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INTRODUCTION |
Cell migration plays a key role in a wide variety of biological
phenomena (1, 2). There are three main types of cell migration:
chemokinesis, chemotaxis, and haptotaxis. Chemokinesis comprises
random, non-directional motility in response to a ligand without any
orienting cues. Chemotaxis is the cell movement toward a positive
gradient of soluble stimulants such as chemokines and growth factors.
Haptotaxis involves cell crawling toward substrate-bound molecules such
as various extracellular matrix proteins. Cell migration is the result
of a series of complicated, integrated processes and is controlled by
many kinds of intracellular molecules (1, 2). These molecules include
Rho small G protein family members,
PI3-kinases,1 MAP kinases
(Erk1 and Erk2), and protein kinase C.
Midkine (MK) was first identified as the product of a retinoic
acid-responsive gene in embryonal carcinoma cells (3, 4). MK and
pleiotrophin (PTN, also called HB-GAM for heparin-binding growth-associated molecule) comprise a family of heparin-binding growth/differentiation factors and are not related to other
heparin-binding growth factors such as fibroblast growth factor or
hepatocyte growth factor (5, 6, 7). MK has been reported to promote neuronal survival and neurite outgrowth (8, 9) and to play roles in
carcinogenesis (10, 11) and tissue remodeling (12, 13).
Using MK knock-out mice, it was demonstrated that MK is involved in
neointima formation in a model of restenosis after angioplasty (14).
Neointima is the basic lesion in both atherosclerosis and restenosis
after angioplasty (15). A variety of stresses to the arterial
endothelium can induce the migration of smooth muscle cells from the
media into the space between the endothelium and internal elastic
lamina to form a neointima. One of the most important molecules in this
process is PDGF-BB, which is responsible for the migration of smooth
muscle cells (16). Macrophages recruited into the arterial wall also
play a critical role in this lesion formation (17). Neointima formation
and macrophage recruitment to the arterial wall were suppressed in
MK-deficient mice (14). Because MK induces the migration of both smooth
muscle cells and macrophages in vitro (14), it was concluded
that the cell migration-inducing activity of MK is crucial for the
suppression of neointima formation.
These findings also suggest a possible interaction between MK and PDGF
in smooth muscle cell migration. In addition, remodeling after bone
fractures also supports the interaction of MK and PDGF, because MK
expression and PDGF accumulation are induced during this process (13).
PTN/HB-GAM promotes the migration of osteoblast-like cells, including
UMR106 cells, which provides further evidence (18).
MK and PTN/HB-GAM induce the migration of cortical neurons (19, 20). MK
also induces the migration of neutrophils (21). However, only a limited
body of information concerning the signaling involved in MK-mediated
cell migration is available. We conducted the present study to
elucidate the molecular components essential for MK-mediated cell
migration, and to test our hypothesis that MK and PDGF could cooperate
in cell migration.
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EXPERIMENTAL PROCEDURES |
Cell Line, Reagents, and Antibodies--
A rat osteoblast-like
cell line, UMR106 (ATCC No. CRL 1661), was purchased from the American
Type Culture Collection. Heparin, chondroitin sulfate A, C, D, and E,
chondroitinase ABC, AC II, and B, and heparitinase were purchased from
Seikagaku, Japan. Dermatan sulfate, phosphatidylinositol, and
phosphatidylinositol 4-monophosphate were obtained from Sigma. The
inhibitors for Src (PP1), protein kinase C (Ro318220), and a MAP kinase
kinase, MEK (PD98059), were from Alexis Biochemicals,
Calbiochem-Novabiochem and Biomol Research Laboratories, respectively.
The other inhibitors for protein kinase C (H7 and calphostin C),
PI3-kinase (wortmannin), and phospholipase C (U-73122) were products of
Sigma. Recombinant human PDGF-BB was obtained from PeproTech, England.
The procedure for producing recombinant human MK with yeast has already
been described (14), and in this paper MK means human MK produced by
yeast unless specified otherwise. Recombinant mouse MK was expressed
with baculovirus and purified as described previously (9). Chemically
synthesized human MK was purchased from the Peptide Institute, Japan.
The procedure for producing recombinant human PTN/HB-GAM with yeast was
the same as that for MK described previously (14). The polyclonal
anti-PI3-kinase (p85
) antibody and anti-phospho-AKT-(Ser-473)
antibody were purchased from Upstate Biotechnology and Cell Signaling
Technology, respectively. The rabbit anti-phosphorylated Erk antibody
was from New England BioLabs. The monoclonal anti-Erk2 antibody and
monoclonal anti-RPTP
(PTP
) antibody were purchased from
Transduction Laboratories. The rabbit polyclonal anti-PTP
antibody
(anti-6B4) was prepared as described previously (22).
Cell Migration Assay, PTP
Extraction, and Chondroitinase
Digestion--
The migration assay was performed as described
previously (14, 19, 20) using Chemotaxicell (Kurabo, Japan; 8-µm
pores). PTP
extraction and chondroitinase digestion were performed
as described previously (23).
RT-PCR--
Five µg of total RNA from 15-day-old rat brain or
cultured cells was used for reverse-transcription with
TrueScript (Sawady, Japan). The primers used for PCR were:
5'-GTTCTCAACACATCCCTGAATCCTACTTCCCA-3' and
5'-CTTTAGTGATTCTTCTGAACCTGATGGAGCCGA-3'. The 474-bp PCR product corresponded to the 1587-2061 fragment of rat PTP
(GenBankTM/EBI no. U09357), which was confirmed by DNA sequencing.
Western Blot Analysis--
Polystyrene beads (Polysciences) were
coated with MK (20 µg/ml) or PLL (5 µg/ml, plus 15 µg/ml bovine
serum albumin) at 4 °C overnight or room temperature for 2 h.
The beads were washed with phosphate-buffered saline four times before
use. UMR106 cells starved for 24 h were stimulated with the coated
polystyrene beads. Cells were lysed in a buffer comprising 10 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.5% Nonidet P-40,
150 mM NaCl, 1 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, 0.1 mg/ml aprotinin, 40 nM
leupeptin, and 0.2 mM sodium vanadate. Thirty µg of
protein were separated by 10% SDS-polyacrylamide gel electrophoresis
and then transferred to a nitrocellulose membrane, followed by
detection with anti-phosphorylated Erk, anti-Erk2, anti-phosphorylated
AKT, or anti-AKT antibody.
PI3-kinase Assay--
UMR106 cells were treated as described for
Western blot analysis of Erk phosphorylation. PI3-kinase activity was
measured by in vitro phosphorylation of
phosphatidylinositol, using essentially the same method as described
previously (24).
Immunofluorescence Microscopy--
UMR106 cells, which had been
exposed to MK- or PLL-beads for 15 min, were fixed with 4%
paraformaldehyde and then stained with anti-PI3-kinase (p85
)
antibody/fluorescein isothiocyanate-conjugated anti-rabbit IgG and/or
anti-RPTP
(PTP
) monoclonal antibody/horseradish peroxidase-conjugated anti-mouse IgG antibody/cyanine 5-labeled tyramide. For PTP
staining, the TSA system (PerkinElmer Life Sciences) was employed to enhance the signal.
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RESULTS |
MK Induces Haptotactic Migration of Osteoblast-type
Cells--
PTN/HB-GAM, another member of the MK family, induces
haptotactic migration of several osteoblast cell lines (18). We first investigated whether or not MK had the same activity. When coated on
the lower surface of a filter, MK induced the haptotactic migration of
UMR106 cells, with the maximum level at the concentration of 20 µg/ml
(Fig. 1A). The profile of
MK-induced cell migration was similar to that of the colon carcinoma
cell migration induced by several extracellular matrix proteins, such
as fibronectin, vitronectin, laminin-1, and collagen IV (25). As
chemically synthesized human MK and baculovirus-produced mouse MK
showed the same activity as that of yeast-produced human MK (Fig.
1B), the migratory effect was not caused by impurities in
the MK used. MK contains 30% basic amino acids. However, PLL showed
very weak migratory activity (Fig. 1B), suggesting that the
effect of MK was not because of its high basicity.

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Fig. 1.
MK induces haptotactic migration of UMR-106
cells. A, the migration assay was performed with
filters coated with MK on their lower surface at the indicated
concentrations. UMR106 cells (~1 × 105 cells in 100 µl of 0.3% bovine serum albumin/Dulbecco's modified Eagle's
medium) were added to the upper chamber of Chemotaxicell (a modified
Boyden chamber), followed by incubation for 4 h. Ten fields
at × 400 per filter were counted to obtain the migrated cell
number (1 field = 1/160 of entire surface of filter). The value
shown as the Migrated Cell Number is the mean ± S.E.
(n = 3) per field. A representative of three
independent experiments is shown. B, a filter coated on its
lower surface with PLL, yeast-produced human MK (y-hMK),
chemically synthesized human MK (c-hMK), or
baculovirus-produced mouse MK (mMK), at 20 µg/ml, was used
for the assay. MK was added to the lower chamber in a soluble form at
100 ng/ml (sol. MK). C, the effect of soluble MK
at the indicated concentrations on the migration of UMR106 cells
induced by coated MK on the filters was examined.
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Soluble MK did not induce UMR106 cell migration (Fig. 1B),
even if the filter was precoated with collagen I (data not shown). On
the contrary, when added to the lower chamber, soluble MK inhibited coated MK-induced migration (Fig. 1C). At the concentration
of 10 µg/ml, soluble MK completely abolished coated MK-induced
migration. The same concentration of soluble PLL in the lower chamber
did not inhibit MK-induced migration (data not shown). These findings indicate that the substratum-bound form of MK is active in cell migration. Thus, MK could induce haptotactic, but not chemotactic, migration of UMR106 cells.
Soluble MK and PTN Abrogate Haptotaxis Mediated by MK and
PTN/HB-GAM--
We next addressed the question of whether or not MK
and PTN/HB-GAM share the same cell surface binding site(s). Soluble MK abrogated PTN/HB-GAM-induced haptotaxis and vice versa (Fig.
2A). Coating with a
combination of MK and PTN/HB-GAM did not enhance the migration as
compared with MK or PTN/HB-GAM alone (Fig. 2B). These data
suggest that if the total molar concentration of MK and PTN/HB-GAM
reaches a critical level, the migration activity will be saturated, and
MK and PTN/HB-GAM may function through common molecule(s) on the cell
surface.

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Fig. 2.
Competition between MK and PTN for activating
cell migration. A, the lower surface of filters was
coated with MK (20 µg/ml) (MK-coat) or PTN/HB-GAM (10 µg/ml) (PTN-coat). The migration assay was performed with
medium containing MK (20 µg/ml) or PTN/HB-GAM (10 µg/ml) in the
lower chamber. The symbol 0 means no addition of these
factors. The essential procedure was the same as described in the
legend to Fig. 1. The mean number of migrated cells was ~70 for
0/MK-coat, and 120 for 0/PTN-coat, which corresponded to 10-20% of
the total applied cell number. B, the lower surface of
filters was coated with MK or PTN/HB-GAM alone, or together. MK, coated
with 20 µg/ml MK; PTN, 10 µg/ml PTN/HB-GAM; P-M10, 10 µg/ml MK
and 5 µg/ml PTN/HB-GAM; P-M20, 20 µg/ml MK and 5 µg/ml
PTN/HB-GAM. Values are the means ± S.E.; n = 3.
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Proteoglycan Is Involved in MK-induced Migration--
Heparan
sulfate proteoglycans, such as syndecan-1, -3, and -4, and a
chondroitin sulfate proteoglycan, PTP
, are supposed to act as a
receptor or co-receptor for MK (20, 26, 27, 28). We investigated the
effects of different glycosaminoglycans on MK-mediated migration. In
addition to heparin, dermatan sulfate (chondroitin sulfate B) and
chondroitin sulfate E showed comparable effects. They abolished the
migration at the concentration of 20 µg/ml (Fig.
3A). At 20 µg/ml,
chondroitin sulfate A, C, and D showed only minor effects (Fig.
3A). Treatment with heparitinase to remove the heparan
sulfate on the cell surface did not affect MK-mediated migration (Fig.
3B). On the contrary, chondroitinase ABC digestion and
chondroitinase B digestion down-regulated MK-mediated migration (Fig.
3, C and D), suggesting the involvement of cell surface chondroitin sulfate.

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Fig. 3.
Effect of glycosaminoglycans on MK-induced
cell migration. A, UMR106 cells were preincubated at
37 °C for 30 min with glycosaminoglycans at the concentration of 20 µg/ml or that indicated. The migration assay was carried out with the
same glycosaminoglycan concentration in the lower chamber as in
the upper chamber. CS means chondroitin sulfate. B and
C, UMR106 cells were preincubated at 37 °C with
heparitinase for 90 min (B), or chondroitinase ABC for 30 min (C). D, at 20 milliunits/ml, chondroitinase
ABC and B, but not AC II, inhibited the migration. Values are the
means ± S.E.; n = 3.
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Involvement of PTP
in MK-induced Migration--
As PTP
exhibits high affinity to both MK and PTN/HB-GAM (20, 29), the results
shown in Figs. 1, 2, and 3 suggest that PTP
is involved in
MK-induced haptotaxis. Using RT-PCR, a band corresponding to PTP
was
detected for UMR106 cells and rat brain (Fig.
4A). The PCR product was
confirmed by DNA sequencing (data not shown). No positive band was
detected for L cells or Wilms' tumor cells (Fig. 4A). This
is consistent with the observation that coated MK did not induce the
migration of these cells with a 4-h incubation (data not shown). On
Western blotting with monoclonal anti-RPTP
(PTP
) antibody, which
recognizes the intracellular domain of PTP
, a smear was detected for
the lysate of UMR106 cells (Fig. 4B, bracket,
lane 3). This changed to one band corresponding to about 240 kDa after chondroitinase ABC digestion (Fig. 4B, lane
4, arrow). For rat brain, smears were also detected
(Fig. 4B, lane 1), which shifted to 380 and 220 kDa upon digestion with chondroitinase ABC (Fig. 4B,
lane 2, arrowheads), which represent long- and
short-type receptors, respectively (23). The size difference around
220/240 kDa between the brain and UMR106 cells is probably caused by
differential glycosylation, which is dependent on the cell type (30).
Chondroitinase AC II and B appeared to digest the glycosaminoglycan
chains of PTP
only partially, because the band around 240 kDa was
broader than the band digested with chondroitinase ABC (Fig.
4B, lanes 8, 9, 10). This
is reasonable because chondroitinase AC II and B recognize different
structures (31, 32). Nevertheless, only chondroitinase ABC and B, but not AC II, suppressed MK-induced migration (Fig. 3D),
suggesting that structures susceptible to chondroitinase ABC and B are
important. As chondroitinase B can digest both dermatan sulfate and
E-type dermatan sulfate (32), and chondroitinase ABC can digest all chondroitin and dermatan sulfates (33), this is consistent with the
finding that MK-induced cell migration was inhibited by dermatan sulfate (chondroitin sulfate B) and chondroitin sulfate E (Fig. 3A).

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Fig. 4.
Possible role of PTP-
in MK-induced cell migration. A, 5 µg of total
RNA were used for RT-PCR. L cell, fibroblast cell line; G401 cell,
Wilms' tumor cell line. Upper panel, PTP ; lower
panel, GAPDH. B, Western blot analysis with
anti-RPTP (PTP ) antibody before and after chondrotinase
digestion. Arrowheads and an arrow indicate the
positive bands after chondroitinase ABC digestion. The
bracket indicates the smear detected for UMR106 cells. Note
the nonspecific bands for all lysates of UMR106 and L cells.
C, sodium vanadate (NaVa) inhibited MK-induced
migration of UMR106 cells. D, preincubation with anti-PTP
(anti-6B4) antibodies, but not control rabbit IgG, enhanced MK-induced
migration. Values are the means ± S.E.; n = 3.
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PTP
is also known to be a receptor-type protein-tyrosine
phosphatase. The effect of orthovanadate, an inhibitor of
protein-tyrosine phosphatase, was examined. Sodium vanadate decreased
the migration in a dose-dependent manner (Fig.
4C). Sodium vanadate had no effect on PLL or PDGF-BB-induced
migration of UMR106 cells (data not shown). If the cells were
preincubated with anti-PTP
(anti-6B4) antibodies, which recognize
the ectodomain of the full-length PTP
(23), MK-mediated cell
migration was enhanced (Fig. 4D). This suggests that
ligation of cell surface PTP
with its ligands or antibodies may
transduce signals essential for the migration of UMR106 cells.
MK Induces MAP Kinase and PI3-kinase Activity--
To identify the
intracellular molecules that participate in MK-mediated cell migration,
various specific inhibitors were screened. Tyrosine kinase inhibitors
(genistein and herbimycin A), a Src inhibitor (PP1), protein kinase C
inhibitors (H7, Ro318220, and calphostin C), a PI3-kinase inhibitor
(wortmannin), a MAP kinase kinase (MEK) inhibitor (PD98059), and a
phospholipase C inhibitor (U-73122) effectively inhibited MK-mediated
cell migration (Fig. 5). This probably
indicates that a dynamically concerted signaling interaction is
essential for the cell migration induced by MK.

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Fig. 5.
Effect of different inhibitors on MK-induced
cell migration. UMR106 cells were incubated with the indicated
inhibitors for 30 min. The migration assay was then performed with both
the upper and lower chambers containing the same concentration of the
same inhibitor. A, concentrations of inhibitors: tyrosine
kinase-herbimycin A (herb), 1 µg/ml; tyrosine
kinase-genistein (geni), 50 µg/ml; Src-PP1, 10 nM; protein kinase C-Ro318220 (Ro), 2 µM; PI 3 kinase-wortmannin (wort), 100 nM; MEK-PD98059 (PD), 20 µM;
phospholipase C-U73122 (U-7), 10 µM.
B and C, dose dependence of protein kinase C
inhibitors (B) and MEK, PI3 kinase, and phospholipase C
inhibitors (C). The units are: PD98059, µM;
wortmannin, nM; U73122, µM. Values are the
means ± S.E.; n = 3.
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When UMR106 cells were stimulated with MK-coated polystrene beads, a
transient increase in phosphorylation of MAP kinases (Erk1 and Erk2)
was detected, the maximum level being observed ~20 min after
stimulation (Fig. 6A). At 60 min, it was still higher than the basal level (Fig. 6A). PLL
also enhanced Erk phosphorylation, but the increase was less and the
duration was shorter than in the case of MK (Fig. 6A).
Interestingly, the PI3-kinase inhibitor wortmannin partly blocked
MK-induced Erk phosphorylation, whereas the protein kinase C inhibitor
Ro318220 did not (Fig. 6B). MK also increased PI3-kinase
activity, the profile being similar to that in the case of PDGF-BB
(Fig. 7A). Neither Ro318220
nor MEK inhibitor PD98059 affected MK-induced PI3-kinase activation (Fig. 7B).

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Fig. 6.
Erk activation after MK stimulation.
UMR106 cells stimulated with polystyrene beads coated with PLL (5 µg/ml) or MK (20 µg/ml) for the indicated times were analyzed by
Western blotting with anti-phosphorylated Erk (P-Erk1/2) and
anti-Erk2 (Erk2) antibodies. The densitometric data obtained
on Western blotting are shown in the graph. In
B, inhibitors were used as described in the
legend to Fig. 5.
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Fig. 7.
PI3-kinase activity after MK and PDGF-BB
stimulation. UMR106 cells were stimulated with PDGF-BB, or
polystyrene beads coated with PLL or MK. PI 3-kinase activity was
measured as described under "Experimental Procedures."
A, representative autoradiography. Lower panel,
quantitative presentation (mean ± S.E.; n = 3).
B, effects of inhibitors.
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MK Recruits PTP
and PI3-kinase--
To further confirm
MK-induced PI3-kinase activation, phosphorylation of AKT was examined.
AKT (protein kinase B) plays important roles in many biological
phenomena, such as cell survival (34, 35). AKT contains a pleckstrin
homology (PH) domain, and is phosphorylated (activated) at Thr-308 and
Ser-473 residues by PDK1, which also carries a PH domain. The PH domain
recognizes a phosphoinositide headgroup, and phosphorylation at the 3 position of inositol ring of phosphatidylinositol in the cell membrane is critical for AKT- and PDK1-binding and recruitment to the cell membrane (35). As this phosphorylation is mediated by PI3-kinase, AKT
is an important PI3-kinase effector, and its activation is often used
as a marker of PI3-kinase activation (34, 35).
MK enhanced AKT phosphorylation (Fig.
8A, left), which is
consistent with the data for MK-induced PI3-kinase activation shown in
Fig. 7A. When the cells were preincubated with anti-PTP
(anti-6B4) antibodies, MK-induced AKT activation was stronger than that
of the cells treated with control IgG (Fig. 8A,
right). This was consistent with that anti-PTP
(anti-6B4)
antibodies enhanced MK-induced migration of UMR106 cells (Fig.
4D). It is of interest that, in addition to the PI3-kinase
inhibitor wortmannin, the protein-tyrosine phosphatase inhibitor
orthovanadate and Src inhibitor PP1 blocked MK-induced AKT activation
(Fig. 8A, right), but neither protein kinase C inhibitor Ro
318220 nor MEK inhibitor PD98059 affected it (data not shown). As the
effect of orthovanadate on MK-induced AKT phosphorylation suggested the
possibility of a close connection between PTP
and PI3-kinase, we
localized PTP
and PI 3-kinase after MK beads stimulation on UMR106
cells. MK beads induced PI3-kinase recruitment to the sites of the
beads (Fig. 8B, left two panels). Furthermore, MK-beads
induced colocalization of PI3-kinase and PTP
(Fig. 8B, lower
two panels).

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Fig. 8.
Involvement of PI3-kinase and
PTP in MK signaling. A, left;
UMR106 cells were exposed to MK- or PLL-beads at
37 °C for 15 min. Cell extracts were then analyzed for
phosphorylated AKT (Ser-473) (p-Akt) or AKT expression by
Western blotting. The densitometric ratio (p-Akt versus Akt)
was calculated for each condition, and the relative density of p-Akt is
shown at the bottom. A, right; UMR106 cells were
incubated with either anti-PTP (anti-6B4) antibodies or
control rabbit IgG together with the indicated inhibitor at 4 °C for
2 h. The cells were then exposed to MK-beads in the presence of
indicated inhibitor at 37 °C for 15 min. B, UMR106 cells
were exposed to MK- or PLL-beads at 37 °C for 15 min. The
localization of PI3-kinase and PTP was examined by
immunofluorescence microscopy (PI3-kinase, FITC; PTP , cyanine 5)
using a confocal microscope (MRC-1024, Bio-Rad). The lower two
panels show the same cell that was double-stained with
anti-PI3-kinase and anti-RPTP (PTP ) antibodies. Arrows
and asterisks indicate cells examined and beads,
respectively.
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MK Has a Synergistic Effect with PDGF-BB on Cell
Migration--
The hypothesis that MK and PDGF can cooperate in cell
migration in some in vivo situations was examined next. On
filters coated on both their upper and lower surfaces with collagen I,
PDGF-BB induced chemotactic migration of UMR106 cells (Fig.
9A). PDGF-induced migration
was suppressed by Ro318220 and wortmannin (Fig. 9A). But
PD98059, which inhibited MK-mediated cell migration (Fig. 5), did not
affect PDGF-mediated migration (Fig. 9A). Whereas heparin
abolished MK-mediated cell migration (Fig. 3A), it rather enhanced PDGF-mediated cell migration (Fig. 9A). If the
lower surface of the filters was coated with MK instead of collagen I,
the chemotactic activity of PDGF-BB was dramatically enhanced (Fig.
9B). The effects of MK and PDGF-BB were synergistic, because the migrated cell number was much higher than the total when MK and
PDGF-BB were used separately. The migration induced by the combination
of MK and PDGF-BB was susceptible to inhibitors of PI 3-kinase and
protein kinase C, but not to the MEK inhibitor (Fig. 9C).
This profile resembles that of the migration induced by PDGF-BB alone,
but not that in the case of MK alone.

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Fig. 9.
Synergistic effect of MK and PDGF-BB on cell
migration. A, PDGF-BB-induced migration was inhibited
by protein kinase C and PI3-kinase inhibitors. PDGF-BB (20 ng/ml) was
added to the lower chamber. Concentrations of reagents: PD98059, 20 µM; Ro318220, 2 µM; wortmannin, 100 nM; heparin, 20 µg/ml. B, MK and PDGF-BB
showed synergistic effect to induce UMR106 cell migration. The filter
of Chemotaxicell was coated with collagen I on both its upper and lower
surfaces or with MK on only its lower surface. Migration was then
monitored in the presence or absence of PDGF-BB in the lower chamber.
C, MK/PDGF-BB-mediated migration was not affected by MEK
inhibitor. PD98059, Ro318220, and wortmannin were used as described in
A.
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DISCUSSION |
MK has been reported to be induced in areas of a variety of types
of tissue injury, such as cerebral and heart infarction, bone
fractures, skin burns, and arterial endothelial injury (12, 13, 14, 36,
37). In the case of arterial endothelial injury, we found not only the
induction of MK expression in wild-type mice, but also dramatically
suppressed neointima formation in MK-deficient mice (14). The
administration of the MK protein to MK-deficient mice caused resumption
of neointima formation (14). Thus, MK seems to be vital for tissue
remodeling. The present study revealed another important aspect of the
mode of MK action, namely, that it acts synergistically with PDGF in
cell migration. PDGF was first purified from platelet
granules and has several important activities, such as mitogenesis and chemotaxis. Because tissue injury is usually accompanied by bleeding and/or cessation of blood flow, an abundance of PDGF can be found in these
areas. PDGF expression is also induced during the process of wound
healing (38). Furthermore, PDGF induces the migration of smooth muscle
cells (16, 39) and osteoblasts (40), and the migration of both is also
induced by MK (Ref. 14 and the present study). Taken together, the
synergism between MK and PDGF appears to be pivotal in many in
vivo situations.
Several possibilities should be considered for the mechanism underlying
the synergism between MK and PDGF. The present study revealed a
difference in the signaling mechanism between MK and PDGF. Heparin
inhibited MK-induced cell migration but rather enhanced PDGF-induced
cell migration (Figs. 3A and 9A). MAP kinases
were essential for MK-mediated cell migration but not for PDGF-induced cell migration (Figs. 5 and 9). Orthovanadate inhibited MK-induced cell
migration but not PDGF-induced cell migration (Fig. 4C and data not shown). Because distinct signaling molecules are used for cell
migration, depending on the ligand (41, 42), the synergism between MK
and PDGF might be attributed to the integration of distinct signaling
pathways. Alternatively, induction of MK receptor(s) by PDGF or vice
versa should also be considered, as in the case of the induction of the
Interleukin-1 receptor or PDGF receptor by the neuropeptide substance P
in their synergism in bone marrow fibroblast proliferation (43). In
addition, a third cell surface molecule, such as integrin
1, might be involved in the synergism, like in the case
of the synergism between lysophosphatidic acid and epidermal growth
factor or PDGF in cell migration (44).
MAP kinases (Erk1 and Erk2) can activate myosin light chain kinase and
induce changes in the cytoskeletal structure, leading to cell migration
(45). In this context, it is noteworthy that MK enhances collagen gel
contraction by dermal fibroblasts (46). MK-induced MAP kinase
activation appeared to be at least partly regulated by PI3-kinase (Fig.
6B). PI3-kinase functions as an early intermediate in
G
-mediated MAP kinase activation (47). Several papers have
reported that PI3-kinase acts upstream of MAP kinase, e.g.
in insulin signaling in 3T3-L1 adipocytes and PDGF signaling in Swiss
3T3 cells (48, 49).
The present study demonstrated that MK recruited PTP
and PI3-kinase
(Fig. 8B). Furthermore, MK-induced PI3-kinase activation was
inhibited by the Src inhibitor PP1 and protein-tyrosine phosphatase inhibitor orthovanadate (Fig. 8A). Src activation is
sometimes needed for PI3-kinase activation (50) and requires
dephosphorylation at its C-terminal phosphotyrosine, which can be
mediated by PTP
(51). Taken together, the present results suggest a
possible MK signaling cascade, that is, MK binds and activates PTP
,
which then activates Src and PI3-kinase and further activates MAP
kinases. However, details of the precise mechanism underlying the
interaction between these molecules remain to be elucidated, and other
unidentified important molecules may be involved in MK signaling.
With regard to the sugar structure essential for the binding, the MK
and heparin interaction needs all the three sulfate groups in the
heparin disaccharide unit (2-O-, N-, and
6-O-sulfation) (52). Dextran sulfate, which has 1.5 sulfate
residues per sugar residue, strongly inhibits MK-sulfatide binding
(53). In the case of MK binding to PG-M/versican, a matrix chondroitin
sulfate proteoglycan, disulfated disaccharides were identified (54). Furthermore, chondroitin sulfate E specifically inhibits
MK-dependent neuronal cell adhesion (55). PTP
harbors
chondroitin sulfate chain, which is important in MK-induced migration
of neurons (20). The present study revealed that the chondroitin
sulfate chain in PTP
is also important in migration of
osteoblast-like cells. Differential susceptibility of the migratory
activity to chondroitinases with different specificities gave further
insights into the nature of chondroitin sulfate chain in PTP
.
Digestion with chondroitinase B abolished MK-dependent
migratory activity, whereas chondroitinase AC II did not. The former
enzyme acts on chondroitin sulfate chain with the
L-iduronic acid residue, namely dermatan sulfate, whereas the latter does not. Thus, it is concluded that chondroitin sulfate, which is important in MK-signaling in PTP
, has a dermatan sulfate domain. The finding that the MK-induced migration is inhibited by
dermatan sulfate is consistent with the view. The MK activity was also
inhibited by chondroitin sulfate E, which is an oversulfated chondroitin sulfate with 4,6-disulfo-N-acetylgalactosamine
residue. Taken together, most probably the chondroitin sulfate chain in PTP
, to which MK binds, has an oversulfated structure in a dermatan sulfate domain. Indeed, E-type structure with a dermatan sulfate domain
was found in PG-M/versican, which was isolated from mouse embryos and
has MK binding activity (54).
The characteristics of MK-induced migration of UMR106 cells are very
similar to those of MK- and PTN/HB-GAM-induced neuronal migration in
that PTP
is involved in haptotactic migration (19, 20). In this
context, the effect of anti-PTP
antibodies on MK-mediated cell
migration was unexpected. These antibodies effectively inhibited
PTN/HB-GAM-mediated neuronal migration in the previous study, probably
because of competitive inhibition for the PTN/HB-GAM-binding sites of
cell surface PTP
by the antibodies (20). But in the present study,
the antibodies rather enhanced MK-mediated osteoblast-like cell
migration. One possible interpretation of this is that PTP
on UMR106
cells may physically associate with another unidentified component
necessary for signal transduction and thus can be readily activated by
the oligomerization or conformational change induced by a specific
antibody. On nerve cells, PTP
might need MK to associate with such a
component. Supporting our data, Revest et al. (56) recently
reported that cross-linking of PTP
with antibodies enhances the
protein-tyrosine phosphatase activity of C6 astrocytoma cells.
In this study, we confirmed the involvement of Erk1 and 2 and
PI3-kinase in MK-induced cell migration by detecting their active forms
or activity induced by MK, in addition to by demonstrating the effects
of inhibitors of them. Consistent with our data, Souttou et
al. reported that Erk1 and 2 and PI3-kinase are involved in PTN/HB-GAM-mediated cell proliferation (57). In addition, Src, JAK1,
and 2 and
-catenin have been reported to be involved in PTN/HB-GAM
signaling (58, 59, 60). A study on the possible involvement of these
molecules in MK-induced cell migration is underway in our laboratory.