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Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGF binds to two tyrosine kinase receptors, Flt-1 and Flk-1, with high affinity (5). While the biological function of the VEGF receptor type II, Flk-1, is understood, that of Flt-1 is still unclear (6). It has been recently reported that VEGF is potentially a monocyte chemoattractant (7) and that monocytes express not Flk-1 but Flt-1 (8). Furthermore, macrophages derived from Flt-1 mutant mice indicated deranged chemotaxis in response to VEGF (9). These findings show that the activation of Flt-1 may stimulate the migration of monocyte/macrophage lineages. However, the signaling pathway of Flt-1 was not yet fully understood.
Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that is localized to focal adhesions (10) and involved in the control of several biological processes, including cell spreading, migration, and survival (11). FAK associates with activated growth factor receptors such as platelet-derived growth factor and epidermal growth factor receptors through its N-terminal domain and plays important roles in platelet-derived growth factor-induced and epidermal growth factor-induced cell migration (12). Furthermore, it has been shown that the tubulogenic activity of rat endothelial cells is dependent on the VEGF-Flt-1-FAK pathway (13).
In this study, we tried to elucidate the effects of VEGF on pOCs in vitro and the role of the VEGF signaling pathway in the recruitment of pOCs in rats with adjuvant-induced arthritis (AIA). We demonstrate for the first time the possible involvement of the VEGF-Flt-1-FAK pathway in chemotaxis and the cell proliferation of pOCs in arthritic joint destruction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant mouse VEGF was obtained from Genzyme/Techne
(Minneapolis, MN). Anti-receptor activators of NF-
B (RANK), VEGF,
Flt-1, and Flk-1 Abs were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Anti-FAK and phosphotyrosine (clone 4G10) Abs came
from Upstate Biotechnology (Lake Placid, NY). Anti-phospho-FAK and
proline-rich tyrosine kinase 2 (Pyk2) Abs were from BioSource
(Chicago, IL). A cell proliferation ELISA 5-bromo-2'-deoxyuridine
(BrdU) kit was purchased from Roche (Mannheim, Germany). PD98059 was
obtained from Calbiochem (San Diego, CA), and wortmannin was purchased
from Sigma-Aldrich (St. Louis, MO).
Induction of AIA
Induction of AIA was performed as previously described (14). Briefly, 10- to 12-wk-old female Lewis rats (100 g) were injected s.c. with 300 µl (5 mg/ml) of lyophilized Mycobacterium butyricum (Difco, Detroit, MI) at the base of tail. All time points were considered in relation to the AIA induction day, designated as day 0. Arthritis of the bilateral ankle joints was developed in 100% of the treated animals by day 10.
Immunohistochemistry and immunofluorescence
Immunohistochemistry and immunofluorescence were performed as previously described (15). Briefly, joint specimens were initially decalcified for 2 wk in an EDTA-containing buffer and embedded in paraffin. The endogenous peroxidase activity was quenched by incubating the sections in absolute methanol and 3% hydrogen peroxide. The slides were then incubated with various primary Abs. Biotinylated Abs and peroxidase-conjugated streptavidin were used as second and third reagents, respectively. The signals were detected using 3-amino-9-ethylcarbazole in N,N-dimethylformamide. The slides were counterstained with methylgreen. For immunofluorescence examination, primary Abs were applied simultaneously and incubated overnight at 4°C. The samples were washed in PBS and incubated with FITC- or tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary Abs. Then the sections were mounted and examined by confocal laser scanning microscopy.
Cell lines and culture conditions
The mouse myeloid cell line Raw 264.7 was obtained from American Type Culture Collection (Manassas, VA). The cells were maintained in an anti-MEM (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS. The cells were incubated at 37°C in a humidified atmosphere with a CO2 content of 5%. The cells were harvested by scraping, counted using a hemocytometer, and then used for various experiments.
Immunoprecipitation and immunoblotting
Cells that were growing logarithmically (2–4 x
106) at 
70% confluency were harvested and
solubilized in a lysis buffer (20 mM Tris (pH 7.4), 250 mM NaCl, 1.0%
Nonidet P-40, 1 mM EDTA, 50 mg/ml leupeptin, and 1 mM PMSF). Protein
quantity was determined with a Bradford protein assay (Bio-Rad,
Hercules, CA). The protein samples were boiled for 5 min, and 10 µg
of total protein from each sample was run on 4–12% gradient pre-cast
MOPS-polyacrylamide gels (NOVEX, San Diego, CA) and blotted onto a
nitrocellulose filter. For immunoprecipitations, lysate aliquots were
incubated with anti-FAK Ab overnight at 4°C. Immunocomplexes were
collected on protein A-G Sepharose beads (Santa Cruz Biotechnology).
The beads were washed three times with a lysis buffer, boiled, and
subjected to electrophoresis. After transfer to nitrocellulose
membranes, the filters were pretreated with TBS containing 5% dry milk
and 0.05% Triton X for 2 h at room temperature. They were
then incubated with the appropriate primary Abs for 2 h at room
temperature. After several washes, a HRP-conjugated secondary Ab
(BioSource) was added and incubated at room temperature for 1 h.
After the final wash, the immunoreactivity of the blots was detected
using an ECL system (Amersham, Arlington Heights, IL).
Cell proliferation assay
Raw cells seeded in culture plates were incubated in serum-free medium with or without VEGF for 24 h. The cell growth rate was determined using a cell proliferation kit (Roche) based on the ELISA for a thymidine analog, BrdU, according to the manufacturer’s protocol.
Chemotaxis assay
The chemotaxis assay was performed using transwell chambers
(Costar, Cambridge, MA) as described previously (16, 17, 18).
In brief, cells were suspended in serum-free anti-MEM containing
1% BSA and seeded in the upper chamber. The lower chamber was filled
with serum-free anti-MEM, which was or was not supplemented with
various cytokines (10 ng/ml VEGF, 10 ng/ml TNF-
, 500 pg/ml IL-1,
10 ng/ml M-CSF, and 10 ng/ml monocyte chemoattractant protein-1
(MCP-1)). Polyvinylpyrroidone-free polycarbonate filters with 8-µm
pore size were coated with type IV collagen and inserted between the
two chambers. Then the cells were allowed to migrate for 6 h at
37°C. After the incubation, cells that migrated to the lower side of
the filter were fixed, stained, and counted for five fields per filter
under a microscope.
Flow cytometry
Cells were washed with ice-cold PBS twice and harvested by scraping. The cells were then stained with anti-Flt-1 or Flk-1 Abs. The stained cells were further incubated with FITC-conjugated secondary Abs and analyzed using a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA).
Adenoviral constructs
A replication-defective adenovirus encoding FAK-related
nonkinase (FRNK), Adv-FRNK, was kindly provided by Dr. A. M.
Samarel (19). A replication-defective adenovirus
containing the 
-galactosidase gene, LacZ (Adv-LacZ), was used to
control for the nonspecific effects of viral infection. Adenoviruses
were amplified and purified using HEK293 cells, as previously described
(20). Preliminarily, it was determined that a viral
concentration of >10 multiplicity of infection (moi) produced a
detectable expression of FRNK protein within 48 h. The infected
cells were subjected to immunoblotting, chemotaxis, and cell
proliferation assays.
Assesment of the effect of Adv-FRNK on AIA
Twenty-one rats were immunized with adjuvant (day 0). On days 7 and 14, PBS (n = 7), LacZ (n = 7), and FRNK (n = 7) mice were intra-articularly injected with 50 µl of PBS, adv-LacZ, and adv-FRNK, respectively. The viruses (1 x 108 virus particles per joint) were directly injected into the bilateral joint spaces. On days 7, 14, 21, and 28, hind paws were immersed into the chamber up to the anatomic hair line and edema measurement were made with a volumetric apparatus (MK-550; Muromachi-kikai, Tokyo, Japan). On days 21 and 28, the rats were sacrificed and the therapeutic effects of Adv-FRNK injection were examined. For histological evaluation, the specimens were fixed and decalcified in 10% EDTA for 14 days and embedded in paraffin. The sectioned specimens were subjected to tartrate-resistant acid phosphatase (TRAP) staining and immunohistochemistry. The number of TRAP-positive cells was calculated in three sections of ankle joints from three different specimens in each group. Five visual fields were randomly selected in each section and the number of TRAP-positive cells was counted under a microscope. For radiological examination, both hind paws were dissected on day 28 and examined by soft x-ray (Softex, Tokyo, Japan). The evaluations were performed blindly by the same observer. A 0–3 subjected grading scale (0 = normal, 1 = mild, 2 = moderate, 3 = severe) described previously (21), with modification, was used to evaluate four different parameters including joint space narrowing, subchondral bone erosion, osteoporosis, and periosteal new bone formation. The radiological score refers to the sum of the subjective scores for each of these four parameters (maximum 12).
Statistical analysis
The values of chemotaxis and cell proliferation assays were expressed as the mean ± SD and were statistically analyzed by a Student’s t test. The volume of the hind paw volume, the number of TRAP-positive cells, and the radiological scores were analyzed by Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initially, we analyzed the expression profiles of VEGF and VEGF
receptors in the specimens taken from the ankle joints of rats with
AIA. Arthritis was remarkably induced in the hind feet of the rats 3
wk after immunization by adjuvants. The specimens of ankle joints
showed strong bone destruction and the accumulation of mononuclear
(Fig. 1
b, arrows) and
multinuclear cells (Fig. 1b, arrowheads). TRAP
staining demonstrated the presence of bone-resorbing OCs (Fig. 1c, arrowheads). In serial sections, expressions of VEGF and
Flt-1 were observed mainly in infiltrating mononuclear cells (Fig. 1, d and e, arrows) and TRAP-positive multinuclear
cells (Fig. 1, d and e, arrowheads) that were
near the bone surface. Flk-1 expression was not found in the same cells
(Fig. 1f). Tissue sections stained with preimmune control
IgG showed no nonspecific staining (Fig. 1g). Because mature
OCs and pOCs expressed RANK (22), we next investigated
whether Flt-1 was colocalized with RANK-positive cells in the AIA
joints. The specimens were incubated with anti-RANK (Fig. 1h) and anti-Flt-1 (Fig. 1i) Abs and then
reacted with TRITC- or FITC-labeled secondary Abs, respectively. As
shown in Fig. 1j, RANK and Flt1 were colocalized in
mononuclear (Fig. 1j, arrow) and multinuclear (Fig. 1j, arrowheads) cells in the specimens. These results
suggest that OCs and pOCs in the AIA joints expressed Flt-1.
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We next focused on the involvement of VEGF signaling in pOCs in
vitro. The myeloid Raw cells in mice have been shown to be able to
differentiate into OC-like cells in the presence of RANK ligand
(23, 24). In this study, treatment of Raw cells with RANK
ligand (100 ng/ml) for 6 days induced the formation of
multinuclear TRAP-positive OC-like cells, and the cells were able to
form resorption pits on dentin slices (data not shown). Raw cells also
expressed CD11b, which is a typical marker of macrophages (data not
shown), indicating that Raw cells are in the same lineage as
monocyte-macrophage and can be used as pOCs. FACS analysis demonstrated
that Raw cells expressed Flt-1, but not Flk-1 (Fig. 2a). Expression of VEGF was
also observed in the cells (data not shown). Because tyrosine
phosphorylation was required to activate Flt-1, we next examined the
tyrosine phosphorylation of Flt-1 in Raw cells after treatment with
VEGF. As shown in Fig. 2b, Flt-1 was tyrosine-phosphorylated
by VEGF treatment, and the neutralizing Ab against VEGF effectively
inhibited the phosphorylation of Flt-1.
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Adding VEGF to the lower chamber stimulated the chemotaxis of Raw
cells, and maximal stimulation was observed at 10 ng/ml with a typical
bell-shaped curve. VEGF-driven chemotaxis was inhibited by the
neutralizing Ab to VEGF (Fig. 3a). However, other
bone-resorptive cytokines such as TNF-, IL-1, and M-CSF could not
enhance the chemotaxis of Raw cells. However, the addition of serum or
MCP-1 to the lower chamber enhanced the chemotaxis, as previously
reported (25) (Fig. 3b). We next analyzed the
VEGF-induced cell proliferation of Raw cells by using BrdU
incorporation assay. Stimulation of serum-starved Raw cells with
various concentrations of VEGF for 24 h raised the cell
proliferation dose-dependently in contrast to serum-free control cells
(Fig. 3c). VEGF (50 ng/ml) stimulated the cell proliferation
of Raw cells at a level 3-fold higher that of control cells.
TNF-, IL-1, and M-CSF also stimulated the cell proliferation of Raw
cells (Fig. 3d). These results indicate that VEGF enhanced
the chemotaxis and cell proliferation of Raw cells.
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VEGF binds to its plasma membrane receptors and transmits signals
through the phosphorylation of intracellular proteins. After VEGF
treatment, major tyrosine-phosphorylated bands were found at 180to
200 kDa and 110 to 130 kDa in size (data not shown). It was
reported that 112-kDa Pyk2, a member of the FAK family, acts to
transmit biological signals such as cell adhesion and bone resorption
in mature OCs (26). Therefore, we first postulated that
VEGF treatment might induce tyrosine phosphorylation of Pyk2. However,
Raw cells showed constitutive phosphorylation of Pyk2, and VEGF
treatment did not affect the phosphorylation (Fig. 4a).
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5- to 6-fold compared with that of the control,
suggesting a VEGF-induced association between Flt-1 and FAK (Fig. 4
b). We then investigated the effect of VEGF in the tyrosine
phosphorylation of FAK. VEGF treatment induced the tyrosine
phosphorylation of FAK in Raw cells, and the neutralizing Ab reduced
the phosphorylation (Fig. 4c). It has been reported that
major sites of FAK phosphorylation are tyrosine residues 397, 576, 577,
861, and 925 (28), and that each residue has a specific
function (29). After VEGF stimulation, we observed the
tyrosine phosphorylation of Y397 and Y925 (pY397 and pY925) in FAK
(Fig. 4d), whereas no tyrosine phosphorylation was observed
in Y576, Y577, and Y861 (data not shown). Other cytokines involved in
pathological bone resorption, such as IL-1, TNF-, and M-CSF, did
not induce the phosphorylation of FAK (Fig. 4e). In the
joints of rats with AIA, the expressions of pY397 in FAK were also
observed in the small mononucleated cells near the bone surface (Fig. 5b, arrows), while control IgG
Ab showed no nonspecific staining (Fig. 5c). The expression
pattern of pY925 in FAK was similar to that of pY397 (data not shown).
Immunofluorescence analysis clearly indicated that small RANK-positive
cells, probably pOCs, expressed pY397 in FAK (Fig. 5, d–f, arrows).
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Y397 in FAK has been identified as the binding site for
phosphatidylinositol 3-kinases (PI3K) and functions as a critical
factor for cell motility (30). In this study, pretreatment
of Raw cells with wortmannin, a specific inhibitor of PI3K, reduced
VEGF-driven chemotaxis (Fig. 6a), indicating that PI3K
might be involved in VEGF-Flt-1-FAK signaling in the cells. Recently it
was demonstrated that pY925 in FAK creates a binding site for the SH2
domain of Grb2 (31), and that this interaction may
activate the Ras-extracellular signal-regulated
kinase/mitogen-activated protein kinase (MAPK) pathway
(32). In the present study, PD98059, a specific MAPK
inhibitor, reduced the VEGF-induced cell proliferation in a
dose-dependent manner (Fig. 6b), suggesting the involvement
of the MAPK pathway in the VEGF-induced cell proliferation of
pOCs.
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To further examine the role of FAK in the VEGF-driven chemotaxis
and the cell proliferation of Raw cells, we inhibited the function of
FAK by using a replication-defective adenovirus which contained FRNK
(adv-FRNK). Initially, Raw cells were infected with adenovirus
containing -galactosidase (adv-LacZ). X-gal staining revealed that
>95% of the cells were effectively infected with adv-LacZ (moi =
10) (data not shown). When Raw cells were infected with adv-FRNK
(moi = 10), the cells expressed FRNK (Fig. 7a). The FRNK-infected cells
exhibited a decreased basal level of chemotaxis compared with control
cells. Furthermore, the cells expressing FRNK did not respond to VEGF
treatment (Fig. 7b). Infection by adv-FRNK also remarkably
reduced VEGF-induced cell proliferation. However, adv-FRNK failed to
inhibit the M-CSF-stimulated proliferation of Raw cells (Fig. 7c). Adv-FRNK also abrogated the phosphorylation of Y397 and
Y925 in FAK induced by VEGF (Fig. 7d). The expression of
total FAK was not affected by the infection of viruses (data not
shown).
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The results described above indicate that the activation of the
VEGF-Flt-1-FAK pathway may play an important role in infiltrating the
pOCs in the arthritic joints of rats with AIA. To obtain direct
evidence to support this notion, we injected Adv-FRNK into the inflamed
ankle joints of rats with AIA. On day 21, histopathologic examination
of the joints from rats treated with adv-FRNK showed suppressed
infiltration of mononuclear and multinuclear cells (Fig. 8b) compared with those with
adv-LacZ (Fig. 8a). In addition, the number of the
mononuclear cells with the expression of pY397 in FAK (Fig. 8d, arrows) was notably reduced through treatment with
adv-FRNK, but not with adv-LacZ (Fig. 8c). The serial
sections stained with control anti-IgG Ab showed no nonspecific
signaling (Fig. 8, e and f). The expression of
FRNK proteins in the joints was confirmed 7 days after the injection
(Fig. 8g). On days 21–28, the hind paw volume of AIA rats
was also decreased by adv-FRNK (Fig. 8h). Injection of
adv-FRNK reduced the number of TRAP-positive cells in the arthritic
joints found on day 28 (Fig. 8i). Evaluation by radiological
scores on day 28 confirmed that the injection of adv-FRNK significantly
reduced joint destruction compared with PBS and adv-LacZ injections
(Fig. 8j).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inflammatory cytokines such as TNF-, IL-1, and M-CSF are
involved in the progression of bone destruction (35, 36, 37).
However, the effect of these cytokines on the chemotaxis of pOCs
remains unclear. Our data demonstrated that VEGF and MCP-1 stimulated
the chemotaxis of Raw cells, whereas TNF-, IL-1, and M-CSF did
not. Therefore, we concluded that one of the characteristic biological
effects of VEGF on pOCs might be the stimulation of chemotaxis in pOCs.
Most signals for VEGF-induced cell proliferation of endothelial cells
are mediated by Flk-1 (38). In contrast, VEGF treatment
caused the phosphorylation of Flt-1 but not Flk-1 and subsequently
stimulated the cell proliferation of Raw cells as well as M-CSF,
TNF-, and IL-1, pointing to a new function of Flt-1 in
pOCs.
The kinase activity of FAK is negatively regulated by FRNK via
the dephosphorylation of the tyrosine residues of FAK. Adenoviral
expression of FRNK effectively inhibited VEGF-induced chemotaxis, cell
proliferation, and the tyrosine phosphorylation of FAK in Raw cells.
These results directly demonstrate that tyrosine phosphorylation of FAK
was required for the biological effects of VEGF in Raw cells.
Inflammatory cytokines such as TNF-, IL-1, and M-CSF did not
stimulate the tyrosine phosphorylation of FAK. Therefore, the results
suggest that VEGF specifically causes the tyrosine phosphorylation of
FAK via Flt-1 in Raw cells to occur.
It was reported that, in HUVECs, FAK plays an important role in
VEGF-induced antiapoptosis (39). Thus, it is reasonable to
suppose that the VEGF-Flt-1-FAK pathway may mediate an antiapoptotic
signal in Raw cells and that dephosphorylation of FAK by FRNK induces
apoptosis of the cells. We first infected Raw cells with adv-FRNK or
adv-LacZ (moi = 10) under serum-free conditions with or without
VEGF (50 ng/ml) for 24 h. Then cell viability was evaluated by a
trypan blue dye exclusion assay. However, cell viability was unchanged
by the adv-FRNK infection, and addition of VEGF in the medium did not
affect the results. We also conducted a DNA fragmentation assay and
confirmed that the adv-FRNK infection did not result in DNA
fragmentation (data not shown). As shown in Fig. 6, the MAPK pathway
may be involved in the VEGF signaling in Raw cells. Consistent with
these findings, FRNK effectively reduced VEGF-induced extracellular
signal-regulated kinase activation in the cells (data not shown). Taken
together, our results suggest that FRNK exerts its inhibitory effect on
cell proliferation by attenuating the MAPK activation but not
stimulating apoptosis of the cells.
Immunohistochemistry demonstrated that pY397 in FAK was found mainly in pOCs. Using adv-LacZ, we confirmed that the adenovirus vector can effectively transduce the gene at the site of bone destruction in AIA (data not shown), as previously reported (40). Adv-FRNK injection into the arthritic joints decreased the expression of pY397 in FAK as well as the number of OCs in the specimens, resulting in the reduction of bone destruction. These results suggest that the major targets of adv-FRNK might be pOCs and that adv-FRNK inhibited recruitment and proliferation of pOCs in the inflamed joints. Our present findings support the idea that the VEGF-Flt1-FAK pathway might be involved in the bone destruction associated with AIA. However, in the human monocytic cell line THP-1, integrin-dependent cell adhesion to extracellular matrices such as fibronectin causes a rapid tyrosine phosphorylation of FAK (41). The synovial pannus was rich in fibronectin, which could activate FAK in the monocyte/pOCs in the joint (42). Therefore, adv-FRNK may exert its effect by inhibiting the extracellular matrix-integrin-FAK signaling in pOCs, and further investigation is needed to show the direct link between VEGF and FAK in the arthritic joints.
In conclusion, we indicate a linear signal transduction pathway
of VEGF in pOCs in arthritic joints; VEGF ligates to its receptor
Flt-1, resulting in the tyrosine phosphorylation of FAK, subsequently
inducing chemotaxis and cell proliferation as summarized in Fig. 9.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yukihide Iwamoto, Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582. E-mail address: yiwamoto{at}ortho.med.kyushu-u.ac.jp
3 Abbreviations used in this paper: VEGF, vascular endothelial growth factor; OC, osteoclast; pOC, OC precursor cell; MCP-1, monocyte chemoattractant protein-1; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; moi, multiplicity of infection; AIA, adjuvant-induced arthritis; FAK, focal adhesion kinase; RANK, receptor activator of NF-B; FRNK, FAK-related nonkinase; BrdU, 5-bromo-2'-deoxyuridine; TRITC, tetramethylrhodamine isothiocyanate; TRAP, tartrate-resistant acid phosphatase; Pyk2, proline-rich tyrosine kinase 2.
Received for publication December 26, 2001. Accepted for publication March 20, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced motility and invasion of human osteosarcoma cells: possible involvement of NFB activation. Clin. Exp. Metastasis 18:121.[Medline]
B in osteoclast precursors. Endocrinology 142:1290.v3 integrin, and phosphorylated by src kinase. J. Clin. Invest. 102:881.[Medline]
, a novel phosphatidylinositol 3-kinase catalytic subunit that associates with p85 and is expressed predominantly in leukocytes. J. Biol. Chem. 272:19236. stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J. Exp. Med. 191:275. through upregulation of osteoclast differentiation factor and cyclooxygenase-2. Biochem. Biophys. Res. Commun. 259:97.[Medline]
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