baron, R. and De Camilli, P.
(2000). A functional link between dynamin and the actin
cytoskeleton at podosomes. J. Cell Biol.
150,377
-389.
O'Toole, T. E., Ylänne, J. and
Culley, B. M. (1995). Regulation of integrin affinity states
through an NPXY motif in the ß subunit cytoplasmic domain. J
Biol Chem. 270,8553
-8558.
Patil, S., Jedsadayanmata, A., Wencel-Drake, J. D., Wang, W.,
Knezevic, I. and Lam, S. C.-T. (1999). Identification of a
talin-binding site in the integrin ß3 subunit distinct from the NPLY
regulatory motif of post-ligand binding functions: the talin N-terminal head
domain interacts with the membrane-proximal region of the ß3 cytoplasmic
tail. J. Biol. Chem.
274,28575
-28583.
Pfaff, M., Liu, S., Erle, D. J. and Ginsberg, M. H.
(1998). Integrin ß cytoplasmic domains differentially bind
to cytoskeletal proteins. J. Biol. Chem.
273,6104
-6109.
Quinn, J., Elliott, J., Gillespie, M. T. and Martin, T. J.
(1998). A combination of osteoclast differentiation factor and
macrophage-colony stimulating factor is sufficient for both human and mouse
osteoclast formation in vitro. Endocrinology
139,4424
-4427.
Rottner, K., Hall, A. and Small, J. V. (1999). Interplay between Rac and Rho in the control of substrate contact dynamics. Curr. Biol. 9,640 -648.[Medline]
Sabe, H., Hata, A., Okada, M., Nakagawa, H. and Hanafusa, H. (1994). Analysis of the binding of the src homology 2 domain of csk to tyrosine tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-src. Proc. Natl. Acad. Sci. USA 91,3984 -3988.[Abstract]
Sato, T., del Carmen Ovejero, M., Hou, P., Heegaard, A.-M.,
Kumegawa, M., Foged, N. T. and Delaissé,
J.-M. (1997). Identification of membrane-type matrix
metalloproteinase MT1-MMP in osteoclasts. J. Cell Sci.
110,589
-596.
Schaffner-Reckinger, E., Gouon, V., Melchior, C.,
Plançon, S. and Kieffer, N.
(1998). Distinct involvment of ß3 integrin cytoplasmic
domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal
assembly and phosphotyrosine signaling. J. Biol. Chem.
273,12623
-12632.
Schaller, M. and Sasaki, T. (1997).
Differential signaling by the focal adhesion kinase and cell adhesion kinase
ß. J. Biol. Chem.
272,25319
-25325.
Schaller, M. D., Otey, C. A., Hildebrand, J. D. and Parsons, J. T. (1995). Focal adhesion kinase and paxillin bind to peptides mimicking ß integrin cytoplasmic domains. J. Cell Biol. 130,1181 -1187.[Abstract]
Shen, Y., Schneider, G., Cloutier, J.-F., Veillette, A. and
Schaller, M. D. (1998). Direct association of
protein-tyrosine phosphatase PTP-PEST with paxillin. J. Biol
Chem. 273,6474
-6481.
Sieg, D., Hauck, C. R., Ilic, D., Klingbeil, C. K., Schaefer, E., Damsky, C. H. and Schlaepfer, D. D. (2000). FAK integrates growth-factor and integrin signals to promote cell migration. Nat. Cell Biol. 2,240 -256.
Solari, F., Domenget, C., Gire, V., Woods, C., Lazarides, E.,
Rousset, B. and Jurdic, P. (1995). Multinucleated cells can
continuously generate mononucleated cells in the absence of mitosis: a study
of cells of the avian osteoclast lineage. J. Cell Sci.
108,3233
-3241.
Stickel, S. and Wang, Y.-L. (1987). Alpha-actinin-containing aggregates in transformed cells are highly dynamic structures. J. Cell Biol. 104,1521 -1526.[Abstract]
Tanaka, T., Yamaguchi, R., Sabe, H., Sekiguchi, K. and Healy, J. M. (1996). Paxillin association in vitro with integrin cytoplasmic domain peptides. FEBS Lett. 399, 53-58.[Medline]
Tapley, P., Horwitz, A., Buck, C., Duggan, K. and Rohrschneider, L. (1989). Integrins isolated from Rous sarcoma virus-transformed chicken embryo fibroblasts. Oncogene 4, 325-333.[Medline]
Tarone, G., Cirillo, D., Giancotti, F. G., Comoglio, P. M. and Marchisio, P. C. (1985). Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes. Exp. Cell Res. 159,141 -157.[Medline]
Turner, C. (1994). Paxillin: a cytoskeletal target for tyrosine kinases. BioEssays 16, 47-52.[Medline]
Turner, C. and Miller, J. T. (1994). Primary
sequence of paxillin contains putative SH2 and SH3 domain binding motifs and
multiple LIM domains: identification of a vinculin and pp125FAK binding
region. J. Cell Sci.
107,1583
-1591.
Turner, C., Brown, M. C., Perrotta, J. A., Riedy, M. C.,
Nikolopoulos, S. N., McDonald, A. R., Bagrodia, S., Thomas, S. and Leventhal,
P. S. (1999). Paxillin LD4 motif binds PAK and PIX through a
novel 95-kD ankyrin repeat, ARF-GAP protein: a role in cytoskeletal
remodeling. J. Cell Biol.
145,851
-863.
Väänänen,
H. K. and Horton, M. (1995). The osteoclast clear zone is a
specialized cell-extracellular matrix adhesion structure. J. Cell
Sci. 108,2729
-2732.
Väänänen,
H., Zhao, H., Mulari, M. and Halleen, J. M. (2000). The cell
biology of osteoclast function. J. Cell Sci.
113,377
-381.
Wang, E., Yin, H. L., Krueger, J. G., Caliguiri, L. A. and Tamm, I. (1984). Unphosphorylated gelsolin is localized in regions of cell-substratum contact or attachment of Rous sarcoma virus-transformed rat cells. J. Cell Biol. 98,761 -771.[Abstract]
Weng, Z., Taylor, J. A., Turner, C. E., Brugge, J. S. and
Seidel-Dugan, C. (1993). Detection of src homology 3-binding
proteins, including paxillin, in normal and v-src-transformed Balb/c 3T3
cells. J. Biol. Chem.
268,14956
-14963.
Yasuda, H., Shima, N., Nakagawa, N., Yamaguchi, K., Kinosaki,
M., A. Mochizuki, Yano, K., Goto, M., Murakami, A., Tsuda, E. et al.
(1998). Osteoclast differentiaton factor is a ligand for
osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to
TRANCE/RANKL. Proc. Natl. Acad. Sci. USA
95,3597
-3602.
Yin, H. (1987). Gelsolin: calcium- and polyphosphoinositide-regulated actin-modulating protein. BioEssays 7,176 -179.[Medline]
Ylänne, J., Chen, Y., O'Toole, T.
E., Loftus, J. C., Takada, Y. and Ginsberg, M. H. (1993).
Distinct functions of integrin and ß subunit cytoplasmic domains
in cell spreading and formation of focal adhesions. J. Cell
Biol. 122,223
-233.[Abstract]
Ylänne, J., Huuskonen, J.,
O'Toole, T. E., Ginsberg, M. H., Virtanen, I. and Gahmberg, C. G.
(1995). Mutation of the cytoplasmic domain of the integrin
ß3 subunit. J. Biol. Chem.
270,9550
-9557.
Zambonin-Zallone, A., Teti, A., Grano, M., Rubinacci, A., Abbadini, M., Gaboli, M. and Marchisio, P. C. (1989). Immunocytochemical distribution of extracellular matrix receptors in human osteoclasts: a ß3 integrin is colocalized with vinculin and talin in podosomes of osteoclastoma giant cells. Exp. Cell Res. 182,645 -652.[Medline]