1 Department of Human Anatomy and Histology, University of Bari, Italy
2 M.I.A. Dibit-hsr et University of Milano Bicocca-Monza, Milan, Italy
3 Department of Internal Medicine, University of Bari, Italy
4 Department of Cell Biology and The Scripps Research Institute, La Jolla,
USA
* Author for correspondence (e-mail: faccio{at}pathology.wustl.edu )
Accepted 19 April 2002
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Summary |
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Key words: Osteoclasts, Alpha v beta 3, Integrin, HGF, M-CSF
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Introduction |
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The functional cycle of the OC consists of migration towards bone, followed by adherence to the bone surface, where the cell polarizes and initiates the resorptive process. Motile OCs are non-polarized cells, characterized by the presence at their leading edge of membrane protrusions, called lamellipodia, and, in a row behind the leading edge, by podosome complexes, comprising filaments of actin associated with several actin-binding proteins. The passage from a motile to a resorbing cell involves cytoskeletal reorganization and matrix attachment. Following attachment to the bone surface, podosomes assemble in an actin-rich region of the membrane, forming a tight sealing zone, enclosing the resorptive microenvironment. Following insertion of secretory vesicles, a highly convoluted ruffled membrane, the ruffled border, forms facing the bone surface where resorption will take place, while the basosolateral membrane is involved in membrane trafficking (Fig. 4A).
|
Integrins are believed to play a role in OC activity by mediating matrix
adhesion and regulating the cytoskeletal organization required for both cell
migration and formation of the sealing zone and ruffled border. OCs express
abundant vß3 integrin
(Zambonin-Zallone et al.,
1989
), which recognizes bone matrix proteins
(Davies et al., 1989
;
Duong et al., 2000
;
Lakkakorpi et al., 1991
;
Nesbitt et al., 1993
).
Addition of RGD-containing peptides or disintegrins (such as echistatin or
kistrin), which bind to
vß3, arrest OC
adhesion to bone or cause retraction of attached osteoclasts
(King et al., 1994
;
Nakamura et al., 1996
;
Nakamura et al., 1999
;
Sato et al., 1990
). Moreover,
OCs generated from ß3-deficient mice are dysfunctional in
vitro and in vivo (McHugh et al.,
2000
; Feng et al.,
2001
).
Recent evidence shows that activation of the
vß3 integrin induces the
[Ca2+]I-dependent phosphorylation of Pyk2, a
non-receptor tyrosine kinase involved in formation of the sealing zone.
Activated Pyk2 forms a complex with c-Src and c-Cbl, and these molecules are
all involved in outside-in activation of
vß3
signaling, possibly leading to podosome assembly
(Sanjay et al., 2001
).
Nakamura et al. recently showed that Src-/- prefusion OCs adhered
to, but failed to spread on, vitronectin (Vn)-coated surfaces and that
vß3-integrin-mediated signaling was
inhibited in these cells. Moreover, the same authors showed that the spreading
defect of Src-/- pOCs could be rescued by M-CSF treatment, which
resulted in recruitment of the
vß3 integrin
to adhesion contacts. However, the authors did not directly assess whether or
not M-CSF treatment leads to changes in integrin affinity for ligand.
Cross-talk between integrin-mediated adhesion and growth factors has been
described in many recent studies; however, the underlying mechanisms remain
incompletely understood. PDGF induces the association of its phosphorylated
receptor with integrin vß3 in fibroblasts
plated on vitronectin, which correlates with enhanced PDGF-induced cell
proliferation (Schneller et al.,
1997
). Basic fibroblast growth factor (bFGF) enhances cell
migration of vascular endothelial cells, leading to concentration of
`activated'
vß3 integrin to polarized
lamellipodia (Kiosses et al.,
2001
). Moreover, in transformed cells HGF promotes cell migration
by increasing
vß3 integrin affinity
(Trusolino et al., 2000
).
Recently the crystal structure of vß3
integrin has been solved (Xiong et al.,
2001
; Beglova et al.,
2002
), showing that ligand binding itself alters the conformation
of this integrin, exposing neo-epitopes, known as ligand-induced binding sites
(LIBS). LIBS can also be exposed by intracellular signaling events, a process
known as inside-out signaling. Anti-LIBS antibodies can be used as tools to
identify integrins in the `activated' conformation or to convert an integrin
from the `basal' to the `activated' state. AP5 is an anti-LIBS Ab that
recognizes the ß3 N-terminus and is regulated by
cation-binding at a site distinct from the LIBS
(Honda et al., 1995
). At
normal extracellular calcium levels, AP5 binds to
vß3 only when the integrin is in the
`activated' conformation. However, in low calcium concentrations AP5 can bind
to all forms of the integrin leading to its activation. Once AP5 is bound at
low calcium concentrations,
vß3 remains in
the `activated' state even when the calcium levels are increased. It has also
been possible to generate monoclonal antibodies that specifically recognize
the `activated' conformation of an integrin at the ligand-binding site. WOW1
is one such antibody, in which the heavy chain hypervariable region 3 of PAC1
Fab was replaced with a single
v-integrin-binding domain,
from the multivalent adenovirus penton base, to engineer an antibody that
binds to only activated
vß3
(Pampori et al., 1999
). Thus,
binding of WOW1 can be used as another marker of integrin activation. In
contrast to AP5 and WOW1, AP3 is a monoclonal Ab, which binds to the
vß3 integrin regardless of its
conformational state. In this paper we defined the fraction of integrin that
is recognized by AP5 and/or WOW-1 as the `activated' conformation and the
fraction of integrin that binds to AP3 and/or 1A2, but not to AP5 or WOW-1, as
the `basal' or `non-activated' conformation.
This study focuses on the functional implications of
vß3 conformation in human OCs. We utilized
different sources of human osteoclasts, giant cell tumors of bone-derived cell
line, bone-marrow-derived osteoclasts and osteoclastoma cells, all of which
have been shown to express osteoclastogenic markers. We demonstrate distinct
localization of the `basal' and `activated' integrin in resting and resorbing
OCs. We find that during bone resorption `activated'
vß3 is mainly localized in the ruffled
border, whereas the `basal' conformation is responsible for the adhesive
properties of the sealing zone. Moreover we demonstrate that HGF and M-CSF,
two growth factors that regulate osteoclastogenesis and osteoclast survival
(Fuller et al., 1993
;
Grano et al., 1996
), modulate
the activation state of
vß3. Growth factor
treatment alters the affinity of
vß3 toward
its ligands, promoting OC migration to the
vß3 ligand osteopontin and enhancing bone
resorption. These data offer a potential explanation for the diverse responses
of OC to the same ligand.
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Materials and Methods |
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Primary osteoclasts
Human osteoclasts were obtained as described previously by mechanical
disaggregation of small surgery specimens from osteoclastomas
(Grano et al., 1996) or from
human bone marrow cultures. Briefly, human bone marrow samples obtained by
orthopedic surgical procedures were subjected to Ficoll-Hypaque (Ficoll, Sigma
Chemical Co.; Hypaque 76, Nycomed, Princeton, NJ) gradient purification, and
cells at the gradient interface were collected and cultured overnight in the
presence of 20 ng/ml of recombinant human M-CSF (R&D Systems Inc.,
Minneapolis, MN). Non-adherent cells were plated onto coverslips or dentine
slices in the presence of alpha-MEM supplemented with 20% Fetal Calf Serum
(FCS, Gibco Limited, Uxbridge, UK), and Vitamin D3 10-9 M at
37°C in a water saturated atmosphere with 5% CO2. After 3 weeks
multinucleated TRAP-positive human osteoclasts appeared.
Osteoclast-like cell line
An osteoclast-like cell line, GCT 23, cloned from a human giant cell tumor
of bone has been extensively characterized for its osteoclast phenotype
(Grano et al., 1994). Cells
used for the experiments were all within nine and 14 passages. All the cells
were maintained in Iscove's medium (IMEM) supplemented with 10% fetal calf
serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml
amphotericin and 50 IU mycostatin at 37°C, in a water saturated atmosphere
with 5% CO2 and fed by medium replacement every 2-3 days.
Murine osteoclasts
Macrophages were isolated from bone marrow of four- to eight-week-old mice,
cultured overnight in -MEM containing 10% heat-inactivated FBS and
subjected to Ficoll-Hypaque (Ficoll, Sigma Chemical Co.; Hypaque 76, Nycomed)
gradient purification. Cells at the gradient interface were collected and
cultured in the presence of 10 ng/ml recombinant M-CSF (R&D Systems Inc.)
and 100 ng/ml RANKL for 3-4 days.
Antibodies and proteins
Recombinant osteopontin (OPN) was the kind gift of K. O. Johanson
(SmithKline Beecham, King of Prussia, PA). Laminin from human placenta (LN-2)
was from Sigma Chemical Co (St Louis, MO). All the antibodies were protein-A
purified IgGs from ascites fluid. LM609 is a blocking anti
vß3 antibody
Cheresh, 1991
) and was kindly
supplied by David Cheresh (The Scripps Research Institute, La Jolla, CA);
WOW1, a monoclonal antibody which specifically binds the activated
conformation of
vß3, was generously provided
by S. Shattil (The Scripps Research Institute, La Jolla, CA); AP3, a
monoclonal antibody which recognizes all forms of ß3, and AP5,
a monoclonal antibody against the activated conformation of
ß3, have been extensively characterized
(Honda et al., 1995
); 1A2, an
mAb against human ß3 was a gift of S. Blystone, Department of
Cell and Developmental Biology, State University of New York (SUNY), Upstate
Medical University at Syracuse, NY.
Monoclonal antibodies against the v integrin subunit were
purchased from Telios Pharmaceuticals, Inc (San Diego, CA). Human recombinant
macrophage colony stimulating factor (M-CSF) was from Genetics Institute
(Cambridge, MA). Affinity-purified HGF was a kind gift of P. M. Comoglio
(IRCC, University of Torino, Italy).
Immunofluorescence
Freshly isolated human osteoclasts were plated onto bone slices,
hydroxyapatite-coated coverslips (Osteologic, Millenium Biologix Inc.,
Kingston, Ontario, Canada) or glass coverslips in -MEM supplemented
with 20% FHS (fetal horse serum) at 37°C in a humidified atmosphere
containing 5% CO2. After 24 hours, cells were incubated with HGF or
M-CSF in IMEM supplemented with 0.5% BSA for 30 minutes at 37°C. Control
cells were incubated with
-MEM + 0.5% BSA alone. After incubation,
cells were washed three times with PBS and fixed in 3% paraformaldehyde, 2%
sucrose in PBS pH 7.6 for 10 minutes at room temperature. After rinsing in
PBS, cells were made permeable to antibodies by soaking coverslips for 3
minutes at 0°C in Hepes-Triton X-100 buffer (20 mmol/l Hepes pH 7.4, 300
mmol/l sucrose, 50 mmol/l NaCl, 3 mmol/l MgCl2 and 0.5% Triton
X-100).
For indirect immunofluorescence, the primary antibody was layered on fixed and permeabilized cells and incubated in a humidified chamber for 45 minutes at 37°C. The distribution of AP5 and WOW1 were also evaluated in live cells using Abs incubated for 45 minutes at 4°C to prevent receptor internalization. Cells were then fixed and permeabilized as described previously. After rinsing in PBS (pH 7.6), coverslips were incubated with the appropriate Cyanine-3 conjugated secondary Ab (Cy-3, Chemicon, CA) and with 10 µg/ml fluorescein-labeled phalloidin (F-PHD, Sigma) for 45 minutes at 37°C. Stained coverslips were than mounted in 20% Mowiol 4-88 (Calbiochem-Novabiochem Corporation, La Jolla, CA). Observations were performed by epifluorescence in a Zeiss axioplan microscope.
Osteoclasts plated onto bone slices were viewed with a confocal microscope in the M.I.A. laboratory at DIBIT San Raffaele in Milan, Italy.
Flow cytometry
GCT23 cells were removed from growth plates by a brief treatment with
Trypsin/EDTA (Sigma) and washed three times in a calcium-free buffer based on
Hanks Balanced Salt Solution (HBSS). Pretreated cells were incubated with HGF
or M-CSF in IMEM supplemented with 0.5% BSA for 30 minutes at 37°C;
control cells were incubated with IMEM alone. After incubation with or without
the growth factors, cells were washed twice with HBSS buffer supplemented with
calcium and magnesium and incubated with the mAb AP5 (50 µg/ml) against the
activated ß3 integrin subunit in high calcium buffer for 45
minutes on ice. A positive control was performed by incubating cells with AP5
in HBSS calcium-free buffer. Cells were then rinsed and treated with
FITC-conjugated goat-anti-mouse serum (Sigma) for 30 minutes on ice. Finally,
cells were suspended in 0.5 ml of HBSS buffer in the presence of propidium
iodide (500 ng/ml, Sigma) added immediately prior to analysis on a
Becton-Dickinson FACScan. Appropriate forward- and side-scatter gates were set
for these cells, and only those cells excluding propidium iodide were included
in the analysis of FITC fluorescence.
In vitro bone resorption assay
Murine osteoclasts from bone marrow macrophages were generated on whale
dentin slices or on BD BioCoatTM OsteologicTM Bone Cell
Culture System (Millenium Biologix) as described above. After 3 days in
culture in the presence of 25 ng/ml M-CSF and 100 ng/ml RANKL, the culture
media was changed and HGF (50 ng/ml), M-CSF (100 ng/ml) or vehicle alone were
added for the next 48 hours. Cells on dentine slices were fixed in 4% PFA for
10 minutes at room temperature and TRAP stained. Resorptive pits were analyzed
by light microscopy and pictures were taken with a 16x objective.
At the same time point, osteoclasts plated onto dentine slices were removed by a brief treatment with domestic bleach, and resorptive areas were analyzed by light microscopy.
Migration assay
Haptotactic migration assays were performed in transwell plates with an 8
µm pore size (Costar, Cambridge, MA) as described previously
(Pelletier et al., 1996).
Briefly, the lower side of the membrane was coated with OPN or LN at the
concentration of 10 µg/ml for 2 hours at room temperature. Both sides of
the membrane were blocked with 2% heat-denatured BSA for 1 hour. Cells were
preincubated as described above, and 104 cells, diluted in IMEM
supplemented with 0.5% FCS, were added per well. Following incubation at the
indicated times at 37°C, cells were stained with ADiff-Quick@ (Dade
Diagnostics, Aquada, PR). The cells attached to the top surface of the
membrane were removed with cotton swabs. Cells that migrated to the lower side
were viewed at 300x magnification, and the number of cells per field was
counted. Results represent the averages from 15 fields±s.e. of a
representative experiment. For inhibitory experiments, cells, after
pre-incubation with AP5, HGF or M-CSF, were incubated for an additional 30
minutes at 37°C with the function-blocking mAb LM609 or the PI3-kinase
inhibitor Wortmannin (Sigma).
Statistics
Statistical analysis was performed by unpaired Student's t-test.
The results are expressed as average±s.e.
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Results |
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|
vß3 conformations and localization
in human osteoclasts
Having established the presence of the
vß3 integrin in different conformational
states in human osteoclast-like cells GCT 23, we analyzed their expression in
human osteoclasts. Because FACS analysis detects the presence of the activated
vß3 but not its localization, we turned to
immunofluorescence as a method to detect the distribution of the two
conformations of
vß3. We used the mAb AP3,
which binds to epitopes within residues 287-489
(Honda et al., 1995
) and
recognizes ß3 in both conformational states. In non-polarized
primary OCs plated on glass, AP3 highlights
vß3 on the OC surface in the podosomes
(Fig. 2A,B) and in the motile
parts of the plasma membrane, called lamellipodia
(Fig. 2A,B arrow). At higher
magnification (Fig. 2A,
insert), AP3 demonstrates that
vß3 is
organized in rosette-like structures that surround a core of actin filaments
in the podosomes (Fig. 2B,
insert). We have also seen this particular organization of the
ß3 integrin around the podosomal actin core in avian OCs
(Marchisio et al., 1984
).
Similar results were obtained using the mAb 1A2, which binds to an external
domain of the ß3 subunit (not shown), and using an
anti-
v mAb, which exhibited a complete overlap with the
ß3 distribution (data not shown). By contrast, the `activated'
conformation of the ß3 chain, which is recognized by AP5, was
primarily detected at the edge of the cell, where it colocalized with a
meshwork of actin filaments in lamellipodia and membrane ruffles
(Fig. 2C,D, insert) but was
mostly absent from the podosomes. To confirm that the different distribution
of the `activated' receptor was not caused by an artifact dependent on
fixation procedures or to non-specific binding of AP5, cells were stained with
a second Ab that recognizes `activated'
vß3,
WOW1, before and after fixation. In Fig.
3, WOW1 staining of human osteoclasts shows a complete concordance
with the AP5 distribution. In both live and fixed cells, the `activated'
receptor colocalized at the cell edge with the cortical actin (A-B and D-E
arrows), and, in contrast to the `basal' receptor, was absent from the
podosomes (C,F, asterisk). On the basis of these findings, we performed all
the following immunofluorescence staining experiments on fixed cells to avoid
possible modifications, such as receptor internalization, during the Ab
incubation.
|
|
As shown in Fig. 4A,
resorbing OCs are polarized cells that adhere to the bone through the sealing
zone (SZ) and form a specific membrane domain, the ruffled border (RB), which
is involved in the dissolution of mineralized bone matrix. Having shown that
the two different conformational states of the integrin coexist in
non-resorbing OCs plated on coverslips, we examined the distribution of the
two conformational states of vß3 during bone
resorption. Human OCs were plated onto bone slices, stained with AP3 or AP5
mAbs and observed by confocal microscopy. In the cross-section from the
sealing zone, total
vß3, as visualized with
AP3 (Fig. 4B), appeared as a
circle at the cell periphery (SZ) that perfectly colocalized with the actin
ring (Fig. 4C), suggesting that
the receptor participates in recognition of bone matrix. The cross-section
shown in panels D and E revealed the
vß3
distribution in the ruffled border (Fig.
4D) where actin filaments were also detected
(Fig. 4E). The same cell has
been analyzed through an optical yz plane (F-G). In this cross section, the
overlap of
vß3 and actin in the sealing zone
(SZ), as well as their localization in the ruffled border (RB), was evident.
By contrast, the receptor in the `activated' conformation, detected by AP5,
only partially colocalized in a punctuate pattern with actin in the sealing
zone (Fig. 5A,B), a
distribution confirmed by analysis of the same cell through the yz plane
(Fig. 5E,F in SZ). The
`activated' integrin was strongly detected in the ruffled border
(Fig. 5C,D;
Fig. 5E,F in RB) with a similar
distribution to the AP3 staining. On the basis of these observations we can
conclude that the `activated' form is present mainly at the edge of the
ruffled border, whereas the `basal' conformation is also present in the
sealing zone. These findings suggest that the two conformational states of
vß3 are differently distributed, indicating
that they may exert distinct effects on OC activity.
|
HGF and M-CSF induce changes in vß3
distribution in non-resorbing OCs
We next determined whether both `basal' and `activated' conformation of
vß3 could be modulated by growth factor
treatment. Human OCs plated on coverslips were treated with HGF, M-CSF or
vehicle alone and immunostained with the mAb AP3, which recognizes both
vß3 conformational states. In untreated
cells,
vß3 was organized in rosette-like
structures (Fig. 6A-C), as
described previously (Fig.
2A,B). By contrast, pre-incubation with HGF or M-CSF for 30
minutes at 37°C alters the distribution of
vß3, shifting the integrin away from
podosomes to the protrusive edges of the plasma membrane
(Fig. 6D-F and 6G-J). The
distribution of the `activated'
vß3 detected
by AP5 and WOW-1 mirrors immunostaining with AP3 (not shown), indicating that
the majority of the integrin has been activated by the growth factors.
|
Flow-cytometric analysis revealed that PI 3-kinase is required for
growth-factor-mediated vß3 activation in
osteoclast-like cells (not shown). In mature OCs, PI 3-kinase associate with
vß3
(Hruska et al., 1995
;
Lakkakorpi et al., 1997
). In
our system, adding the PI 3-kinase inhibitor wortmannin before growth factor
stimulation blunted the
vß3 redistribution
(Fig. 6K-M). The receptor was
still organized in podosomes and failed to localize at the cell edge. These
results suggest that PI 3-kinase is important for the activation and
redistribution of
vß3 under growth factor
stimulation. Thus, HGF and M-CSF mobilize
vß3 from the cytoskeleton, inducing its
relocalization in a PI-3-kinase-dependent manner.
Growth factors change the distribution of activated
vß3 in resorbing OCs
Given the changes in vß3 conformation in
non-resorbing cells in response to HGF and M-CSF, we next turned to the
effects of these cytokines on resorbing OCs. Human OCs were plated onto
hydroxyapatite-coated coverslips (BD BioCoatTM OsteologicTM), which
efficiently substitute for bone slices. Although this inorganic substrate does
not itself contain the organic matrix components found in bone, OC resorptive
activity does depend on the presence of
vß3
integrin (R.F. and D. Novack, unpublished). The necessary organic components
may be secreted by the osteoclastic cells and/or contained in the
serum-supplemented media. Mature human osteoclasts were treated with growth
factors or vehicle and immunostained with AP5. As we saw in OCs on bone
(Fig. 5), high affinity
vß3 is confined mainly to the ruffled border
and is distributed with a punctuate pattern in the sealing zone in the absence
of exogenous growth factors (Fig.
7A,C). Treatment with HGF (Fig.
7D,F) or M-CSF (not shown) for 30 minutes modified the integrin
conformation, including the fraction in the sealing zone that appeared
brightly stained by AP5 (Fig.
7D). However, the `activated' integrin only partially colocalized
(yellow) with the actin ring, as demonstrated by the merge between
vß3 staining in red and actin in green
(Fig. 7F). The `activated'
integrin after growth factor treatment was also detected in small protrusions
at the cell periphery external to the actin ring. Longer incubation (8 hours)
yielded dramatic morphological changes in resorbing OCs. The cells started to
migrate and the integrin was eventually found in more organized motile areas
of the plasma membrane, such as lamellipodia and fillopodia (not shown).
|
HGF and M-CSF increase bone resorption
To assess the functional implications of HGF- and M-CSF-induced alterations
in vß3 activation, we determined the effect
of the growth factors on osteoclastic bone resorption. Murine bone marrow
macrophages were plated onto whale dentine slices or hydroxyapatite-coated
slides and cultured in the presence of 100 ng/ml RANKL and 25 ng/ml M-CSF.
After 3 days, the presence of mature OCs was confirmed by TRAP staining of
parallel cultures. At this time, the culture media was replaced with a fresh
one containing 50 ng/ml of HGF or 100 ng/ml M-CSF. After an additional 48
hours, cells on dentine slices were fixed and TRAP stained, and the bone
resorptive lacunae were analyzed by light microscopy
(Fig. 8A-C). Compared with
untreated cells (A), HGF (B) and M-CSF (C) treatment increased the pit number
2.6-and 2.3-fold, respectively. To evaluate the resorbed area,
hydroxyapatite-coated slides were treated with bleach to remove cells, and the
cleared area was quantified. Unstimulated OCs resorbed 30% of the total area
(Fig. 8D), whereas HGF (E) and
M-CSF (F) increased the resorptive area to 65% and 58%, respectively.
|
Activated vß3 increases osteoclast
migration to OPN in a PI-3-kinase-dependent manner
Normal resorptive function of OCs depends in part upon their ability to
migrate over the bone surface to initiate new sites of bone resorption. Having
shown an increased bone resorption in OCs treated with HGF and M-CSF, we
determined whether it was correlated with an increased motility. Haptotactic
migration of human osteoclast-like cells was measured in a transwell assay in
which the bottom of the membrane had been coated with OPN (10 µg/ml).
Cells, which had migrated through the membrane to the OPN-coated surface, were
counted. Pretreatment with either growth factor increased human
osteoclast-like cell migration to levels comparable to those pre-treated with
AP5 in low calcium, which we had previously established promotes motility
(Faccio et al., 1998)
(Fig. 9A). This increase in
cell migration was inhibited by the
vß3
blocking mAb LM609 (Fig. 9A).
In contrast to OPN, human osteoclast-like cell migration toward laminin (LN),
a substrate not recognized by
vß3, was not
affected by HGF or M-CSF pretreatment nor inhibited by LM609
(Fig. 9B), indicating a
specific role for the two growth factors in
vß3-dependent migration.
|
Having established that the PI-3-kinase inhibitor wortmannin blocks
growth-factor-mediated redistribution of vß3
(Fig. 6K-M), we next examined
whether this treatment would also affect growth-factor-mediated OC migration.
After pre-incubation with HGF, M-CSF or AP5 in the presence of low calcium, as
a control for integrin activation, wortmannin (200 nM) was added to the medium
and cells were allowed to migrate toward OPN or LN. As seen in
Fig. 9A, wortmannin
substantially reduced cell migration towards OPN to levels comparable with
anti-
vß3 mAb-mediated inhibition. The
inhibitor was completely ineffective in the altering migration toward LN
(Fig. 9B). This result and the
previous morphologic observations suggest that PI 3-kinase activity is
required for growth-factor-induced cell migration via recruitment of the
`activated'
vß3 to the motility related
sites.
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Discussion |
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In the current study we utilized two different antibodies, WOW-1 and AP5,
to detect the `activated' vß3 integrin.
WOW-1, a monovalent Ab, binds only to unoccupied, activated integrins and is
insensitive to changes in integrin clustering. AP5, in contrast, binds to the
N-terminal domain of the ß3 subunit stabilizing and/or
inducing its active conformation. Certain cations (e.g. Ca2+) can
compete with AP5 in binding to the receptor when in the inactive conformation.
The advantage of using AP5 is that it can be used to detect the fraction of
integrin that is `activated' (at high calcium) and the fraction of
`activatable' integrin (at low calcium).
The crystal structure of the vß3 integrin
has been solved recently by two different groups
(Beglova et al., 2002
;
Xiong et al., 2001
). The
C-termini of the
v and ß3 extracellular
domains are very close together in the
vß3
structure (Xiong et al.,
2001
), mimicking the close association in the juxtamembrane region
that maintains integrin in the inactive state. In this genuflected
conformation, the activation epitopes are also masked. Integrin activation has
been correlated with flexibility at the `genu'. Therefore an extended form,
which is commonly seen in electron micrographs of integrins, including
integrin simultaneously bound to ligand and activating antibody, exposes a
much broader surface, including several activating epitopes. AP5, a monoclonal
activating antibody used in the current study, binds to the first six amino
acids in the N-terminal end of the ß3 subunit, which are
localized in the PSI (plexin-semaphorin integrin) domain
(Honda et al., 1995
).
Unfortunately the structure of this terminal region, localized in the genu
motif, has not been solved. It is possible that the binding of AP5 is
completely masked in the genuflected conformation (inactive) but is exposed in
the extended configuration (active), as demonstrated for other activating
antibodies (Beglova et al.,
2002
).
In the current paper we have identified the localization of two different
vß3 conformations in resting and resorbing
human osteoclasts derived from several sources (bone marrow culture,
osteoclastoma and a giant cell tumor of bone-derived cell line). We have
utilized two antibodies to detect the `activated'
vß3 conformation, AP5 and WOW-1, which
recognize different epitopes localized in the ß3 subunit, and
both have shown same pattern of distribution in both live and fixed cells.
Both antibodies specifically bind to the fraction of integrin organized at the
cell edge, where it colocalizes with filaments of cortical actin. By contrast,
the antibodies to total
vß3, AP3 and 1A2
identify an additional pool of `basal' integrin in the podosomes and in the
sealing zone, where it is associated with the actin ring. It is not clear why
the `basal' and not `activated' integrin is found in the sealing zone where
there is very tight membrane apposition to the bone surface, as observed in
electron micrographs of resorbing OC. It is possible that the extended
conformation of activated integrin, seen in the crystal structure, is too
large to fit in this space. The concentration of integrin in the sealing zone
is great, so even with `non-activated' integrin, the avidity is high at this
site.
Even in the absence of growth factors, the integrin exists in both `basal'
and `activated' forms. Growth factor treatment, which promotes cell motility,
induces both redistribution and activation of the
vß3 integrin. The integrin moves from the
podosomes, in which the receptor is found in a `basal' conformation, to the
new forming leading edges, where the receptor is in an `activated' state.
These changes in the integrin are accompanied by functional changes in the OC
(increased migration and resorption) and suggest that the two pools of
integrin serve different functions.
Evidence that integrins and growth factor receptors share common signaling
pathways (Clark and Brugge,
1995), suggests that signals induced by growth factors could be
responsible for integrin activation
(Trusolino et al., 2000
;
Trusolino et al., 1998
). We
studied the effects of HGF and M-CSF because of their ability to influence
osteoclast differentiation, survival and activity. Flow cytometric analysis
confirms the ability of the two growth factors to induce integrin activation
as measured by binding of the mAb AP5 in the presence of divalent cations.
Following growth factor exposure, cytoskeletal rearrangements occur,
allowing lateral movement of the integrin and leading to
vß3 translocation to the motile regions of
the plasma membrane. In resorbing OCs, growth factor treatment causes dynamic
reorganization of the `activated' integrin into the marginal area of the cell
around the clear zone in fillopodia-like structures. Consequently, this
integrin reorganization supports the translocation of OCs to new sites of the
bone surface to be resorbed and explains the increased bone resorption found
in the presence of HGF and M-CSF (Fig.
8). Migration assays confirm the pivotal role of the activated
receptor during this process. HGF or M-CSF pre-incubation induces a
vß3-dependent and-specific increase in
osteoclast motility toward osteopontin but has no effect on cell migration
toward LN. Nakamura et al. show that M-CSF treatment causes recruitment of the
vß3 integrin to adhesion contacts and
induces stable association with PLC-
, PI 3-kinase and Pyk2
(Nakamura et al., 2001
). In
that system the authors did not address the question of whether the
`activated'
vß3 is mediating the recruitment
of this molecular complex. In our study we found that the growth factor
treatment induces activation of most
vß3
expressed in OCs and that PI 3-kinase is required for this effect.
PI 3-kinase has been implicated in integrin activation in leukocytes and
platelets (Shimizu et al.,
1995; Kovacsovics et al.,
1995
; Zell et al.,
1996
; Zhang et al.,
1996
). In avian OCs,
vß3 is
associated with the signaling PI 3-kinase, c-Src and FAK
(Hruska et al., 1995
). In that
system, interaction of
vß3 with osteopontin
increases PI 3-kinase activity and association with Triton-insoluble gelsolin
(Chellaiah and Hruska, 1996
).
In generated murine osteoclasts, PI 3-kinase translocates to the cytoskeleton
upon osteoclast attachment to the bone surface
(Lakkakorpi et al., 1997
). We
also found that the functional consequences of
vß3 activation are PI 3-kinase dependent.
First, pre-incubation with wortmannin inhibits cell migration towards the
vß3 ligand OPN but not towards LN. Second,
inhibition of PI 3-kinase activity is accompanied by the inability of
HGF/M-CSF to recruit
vß3 integrin to
motility related sites. Phosphatidylinositides generated by PI 3-kinase can
activate GTP-GDP exchangers for the small GTPases Rho and Rac, which, in turn,
control organization of the actin cytoskeleton, inducing formation of focal
adhesion, podosomes, membrane ruffles and lamellipodia
(Chellaiah et al., 2000
;
Clark et al., 1998
;
Ory et al., 2000
;
Ridley et al., 1999
). Finally,
PI 3-kinase mediates M-CSF- and HGF-driven intracellular signaling. Upon HGF
stimulation, the HGF receptor c-met exhibits tyrosine kinase
activity, including receptor autophosphorylation, and activates a series of
signal transducing proteins. Autophosphorylated c-met associates with
PI 3-kinase in vivo and in vitro (Graziani
et al., 1991
; Ponzetto et al.,
1993
). Husson et al. also showed the formation of a multiprotein
complex between c-fms, c-Cbl, PI 3-kinase and Grb2 upon M-CSF stimulation
(Husson et al., 1997
). In OCs,
a functional association between PI 3-kinase and c-Src has been shown during
M-CSF-induced spreading (Grey et al.,
2000
).
In conclusion, our results indicate that the equilibrium between two integrin conformations is modified by growth factors, resulting in altered inside-out signaling and thus modulating the resorptive capability of the OCs. Although it is clear that HGF and M-CSF govern integrin activity, the precise mechanisms by which PI3-K and other signaling molecules interact with growth factors to modulate integrin conformation are still to be determined.
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Acknowledgments |
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References |
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