From the Departments of Bone Biology and Osteoporosis
and
Virus and Cell Biology, Merck Research Laboratories,
West Point, Pennsylvania 19486 and the ¶ Institute of Biomedicine,
Department of Anatomy, University of Turku, FIN-20520
Turku, Finland
Received for publication, September 12, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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Osteoclast activation is initiated by adhesion to
bone, cytoskeletal rearrangement, formation of the sealing zone, and
formation of the polarized ruffled membrane. Previous findings suggest
that protein-tyrosine kinase 2 (PYK2), a cytoplasmic kinase
related to focal adhesion kinase, participates in these events. This
study examines the role of PYK2 in adhesion-mediated signaling and
osteoclast function, using PYK2 antisense. We produced a recombinant
adenovirus containing a 300-base pair reversed 5'-coding region
of PYK2 and used full-length PYK2 as a control. Murine osteoclast-like
cells or their mononuclear precursors were generated in a co-culture of
bone marrow and osteoblasts. Infection with antisense adenovirus significantly reduced the expression of endogenous PYK2 protein relative to uninfected cells or to cells infected with sense PYK2 and
caused: 1) a reduction in osteoclast formation in vitro; 2) inhibition of cell spreading and of actin ring formation in osteoclasts plated on glass or bone and of attachment and spreading of osteoclast precursors plated on vitronectin; 3) inhibition of bone resorption in vitro; 4) marked reduction in p130Cas
tyrosine phosphorylation; and 5) no change in
Osteoclasts are multinucleated, terminally
differentiated cells that degrade mineralized matrix. Osteoclast
adhesion to bone matrix is an essential prerequisite for osteoclast
differentiation, migration, and polarization, including formation of a
tight sealing zone and directional secretion of protons and lysosomal
proteases into the resorption lacuna (1, 2). Integrins have been
suggested to mediate osteoclast adhesion to the matrix and regulate the cytoskeletal organization required for migration and formation of the
sealing zone (3, 4). The proline-rich tyrosine kinase (PYK2; also known as cell adhesion
kinase In osteoclasts, PYK2 tyrosine kinase has been suggested to mediate
integrin-initiated Src-dependent signaling, following
adhesion (5). Furthermore, PYK2 localizes to podosomes and the sealing zone, the primary adhesion structures in osteoclasts during resorption (17). It has been shown that Src deficiency is associated with osteopetrosis in mice, because of loss of osteoclast function (21).
Recently, targeted disruption of Antibodies--
Anti-PYK2 polyclonal antibodies were previously
described (17). Antibodies to phosphotyrosine (monoclonal antibody
4G10) and anti-c-Src were from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-paxillin was from Transduction Labs (Lexington, KY). Monoclonal antibody to adenoviral penton protein (clone 1431) was from
Accurate Chemicals. Polyclonal antibodies raised against human
Cell Culture--
Murine osteoclast-like multinucleated cells
(OCLs) were prepared on collagen gels as reported (24) with some
modifications. Briefly, the osteoblastic MB1.8 cells were plated at
1 × 104 cells/cm2 on culture dishes
precoated with 5 ml of 0.2% collagen gel matrix (Nitta Gelatin Co.,
Osaka, Japan). After 24 h, bone marrow cells isolated from tibia
of 6-8-week old Balb/c mice were added (2.5 × 104
cells/cm2) to the monolayer of MB1.8 cells. The co-culture
was maintained in Construction of Recombinant Adenoviruses--
The recombinant,
replication-deficient adenovirus vectors were constructed using
p Infection of Osteoclast-like Cells--
The co-cultures of MB1.8
cells and murine bone marrow cells were infected with recombinant virus
at different multiplicities of infection (MOI): 1000, 100, 10, 0.1, and
0.01. Viral stock was diluted with Cell Attachment and Spreading--
Polystyrene dishes (35 mm,
Becton Dickinson) were coated overnight at 4 °C with 10 µg/ml
human vitronectin (Life Technologies, Inc.). Uninfected pOCs or cells
infected with Ad-PYK2/wt or Ad-PYK2/AS were isolated as described
above. For biochemical analyses, cells were either kept in suspension
(0.5 × 106 cells/ml) or allowed to attach to
vitronectin-coated plates for 1 h at 37 °C. Cells were
solubilized in modified RIPA buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 1% Nonidet P-40, 0.2% sodium deoxycholate, 1 mM EDTA, 50 mM sodium fluoride,
1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml
leupeptin) and prepared for immunoprecipitation. To quantify the planar
area of cell spreading, cells were allowed to adhere and spread on
vitronectin for 1 h. After fixing with 4% paraformaldehyde in
phosphate-buffered saline, the periphery of each cell was outlined, and
the total planar area was calculated using an image analyzing system
(Empire Imaging System, Milford, NJ).
Assessment of Osteoclast Apoptosis--
Because it is difficult
to determine osteoclast apoptosis in the co-culture system in the
presence of serum, growth factors and extracellular matrix proteins
derived from MB1.8 cells, pOCs (25,000 cells/well) were therefore
isolated from uninfected co-cultures or cultures infected with
Ad-PYK2/wt or Ad-PYK2/AS for 4 days. Purified pOCs were then plated on
vitronectin-coated glass coverslips for 1 h in the absence of
serum. Cells were then fixed with 4% paraformaldehyde in
phosphate-buffered saline and stained with Oregon green-labeled
phalloidin and Hoechst 33342 stain (Molecular Probes Inc., Eugene, OR).
Apoptotic pOCs were assessed by cells showing nuclear condensation and
fragmentation. Results are expressed as the percentages of apoptotic
cells over total counted pOCs (n = 100), viewed with a
20× objective and a Zeiss Axiophot epifluorescence microscope.
Immunoprecipitation--
Cell extracts were prepared with
modified RIPA buffer and immunoprecipitated with antibodies to
PYK2, pp60c-Src, p130Cas, or paxillin.
Immunoprecipitation was carried out for 4 h at 4 °C, followed
by addition of protein G-Sepharose. Immunoprecipitated proteins were
separated on SDS-polyacrylamide gel electrophoresis and transferred to
Immobilon-P membranes. Blots were first immunoblotted with horseradish
peroxidase-conjugated anti-phosphotyrosine monoclonal antibody 4G10 and
then with the respective immunoprecipitating antibodies, followed by
horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG. Blots
were developed by ECL (Amersham Pharmacia Biotech). Image densitometry
(model GS-700; Bio-Rad) was used to estimate the phosphotyrosine
contents and the protein levels of each protein, from which the
specific activity of tyrosine phosphorylated protein was calculated.
Relative specific activity of phosphorylated protein was normally
determined from triplicate experiments.
Pit Assay--
Pit-forming activity of OCLs was determined as
described (3). Aliquots (1,000 cells/slice) of the OCLs were placed on
dentine slices in 96-well culture plates. After 20 h, dentine
slices were either stained with Mayer's hematoxylin for resorption
pits. Pit area was measured by image analysis. The results were
expressed as the means ± S.D. (n = 5) of resorbed
area per whole dentine surface area.
Immunofluorescence Microscopy--
Immunofluorescent labeling of
actin rings in OCLs was performed essentially as previously described
(17). OCLs were plated on serum-coated glass coverslips or on bone
slices for 20 h and fixed in 4% paraformaldehyde. Cells were then
double-stained for TRAP and F-actin (3). PYK2 was visualized using the
affinity purified anti-PYK2 antibodies, followed by
tetramethylrhodamine isothiocyanate-donkey anti-rabbit IgG.
Actin was stained with Oregon green 514 phalloidin (Molecular Probes).
Paxillin was stained with monoclonal antibody 349 and visualized using
fluorescein isothiocyanate goat anti-mouse IgG. The
Efficiency of Adenovirus-mediated Gene Transfer in Osteoclast-like
Cells--
The co-cultures of murine osteoblastic MB1.8 cells and bone
marrow cells were first infected with Ad- Infection of Osteoclast-like Cells with Adenovirus Expressing PYK2
Antisense Reduces PYK2 Protein Expression--
To analyze the effect
of Ad-PYK2/AS on PYK2 expression in OCLs, we infected the co-cultures
with adenoviral vectors (MOI = 10) expressing either full-length
PYK2 (Ad-PYK2/wt) or PYK2 antisense (Ad-PYK2/AS) at day 3 or 4 of the
co-cultures. OCLs were then purified at day 7, as described under
"Materials and Methods." Approximately equal amounts of protein
were isolated from untreated and infected OCLs, as illustrated by
Western blotting with anti- Adenovirus Expressing Antisense PYK2 Inhibits the Formation of
Osteoclast-like Cells--
To determine the effect of Ad-PYK2/AS on
the formation of osteoclast-like cells in vitro, we infected
the co-culture system at day 3 with increasing concentrations of
adenoviral vectors (wt and AS) and quantitated both the number of
TRAP(+) cells and the area of TRAP(+) cells in these cultures at day 7. As shown in Fig. 3A, infection with Ad-PYK2/wt had no effect
on the formation of multinucleated TRAP(+) cells, even at high virus
concentrations (MOI = 1000). In contrast, infection with
Ad-PYK2/AS resulted in significant inhibition of osteoclast formation
with an EC50 of 50 MOI (Fig. 3B). More
importantly, Ad-PYK2/AS appeared to be more potent in reducing the area
of TRAP(+) cells with an EC50 of around 2-5 MOI. This
suggested that the primary effect of PYK2 antisense might be inhibition
of OCL spreading and possibly of OCL migration prior to fusion. As
shown in Fig. 3C, Ad-PYK2/wt infected OCLs appeared as
multinucleated TRAP(+) cells (Fig. 3C, panel b),
not different from OCLs in normal control cultures (Fig. 3C,
panel a). However, OCLs treated with Ad-PYK2/AS were small and retracted and were densely stained for TRAP activity.
Adenovirus Expressing Antisense PYK2 Inhibits pOC Cell Spreading
without Affecting pOC Apoptosis--
To further confirm the role of
PYK2 in the regulation of osteoclast spreading, we studied pOCs
isolated from co-cultures, which were either untreated or infected with
Ad-PYK2/wt or with Ad-PYK2/AS. As shown in Fig. 2, adenoviral gene
transfer did not alter the expression of the
Because Ad-PYK2/AS inhibited osteoclast spreading and subsequently
reduced the number of TRAP(+)-OCLs in the co-culture system (Fig. 3),
we examined the effect of Ad-PYK2/AS on purified pOCs plated on
vitronectin-coated dishes for 60 min under serum-free conditions.
Because it is difficult to assess adhesion-dependent osteoclast apoptosis in the co-culture systems where osteoclastogenesis were influenced by serum and growth factors, cytokines, and
extracellular matrix proteins derived from the osteoblastic/stromal
MB1.8 cells, we therefore examine apoptotic events in pOCs isolated
from co-cultures uninfected and infected for 4 days prior to optimal
fusion (25). Typical preparations of pOCs contain mainly mononuclear
cells (~80%) and cells with two to four nuclei (Fig. 4C).
Uninfected pOCs and pOCs infected with Ad-PYK2/wt appeared as spread
cells with organized podosomal adhesion contacts and with flattened nuclei (Fig. 4C, panels a-d). On the other hand,
pOCs infected with Ad-PYK2/AS were associated with reduced adhesion and
cell rounding and compacted nuclei (Fig. 4C, panels
e and f). Because of thick rounding cell shape, pOCs
expressing PYK2 antisense often appeared to have overlapping nuclei at
one plane of focus. However, we did not detect significant increase in
number of apoptotic cells associated with chromatin condensation and
nuclear fragmentation in pOCs infected with Ad-PYK2/AS. We indeed found
that during the initial phase of cell adhesion, the percentage of
apoptotic pOCs are 4.8% in uninfected pOCs, 5.7% in cells infected
with Ad-PYK2/wt, and 6.2% in pOCs infected with Ad-PYK2/AS.
Adenovirus Expression of Antisense PYK2 Inhibits Tyrosine
Phosphorylation of p130Cas and Modulates the Tyrosine
Phosphorylation of Paxillin--
We have previously shown that PYK2
and p130Cas form a stable complex independent of c-Src
(29). However, in Src(
On the other hand, the tyrosine phosphorylation levels of
p130Cas were greatly reduced in pOCs treated with
Ad-PYK2/AS, as compared with controls or to pOCs infected with
Ad-PYK2/wt (Fig. 5C, upper panel). The kinetics
of p130Cas tyrosine phosphorylation were also faster in
pOCs overexpressing wild type PYK2 (maximal phosphorylation at 15 min).
This is consistent with increased spreading of these cells on VN. Taken
together, these observations suggest that PYK2 might serve as a
rate-limiting signaling molecule on the path to the cytoskeletal
organization required for osteoclast adhesion and spreading.
We also examined the tyrosine phosphorylation of paxillin under the
same conditions. Although paxillin appeared to be phosphorylated normally at 15 and 30 min in pOCs infected with Ad-PYK2/AS, we consistently observed diminished levels of paxillin phosphorylation at
60 min relative to control cells or cells infected with Ad-PYK2/wt (Fig. 5C, lower panel).
Adenovirus Expressing Antisense PYK2 Inhibits Ring Formation in
Osteoclast-like Cells--
Because PYK2 appears to play a role in pOC
spreading, we analyzed the effect of antisense PYK2 on the organization
of actin rings in osteoclasts. OCLs were previously shown to adhere to glass or plastic surfaces via an actin-rich ring structure (24). OCLs
treated with Ad-PYK2/wt or Ad-PYK2/AS were plated on glass coverslips
and costained for F-actin and TRAP activity. As shown in Fig.
6, OCLs expressing wild type PYK2 formed
actin rings similar to control OCLs. On the other hand, the
multinucleated TRAP(+) cells isolated from the co-cultures infected
with Ad-PYK2/AS adhered but did not spread to form actin rings. We then
plated the same number of OCLs on bone slices in the presence of the
osteoblastic MB1.8 cells to investigate distribution of microfilaments
on bone. After 24 h, the cells were fixed, and we analyzed the
organization of the sealing zone in these cells (Fig.
7). OCLs infected with Ad-PYK2/AS
attached to bone surfaces via randomly distributed adhesion contacts
and appeared to have a retracted morphology (Fig. 7, panels
a, b, and g-l) by comparison with the
normal actin-rich sealing zone, formed by normal OCLs (Fig. 7,
panel c) or cells infected with Ad-PYK2/wt during resorption
(Fig. 7, panel d). These cells formed small, punctated, and
disorganized ring structures visualized by staining for F-actin (Fig.
7, panels g and j) and paxillin (Fig. 7,
panel k). We previously reported that
Adenovirus Expression of Antisense PYK2 Inhibits Osteoclastic Bone
Resorption--
Because OCLs expressing antisense PYK2 do not form
normal sealing zones on bone (Fig. 7), we assumed that OCLs with
diminished PYK2 expression will not efficiently resorb bone. The same
number of OCLs, either untreated or treated with Ad-PYK2/wt or
Ad-PYK2/AS, were plated on dentine slices, and the area of resorption
pits made by these cells was determined after 24 h. As shown in
Fig. 8, pit forming activity was
inhibited by 90% in OCLs expressing antisense PYK2. This finding
suggests that PYK2 mediates the cytoskeletal organization required for
osteoclastic polarization and active bone resorption.
This study reports the effective use of a replication-defective
recombinant adenovirus as a vector for expressing PYK2-wt and AS in
osteoclasts and the inhibition of osteoclast adhesion, spreading, and
function by PYK2 AS. Osteoclasts are terminally differentiated cells
that have been notoriously difficult to transfect using conventional
methods. The advantage of recombinant adenovirus is that it normally
has a high efficiency of gene transfer and can infect nonreplicating
cells (27). In addition, adenoviruses enter cells by means of
Despite its structural similarity to FAK, PYK2 appears to have
different or additional cellular roles. In several cell types including
fibroblasts, epithelial, and neuronal cells, PYK2 seems to integrate
adhesion-dependent signaling events with MAP kinase activation (16, 33, 34), with stress-induced c-Jun N-terminal kinase
activation (35, 36), or with Src-mediated MAP kinase activation (37).
PYK2 is also activated by various stimuli that elevate the
intracellular calcium concentration via the nicotine acetylcholine
receptor, the voltage-gated calcium channels, the G protein-coupled
receptors, e.g. bradykinin and angiotensin II, or via
stimuli that promote calcium release from intracellular stores (37,
38). Although FAK was shown to promote cell survival, PYK2
overexpression induced apoptosis in fibroblasts (15). However, in
thymocytes, where PYK2 is physically associated with Janus tyrosine
kinase 1 and participates in interleukin-7-dependent signaling, PYK2 antisense-induced apoptosis (39).
Most information on PYK2 function has been obtained by overexpressing
PYK2 and its mutants in cells, which also express FAK. It is possible
that overexpression of PYK2 and its mutants could have disrupted, in a
dominant negative fashion, not only PYK2-mediated but FAK-mediated
signaling pathways as well. Targeted disruption of FAK in mice results
in embryonic lethality (40), a reflection of its broad cellular
functions (11), whereas PYK2 knockout mice seem to develop normally.
Thus far, the only phenotype identified in PYK2 null mice was mild
osteopetrosis without apparent reduction of
osteoclastogenesis.2 PYK2
expression was elevated in FAK-deficient fibroblasts but did not rescue
the cell migration defects caused by FAK deletion, nor did PYK2
localize to focal adhesion contacts in these cells (33, 40).
Furthermore, tyrosine phosphorylation of wild type PYK2 expressed in
Chinese hamster ovary cells was not altered by plating the cells on
fibronectin. On the other hand, chimeric PYK2, which contained FAK
C-terminal domain, exhibited enhanced tyrosine phosphorylation and
localization to focal adhesion contacts upon attachment to fibronectin
(41). These observations indicate that localization of PYK2 to sites of
integrin receptor clustering seems to play an important role in the
adhesion-dependent activation of this protein-tyrosine kinase.
In this study, we directly examined the role of PYK2 in
Tyrosine phosphorylation of PYK2 is severely reduced in Src-deficient
osteoclasts (17, 29), which are functionally compromised. Osteoclasts
express high levels of c-Src and low levels of other Src family kinases
(45), and Src deficiency is associated with osteopetrosis in mice. Loss
of osteoclast function was shown to be due partly to defective
polarization, leading to lack of ruffled border formation (21, 46).
Furthermore, targeted disruption of We further examined the effect of PYK2 deficiency on known
Our observations strongly suggested that PYK2 play a role in
osteoclastic adhesion and spreading; we examined the effect of PYK2
antisense on osteoclast apoptosis. Because we could not assess osteoclast apoptosis in the co-culture systems where osteoclast formation and survival were influenced by various growth factors, cytokines, and extracellular matrix proteins derived from the osteoblastic/stromal cells, we examine apoptotic events in
osteoclast precursors isolated from co-cultures uninfected and infected
for 4 days (25). Under serum-free conditions, we could not detect a
significant increase in the number of apoptotic pOCs expressing PYK2
antisense. However, we could not rule out the possibility that
apoptotic cells could be lost during the long term co-cultures, because
we observed a significant reduction in TRAP(+) cells in cultures
infected 4 days earlier with high concentration of Ad-PYK2/AS. We
routinely isolated pOCs infected with Ad-PYK2/AS that had 50-70% lower PYK2 protein levels. It is possible that osteoclasts with even
lower levels of PYK2 expression could be detached and undergo anoikis
in culture. A possible role for PYK2 in adhesion-dependent signaling that mediated osteoclast survival in vitro, will
be the subject of future studies. On the other hand, targeted
disruption of In summary, consistent with our previous findings suggesting that PYK2
serves as an adhesion-dependent kinase involved in the
regulation of bone resorption, we demonstrate using adenovirus expressing PYK2 antisense that PYK2 is essential for integrin-mediated cell spreading and actin ring formation in osteoclasts. PYK2 also plays
a central role in the cytoskeletal organization necessary for forming
the osteoclast sealing zone during bone resorption. Taken together
these observations suggest that, similar to
v
3 integrin expression or c-Src tyrosine
phosphorylation. Taken together, these findings support the hypothesis
that PYK2 plays a central role in the adhesion-dependent cytoskeletal organization and sealing zone formation required for
osteoclastic bone resorption.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
v
3 integrin is
highly expressed in osteoclasts in vitro and in
vivo (5). Interference with
v
3
integrin function by blocking antibodies, disintegrins, or small
molecular weight RGD mimetics lead to inhibition of bone resorption
in vitro and in vivo, supporting the key role of
this integrin in adhesion-dependent osteoclast activation
(6-9).
, related adhesion focal tyrosine kinase, or
calcium-dependent tyrosine
kinase)1 and focal adhesion
kinase (FAK) are members of a distinct family of non receptor
protein-tyrosine kinases that are regulated by a variety of
extracellular stimuli (10, 11). Although FAK is widely distributed,
PYK2 is predominantly expressed in the central nervous system and in
hematopoietic lineage cells. The alternative spliced isoform PYK2-H is
specifically expressed in T and B lymphocytes, monocytes, and natural
killer cells (12-15). Similar to FAK, PYK2 lacks SH2 and SH3 domains
but possesses several functional domains, including two proline-rich
regions in its C terminus and several phosphorylated tyrosine residues,
through which specific protein-protein interactions can occur. PYK2 was shown to play an important role in the integration of signals initiated
by a diverse group of extracellular stimuli, including integrin
binding, growth factors, cytokines, chemokines, and certain stress
stimuli (11). In PC12 cells, PYK2 tyrosine phosphorylation and
activation are stimulated by neuronal stimuli and stress signals, leading to modulation of a potassium channel and activation of the
c-Jun N-terminal kinase signaling pathway (16). In addition, stimulation of G protein-coupled receptors induces tyrosine
phosphorylation of PYK2 and complex formation between PYK2 and Src via
the SH2 domain, leading to activation of the MAP kinase signaling
pathway (10, 11). PYK2 was also suggested to participate in the
transfer of signals from the cell surface to the cytoskeleton, because it is tyrosine phosphorylated and activated by adhesion-initiated signaling in osteoclasts, monocytes, platelets, and B lymphocytes (14,
17, 18). PYK2 was also shown to interact with and phosphorylate the
focal adhesion-associated protein paxillin (19, 20).
3 integrin in mice was shown to cause progressive osteosclerosis without an apparent reduction
in the number of osteoclasts on bone (22). Consistent with this
observation, interfering with
v
3 function
in vitro, using the disintegrin echistatin, results in loss
of osteoclast migration and formation of the sealing zone (3). On the
other hand, inhibition of bone resorption in vivo by
echistatin was not accompanied by a reduction in the number of
osteoclasts on the bone surface (23). Because PYK2 is a down stream
mediator of the
v
3-and
Src-dependent signaling pathway in osteoclasts, we
therefore tested whether PYK2 plays a rate-limiting role in osteoclast
activity. In this study, we inhibited PYK2 expression using adenoviral
vectors expressing PYK2 antisense. We demonstrate that reduction in
PYK2 protein level results in inhibition of osteoclast formation
in vitro, primarily because of reduced osteoclast adhesion
and spreading. Moreover, expression of PYK2 antisense inhibits
formation of the sealing zone in osteoclasts and blocks their bone
resorption activity. These findings suggest that PYK2 plays an
essential role in the adhesion-dependent cytoskeletal organization that leads to osteoclast polarization and activity.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3 integrins were a gift from Dr. B. Bednar (Merck).
fluorescein isothiocyanate-conjugated goat anti-mouse IgG and
tetramethylrhodamine isothiocyanate-conjugated donkey
anti-rabbit IgG were purchased from Jackson Labs. (West Grove, PA). All
horseradish peroxidase-conjugated secondary antibodies and protein
G-Sepharose were from Amersham Pharmacia Biotech.
-minimum essential medium containing 10%
fetal bovine serum and 10 nM
1,25(OH)2D3. OCLs were formed within 7 days and
released from the dishes by treatment with 5 ml of 0.2% collagenase
(Wako Pure Chemical Co.) and collected by centrifugation at 250 × g for 5 min. Alternatively, OCLs and mononuclear prefusion
osteoclasts (pOCs) were prepared as described previously (25), except
that at 6 days in culture, pOCs were detached using 10 mM
EDTA solution, after removing MB1.8 cells with collagenase-dispase,
followed by washing three times with
-minimum essential medium.
pOCs, 95% pure as determined by TRAP staining, were used for cell
adhesion assays.
E1sp1 plasmid consisting of the human cytomegalovirus promoter and
the bovine growth hormone polyadenylation site as previously described
(26). Adenovirus (Ad) expressing the Escherichia coli
-galactosidase gene (Ad-
-galactosidase) was used to optimize infection conditions. The p
El-PYK2/wt recombinant plasmid was constructed using the full-length murine PYK2 cDNA, which was cloned from a mouse spleen
-ZAPII cDNA library (Stratagene, La Jolla, CA) using the specific PYK2 probe (17). We also produce a
recombinant adenovirus (Ad-PYK2/AS) carrying an antisense sequence of
PYK2 consisting of 300 base pairs of the reverse 5'-coding region of
the murine PYK2 cDNA. Recombinant viruses were produced in the
human embryonic kidney 293 cell line, purified, and titrated according
to standard methods (27).
-minimum essential medium and
added directly to the co-cultures. Adenovirus was normally added to
cultures at day 3, for 24 h, followed by a change of medium. To
determine the efficiency of infection, cells were infected with
Ad-
-galactosidase for 24 h. At day 7, cells were washed once
with phosphate-buffered saline and fixed with 3.7% formaldehyde
solution in phosphate-buffered saline. The co-cultures were either
stained with X-gal histochemical staining or with tartrate-resistant
acid phosphatase (TRAP), a marker enzyme for osteoclasts (28). The
number of X-gal-positive cells and TRAP-positive cells was counted in
triplicate cultures. Efficiency of adenoviral infected osteoclasts was
expressed as the ratio of X-gal-positive to total TRAP-positive cells.
Infected pOCs or OCLs were harvested from the infected co-cultures at
day 5 or 7, respectively. pOCs were used for biochemical analyses, cell
adhesion, and spreading on vitronectin. OCLs were used in immunostaining and pit formation.
v
3 integrins were immunostained using
polyclonal antibodies as described previously (3), followed by
fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG. Immunofluorescence-labeled cells were photographed through an 100×
objective using a Zeiss Axiophot epifluorescence microscope or with a
Leica TCS SP Spectral confocal laser scanning microscope equipped with
Argon-crypton laser (Leica Microsystems Heidelberg GmBH).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase virus at MOI
of 10 for every 24 h from day 1 to day 5 in culture. At day 7, parallel co-cultures were stained with either X-gal or TRAP stains.
Frequency of adenovirus-mediated gene transfer in OCLs was quantitated
as the ratio of
-gal(+) cells to TRAP(+) cells. As shown in Fig.
1A, 75-80% of the total
TRAP(+) cells were infected with Ad-
-gal when virus was added to
co-cultures at either day 3 or day 4. We also noted that the fraction
of
-gal expressing multinucleated OCLs with three or more nuclei,
was the same as in the TRAP(+) cells, suggesting that adenoviral gene transfer does not affect osteoclast fusion. A dose-response
relationship between viral dose (MOI) and the fraction of infected OCLs
was also observed (Fig. 1B) when Ad-
-gal was added at day
3 and quantitated for
-gal(+) and TRAP(+) cells at day 7. Although
the co-cultures contain a confluent layer of osteoblastic MB1.8 cells,
-gal was expressed preferentially in OCLs and pOCs, as shown in Fig.
1C.
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Fig. 1.
Efficient adenovirus-mediated gene transfer
into osteoclast-like cells. A, co-cultures (in 24-well
plates) were infected with adenovirus expressing -galactosidase
(Ad-
-gal) at MOI of 10 for 24 h at days 1-5. At day 7, the
cultures were fixed and stained with X-gal histochemical staining or
with TRAP staining. Ratios of
-gal positive to total TRAP positive
multinucleated (Multi) OCLs were expressed at different days
of infection. Each condition was quantitated from quadruplicate
samples, and the results were expressed as the means ± S.D.
B, similarly, co-cultures were infected with
Ad-
-galactosidase at day 3, and as described, ratios of
-gal(+)
to total TRAP(+) cells were expressed at increasing MOI. C,
multinucleated and mononuclear (pOCs) osteoclast-like cells were
preferentially infected with Ad-
-gal as observed in the co-culture
system stained with TRAP and X-gal. Bars, 100 µm for
panels a and b and 50 µm for panel
c.
3 integrin antibodies (Fig.
2, lower panel). This
suggested that adenoviral infection did not significantly affect
osteoclast differentiation using markers such as
3
integrin expression (Fig. 2) or TRAP activity (Figs. 1C and
3A). PYK2 protein expression
in OCLs treated with Ad-PYK2/wt at day 3 of the co-culture was
increased about 2-fold, as compared with untreated cells (Fig. 2). In
contrast, there was a significant reduction (50-70%) in PYK2 protein
levels, in OCLs treated with Ad-PYK2/AS, relative to normal controls. We have never observed complete inhibition of PYK2 expression in
osteoclasts exposed to antisense treatment, possibly because cells with
very low PYK2 levels might detach or apoptose. This is supported by
the marked reduction in the number of TRAP(+) cells in co-cultures
treated with Ad-PYK2/AS at MOI = 100 (Fig. 3B).
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[in a new window]
Fig. 2.
Infection of osteoclast-like cells with
adenovirus expressing PYK2 antisense reduces PYK2 protein
expression. OCLs were purified from co-cultures uninfected
(lane C) or infected with adenovirus expressing wild type
PYK2 (Ad-PYK2/wt) or with Ad-PYK2/AS at day 3 or 4 as described under
"Materials and Methods." PYK2 or 3 integrin were
immunoprecipitated from equal protein concentrations of these lysates,
followed by immunoblotting with anti-PYK2 (upper panel) or
anti-
3 integrin (lower panel)
antibodies.
View larger version (55K):
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Fig. 3.
Infection of the co-culture system with
adenovirus expressing PYK2 antisense inhibits osteoclastogenesis.
Numbers and Area of TRAP(+) cells were quantitated from co-cultures
infected with increased concentrations of either Ad-PYK2/wt
(A) or with Ad-PYK2/AS (B). Each condition was
run in quadruplicate samples, and the results are expressed as the
means ± S.D. C, morphological analyses of co-cultures
infected with Ad-PYK2/wt and Ad-PYK2/AS . Note that area of TRAP(+)
cells was more sensitive to the increase in expression of PYK2
antisense than the number of these cells. Bars, 50 µm.
3 integrin.
We plated the pOCs on vitronectin-coated dishes under serum-free
conditions and determined the increase in cellular planar area with
time. Planar area of either untreated or pOCs infected with Ad-PYK2/wt
increased ~4-fold within 60 min (Fig.
4A). However, pOCs infected
with Ad-PYK2/AS failed to spread on vitronectin (Fig. 4, A
and B). On the other hand, several TRAP(
) cells
occasionally found in this pOC preparation normally spread on VN (Fig.
4B, panel c, arrowhead). We observed a
small increase in the rate of cell spreading in pOCs infected with
Ad-PYK2/wt (Fig. 4A), consistent with the 2-fold increase in
PYK2 protein expression in OCLs infected with Ad-PYK2/wt (Fig. 2).
These cells seem very well spread by comparison with the normal controls (Fig. 4B, panels a and
b).
View larger version (31K):
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Fig. 4.
Adenovirus expressing PYK2 antisense inhibits
prefusion osteoclast-like cell spreading without affecting
apoptosis. pOCs were purified from control co-cultures
(cont.) or from cultures infected with either Ad-PYK2/wt or Ad-PYK2/AS. To examine the
planar area of cell spreading, pOCs were allowed to adhere and spread
on VN-coated surfaces for 1 h, fixed, and quantitated for cell
area (A) and stained for TRAP activity (B), as
described under "Materials and Methods." Each condition was
quantitated from quadruplicate samples. The results were expressed as
the means ± S.D. The arrowhead points to a TRAP( )
cell spreading on VN. C, pOCs were isolated from uninfected
cultures (panels a and b) or cultures infected
with either Ad-PYK2/wt (panels c and d) or
Ad-PYK2/AS (panels e and f) for 4 days. Cells
were then allowed to attach to VN-coated glass coverslips for 60 min as
described above, followed by staining for F-actin using Oregon-green
phalloidin (panels a, c, and e) and
stained for DNA using Hoechst 33342 (panels b, d,
panels f). Noted that nuclei in pOCs expressing PYK2 AS are
in different plane of focus (arrowhead), because these cells
have thick rounding cell bodies. Apoptotic cells were carefully
examined for nuclear condensation and fragmentation (arrow).
Bars, 10 µm.
/
) OCLs, tyrosine phosphorylation levels of
PYK2 and p130Cas were greatly reduced (29). In this study,
we examined the kinetics of tyrosine phosphorylation of c-Src,
p130Cas, and paxillin in pOCs which were either untreated
or infected with Ad-PYK2/wt or Ad-PYK2/AS and were allowed to attach to
VN-coated dishes. The level of tyrosine phosphorylated proteins was
determined in cells adhering for 15, 30, or 60 min and was compared
with cells maintained in suspension. First, tyrosine phosphorylated proteins were compared in total cell lysates isolated from uninfected pOCs and cells infected with either Ad-PYK2/wt and Ad-PYK2/AS, adhering
on VN for 60 min (Fig. 5A). At
equal amounts of protein, the level of tyrosine phosphorylated proteins
was generally lower in cells expressing PYK2/AS as compared with the
those in controls. As previously reported (17), PYK2 became
significantly tyrosine phosphorylated upon pOC adhesion to VN-coated
surfaces for 15 min in control cells (Fig. 5B).
Overexpression of PYK2 in pOCs infected with Ad-PYK2/wt was
consistently found to yield a slightly faster rate of tyrosine
phosphorylation relative to normal controls, full phosphorylation being
reached at 15 min. In contrast, the levels of PYK2 expression in pOCs
treated with Ad-PYK2/AS were reduced by 60% as quantitated by
densitometry (data not shown). Consequently, tyrosine phosphorylated
PYK2 was also reduced (Fig. 5B, upper panel).
Tyrosine phosphorylation levels of c-Src in these cells were not
different in pOCs transfected with wild type PYK2 or PYK2 antisense
(Fig. 5B, lower panel). Nor did we find a
significant change in c-Src activity in these cells, as determined by
in vitro kinase assays (data not shown).
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[in a new window]
Fig. 5.
Reduction of PYK2 expression reduces
adhesion-dependent tyrosine phosphorylation of
p130Cas and paxillin without affecting c-Src
phosphorylation. pOCs were purified from control co-cultures
(lane C) or from cultures infected with either Ad-PYK2/wt or
Ad-PYK2/AS. Cells were allowed to attach to VN-coated dishes at
indicated times. Total cell lysates (TCL) of pOCs adhering
on VN for 60 min were blotted with anti-phosphotyrosine antibodies
(A). Lysates were immunoprecipitated with anti-PYK2 or
anti-c-Src antibodies (B) or with anti-p130Cas
or anti-paxillin antibodies (C) and then blotted with
anti-phosphotyrosine antibodies (circled P) and with the
respective immunoprecipitating antibodies.
v
3 integrins preferentially distribute to
the basal plasma membrane of osteoclasts during resorption (3).
Similarly, localization of
v
3 integrins in OCLs infected with Ad-PYK2/wt was normally distributed to the basal
membrane (Fig. 7, panel e). Here, we found that
v
3 integrins appear to cluster into small
punctated structures on the basal plasma membrane in OCLs expressing
antisense PYK2 (Fig. 7, panel h) and partially colocalize
with F-actin (Fig. 7, panel i).
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Fig. 6.
Adenovirus expressing PYK2 antisense inhibits
actin ring formation in osteoclasts during spreading. Uninfected
and infected OCLs were plated on serum-coated glass coverslips for
20 h and then fixed in 4% paraformaldehyde and costained for
F-actin using fluorescein isothiocyanate-phalloidin (upper
panels) and for TRAP activity (lower panels).
View larger version (34K):
[in a new window]
Fig. 7.
Adenovirus expressing PYK2 antisense inhibits
sealing zone formation in osteoclasts during polarization on bone.
Localization of microfilaments and v
3
integrins in uninfected and OCLs infected with Ad-PYK2/AS or Ad-PYK2/wt
were examined after plating on bovine cortical bone slices for 20 h at 37 °C. Pseudocolored confocal microscopic images of osteoclasts
infected with Ad-PYK/AS double stained with anti-adenoviral penton
proteins (panel a, red) and F-actin (panels
a and b, green) as compared with F-actin
localized in a typical sealing zone at the bone surface in normal OCLs
(panel c, green) or in cells infected with
Ad-PYK2/wt (panel d, green). OCLs infected with
Ad-PYK2/wt (panels d-f) showed a sealing zone (panel
d), localization of
v
3 integrins at
the basal membrane (panel e, red) and an overlay
image of F-actin and
v
3 localizations
(panel f). The cells expressing PYK2/AS were also double
stained for F-actin (panels g and j,
green) and
v
3 integrins
(panel h, red) or paxillin (panel k,
red). Colocalization is seen as yellow in overlay
images (panels i and l), respectively. Note small
actin rings or aggregation of F-actin at bone surface in osteoclasts
expressing PYK2/AS (arrows in panels b,
g, and j) by comparison with well defined F-actin
rings in an osteoclast expressing Ad-PYK2/wt (panel d) or in
wild type osteoclasts (panel c).
v
3 integrins do not colocalize with actin
at the bone surface, whereas paxillin and F-actin colocalize in
podosome-containing rings at bone surface (arrows in
panels j-l). On the other hand,
v
3 integrins colocalize with actin in
punctated structures at the basal membrane (arrowheads in
panels g-i). Images merged from optical sections from 4.5 µm (panel a and b), 10.5 µm (panel
c), 5.4 µm (panels d-f), 6.8 µm (panels
g-i), and 5.8 µm (panels j-l) thickness are shown.
Bars, 10 µm.
View larger version (11K):
[in a new window]
Fig. 8.
Adenovirus expressing PYK2 antisense inhibits
bone resorption. Aliquots (20,000 cells/slice) of uninfected
(cont) or infected pOCs with either Ad-PYK2/wt
(wt) or Ad-PYK2/AS (AS) were plated on dentine
slices for 24 h at 37 °C. Resorption pit area were measured
using an image analyzing system. The results were expressed as the
means ± S.D. (n = 5) of resorbed area per whole
dentine surface area.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
v
3 or
v
5
integrins, which serve as receptors for the adenoviral penton (30). We
have previously shown that expression of
v
3 integrins in pOCs is induced around day 3 or 4 of the osteoclast-generating co-culture system (31). Consistent with this observation, we found, using
-galactosidase expressing virus, that adenovirus efficiently mediated gene transfer into osteoclasts around day 3 of the co-culture. Although osteoblastic MB1.8 cells express
v
5 integrins (31), we
did not observe significant osteoblast infectivity with this adenoviral
vector at low virus concentrations (MOI = 10); however, at higher
virus concentrations (MOI = 100), a few MB1.8 cells were infected
(data not shown). Thus, at MOI = 10 the adenovirus vector appears
to mediate in our co-culture system gene transfer preferentially into
OCLs, in agreement with previous reports on the use of recombinant adenovirus to transfer epidermal growth factor receptor and Csk into
osteoclast-like cells (28, 32).
v
3 integrin-dependent
regulation of osteoclastic cytoskeletal organization and bone
resorption. We previously found that in contrast to fibroblasts, osteoclasts and osteoclast-like cells express high levels of PYK2 both
in vivo and in culture and very little FAK protein (17). FAK-independent integrin-stimulated signaling events have also been
demonstrated in other hematopoietic cells, where FAK protein expression
is low (42-44). PYK2 acted in osteoclasts as a cell adhesion-dependent kinase, being rapidly tyrosine
phosphorylated upon ligand engagement of
v
3 integrins (17). In addition, PYK2
localizes to podosomal adhesion contacts of migrating osteoclasts (17).
Furthermore, the involvement of PYK2 in the cytoskeletal organization
associated with osteoclastic bone resorption is consistent with PYK2
localization in the sealing zone structures, a prerequisite for cell
polarization (17).
3 integrin in mice
induces progressive osteoclerosis, without an apparent reduction in
osteoclast number (22). Here, we demonstrated that similar to Src
deficiency, significant reduction in PYK2 expression in osteoclasts
in vitro interfered with the cytoskeletal organization,
required for formation of the osteoclast sealing zone on the bone
surface. Expression levels of
v
3
integrins and of PYK2 were not altered in Src-deficient osteoclasts
(17, 47). Similarly, we show here that the expression of integrin receptors, c-Src, and other PYK2-associated molecules such as p130Cas and paxillin were not changed in osteoclasts
expressing PYK2 AS.
v
3 integrin-mediated signaling events in
osteoclasts. Our previous findings suggested that the
adhesion-dependent increase in PYK2 tyrosine
phosphorylation in osteoclasts may occur downstream of
v
3 integrin-dependent Src
activation (17). The findings of this study are consistent with this
interpretation, because the adhesion-dependent tyrosine
phosphorylation of c-Src was unaffected by PYK2 antisense expression in
osteoclasts. We also found that the in vitro kinase activity
of c-Src was not altered in these cells (data not shown). On the other
hand, we have shown that tyrosine phosphorylated p130Cas is
part of and probably involved in the organization of the podosome-rich ring structure in osteoclasts (24, 29). Similar to PYK2,
p130Cas phosphorylation was also markedly reduced in
osteoclasts derived from Src(
/
) mice (24). Moreover, PYK2 and
p130Cas form a stable complex in osteoclasts. This complex
is independent of tyrosine phosphorylation and is present in Src(
/
)
osteoclasts, in which neither protein is phosphorylated or associated
with the osteoclast adhesion structure (29). Here, we found that suppression of PYK2 expression reduced substantially
p130Cas tyrosine phosphorylation in osteoclasts, suggesting
that PYK2 might indeed directly phosphorylate p130Cas or
serve as an adaptor molecule for recruiting Src kinases to p130Cas. This is consistent with p130Cas being
a substrate for PYK2, as well as for Src kinase (48, 49). We and others
have shown that integrin-mediated PYK2 activation leads to
p130Cas tyrosine phosphorylation in a
Src-dependent manner (29, 50), and PYK2 is constitutively
associated with p130Cas in osteoclasts (29).
3 integrin, c-Src, or PYK2 in mice were
reported to develop osteopetrosis without a reduction in osteoclast
number, suggesting that
3 integrin-mediated signaling
might not play an important role in osteoclast survival in
vivo (21, 22).2
v
3 integrin and to its upstream regulator
c-Src, PYK2 plays a rate-limiting role in osteoclastic bone resorption.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the Visual Communication Department at Merck for preparing the figures for this manuscript and the Department of Anatomy, Turku University for the use of the confocal microscope.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Bone Biology and Osteoporosis Research, Merck Research Laboratories, West Point, PA 19486.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M008368200
2 N. A. Sims, K. Aoki, Z. Bogdanovi, M. Maragh, M. Okigaki, S. Logan, L. Neff, E. DiDomenico, A. Sanjay, J. Schlessinger, and R. Baron, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PYK2, protein-tyrosine kinase 2;
Ad, adenovirus;
AS, antisense;
FAK, focal
adhesion kinase;
OCL, osteoclast-like cell;
pOC, prefusion
osteoclast-like cell;
VN, vitronectin;
MOI, multiplicity of infection;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
TRAP, tartrate-resistant acid
phosphatase;
wt, wild type;
-gal,
-galactosidase.
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