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INTRODUCTION |
Platelet-derived growth factor
(PDGF)1, a mitogen for cells
of mesenchymal origin, is a homo- or heterodimeric protein composed of
disulfide-bonded A- and B-polypeptide chains (see Ref. 1 for a review).
PDGF has been shown to promote chemotaxis of fibroblasts, smooth muscle
cells, and phagocytic cells (2, 3) and to cause reorganization of actin
filaments in the cell (4). The effect of PDGF is exerted by binding to
two structurally similar protein-tyrosine kinase receptors denoted
-
and
-receptors (see Ref. 5 for a review). Ligand binding induces
receptor dimerization and autophosphorylation on specific tyrosine
residues. Src homology 2 (SH2) domain-containing proteins bind in a
specific manner to tyrosine-phosphorylated regions of PDGF receptors
and mediate a number of PDGF-induced intracellular signaling events.
There are nine autophosphorylation sites in the PDGF
-receptor,
which bind to Src family kinases (Tyr-579 and Tyr-581), Grb2 (Tyr-716), the regulatory subunit (p85) of phosphatidylinositol 3-kinase (PI
3-kinase) (Tyr-740 and Tyr-751), Nck (Tyr-751), the GTPase-activating protein of Ras (RasGAP) (Tyr-771), the SH2 domain-containing
phosphatase SHP-2 (Tyr-1009), and phospholipase C-
(PLC-
)
(Tyr-1021) (5). In the PDGF
-receptor, tyrosine residues 754, 762, 768, 988, and 1018 have been identified as autophosphorylation sites
(6-8). Among them, Tyr-762 and Tyr-1018 have been shown to mediate the association of Crk (9) and PLC-
(8), respectively. Tyr-731 and
Tyr-742 are important for the binding of PI 3-kinase (10) and are
likely to be phosphorylation sites, although this has not been directly shown.
Members of the mitogen-activated protein (MAP) kinase family,
extracellular signal-regulated kinase (ERK), stress-activated protein
kinase-1/c-Jun NH2-terminal kinase (SAPK1/JNK), and
stress-activated protein kinase-2 (p38), are central elements that
transduce the signal generated by growth factors, cytokines, and
stressing agents (11-13). It is well known that PDGF activates ERK in
various types of cells expressing PDGF receptors, which leads to the
mitogenic response of the cells. On the other hand, the role of the
other MAP kinases in mediating the cellular function of PDGF remains unclear.
In the present study, we have investigated the functional role of the
other MAP kinases, SAPK1/JNK and p38, in PDGF-mediated cellular
responses. We report that PDGF activates p38 but not SAPK1/JNK.
Furthermore, we show experimental evidence suggesting that PDGF-induced
activation of p38 is mediated through a Ras-dependent pathway and is required for PDGF-induced cell migration as well as
actin reorganization. We conclude that p38 is an important mediator of
the cell motility responses elicited by PDGF.
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EXPERIMENTAL PROCEDURES |
Reagents--
Recombinant human PDGF-BB was purchased from R & D
Systems (Minneapolis, MN). SB203580, a specific inhibitor of p38 MAP
kinase (14), and SB202474, a negative control compound for p38 MAP kinase inhibition studies, were from Calbiochem.
[methyl-3H]Thymidine was from Amersham
Pharmacia Biotech. PD098059, a specific inhibitor of MEK1, was from New
England Biolabs (Beverly, MA). The cDNA encoding Myc-N17Rac1 (a
dominant-negative Rac mutant) (15) in the mammalian expression vector
pEFBOS was provided by Y. Takai (Osaka University School of Medicine,
Japan). The adenoviral vector (AdexCAHRasY57) expressing a
dominant-negative mutant of c-Ha-Ras in which tyrosine replaces
aspartic acid at residue 57 was described previously (16).
Cell Culture, Transfection, and Gene Transfer--
Porcine
aortic endothelial (PAE) cells expressing the wild-type or tyrosine
residue-mutated PDGF
- or
-receptors have been described
previously (4, 17-19). The cells were cultured in Ham's F-12 medium
supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and
40 µg/ml gentamicin. Human skin fibroblasts TIG103 were obtained from
the Tokyo Metropolitan Institute of Gerontology and grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and gentamicin.
Transient transfection of cultured cells with the expression vector
pEFBOS-N17rac1 was performed using Effectene transfection reagent (Qiagen). The cells were used for experiments 48 h after transfection. In vitro gene transfer of rasY57
into cultured cells was carried out by incubation with AdexCAHRasY57 at
100 multiplicity of infection in F-12 medium for 2 h at 37 °C
under gentle agitation. The cells were washed twice with
phosphate-buffered saline, and virus-containing medium was replaced
with fresh medium supplemented with 10% fetal bovine serum. The cells
were used for experiments 48 h after the transfer.
Assays for ERK, SAPK1/JNK, and p38--
The activity of ERK,
SAPK1/JNK, and p38 was measured using a p44/42 MAP kinase assay kit, a
SAPK1/JNK assay kit, and a p38 MAP kinase assay kit, respectively (New
England Biolabs) according to the manufacturer's instructions. Cells
in a 100-mm Petri dish were serum-starved for 20 h in F-12 medium
supplemented with 1 mg/ml bovine serum albumin. Then the cells were
treated with the indicated concentrations of PDGF-BB at 37 °C for
0-120 min or with 10 µg/ml anisomycin (Sigma) for 10 min, rinsed
once with ice-cold phosphate-buffered saline, and lysed. For
immunoprecipitations, 200 µl of the lysates were incubated with p38
MAP kinase antibody (1:50 dilution) or with phospho-p44/42 MAP kinase
antibody (1:100 dilution) overnight at 4 °C, followed by incubation
with Protein A-Sepharose 6MB (Pharmacia) for 1 h at 4 °C. For
precipitation of SAPK1/JNK, the lysates were incubated with a c-Jun
fusion protein as a substrate linked to Sepharose beads overnight at
4 °C. For kinase assays, the beads were incubated at 30 °C for 30 min with 200 µM ATP in the presence of an ATF-2 fusion
protein as a substrate for p38 or an Elk-1 fusion protein as a
substrate for ERK. The reaction was terminated by adding 25 µl of
SDS-sample buffer. The samples were boiled for 5 min and subjected to
SDS-polyacrylamide gel electrophoresis. For immunoblotting, the samples
were electrically transferred from the acrylamide gel onto a
nitrocellulose filter (Hybond-ECL, Amersham) followed by blocking in
phosphate-buffered saline containing 3% bovine serum albumin and 0.1%
Tween 20. The blots were incubated with phospho-ATF-2 antibody (1:1000
dilution), phospho-Elk1 antibody (1:1000 dilution), or phospho-c-Jun
antibody (1:1000 dilution) overnight at 4 °C. The blots were washed
and then incubated with horseradish peroxidase-conjugated sheep
anti-rabbit immunoglobulins (1:2000 dilution) for 1 h at room
temperature. After washing, the sites of antibody binding were detected
with an enhanced chemiluminescence system (Amersham).
Assays for Cell Motility Responses--
A chemotactic response
was assayed essentially as described (6) using a 96-well
microchemotaxis chamber (Neuro Probe, Gaithersburg, MD). A
polycarbonate membrane (polyvinylpyrrolidone-free, pore size, 8.0 µm)
was coated with 100 µg/ml type I collagen (Vitrogen 100, Collagen
Biomaterials, Palo Alto, CA) before use. Actin reorganization and
membrane ruffling were analyzed essentially as described by Mellström et al. (20). Experiments were performed in
triplicate and were repeated at least three times.
[3H]Thymidine Incorporation Assay--
The ability
of PDGF-BB to stimulate DNA synthesis, measured by the incorporation of
[3H]thymidine into trichloroacetic acid precipitable
materials of cultured cells, was determined as described previously
(21).
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RESULTS |
PDGF-BB Activates p38 MAP Kinase Pathway but Not SAPK1/JNK
Pathway--
We first examined whether PDGF-BB activates three members
of the MAP kinase family, ERK, SAPK1/JNK, and p38 in PAE cells
expressing the wild-type PDGF
- or
-receptors. The cells were
serum-starved and stimulated with PDGF-BB. After stimulation, ERK and
p38 were collected by immunoprecipitation, and SAPK1/JNK was collected by adsorption to the c-Jun fusion protein and subjected to an in
vitro kinase assay in the presence of the respective substrates, Elk-1 for ERK, ATF-2 for p38, and c-Jun for SAPK1/JNK. Phosphorylation of the substrates was detected by immunoblotting with the specific antibodies against the phosphorylated substrates. As shown in Fig.
1, PDGF-BB stimulation increased
phosphorylation of Elk-1 (upper panel) and ATF-2
(lower panel) in both the cells expressing the wild-type
- and
-receptors. PDGF-BB-induced activation of ERK, and p38 was
clearly detected at 10 min after stimulation. ERK activity but not p38
activity still remained elevated 120 min after stimulation. In
contrast, PDGF-BB stimulation failed to phosphorylate c-Jun
(middle panel) in either the wild-type
- or
-receptor-expressing cells. These data clearly indicate that PDGF-BB
stimulation of the PDGF
- as well as
-receptors leads to the
activation of both ERK and p38 MAP kinase pathways but not the
SAPK1/JNK pathway. It was also found that SAPK1/JNK and p38 were
strongly activated by treatment with anisomycin (Fig. 1,
middle and lower panels), in agreement with
previous reports (22, 23) that anisomycin activates two MAP kinase
subtypes associated with the stress response but not ERK. Fig.
2 shows the time course of p38 activation
by PDGF-BB in PAE cells expressing the wild-type PDGF
- or
-receptors. PDGF-BB-induced activation of p38 turned out to be
transient; it was maximal at 10 min after stimulation and declined to
basal level by 30 min in both the
- and
-receptor-expressing
cells. The dose response of PDGF-BB-induced activation of p38 was shown
in Fig. 3. The maximal activation of p38
was achieved at the concentrations around 20-50 ng/ml PDGF-BB in both
the
- and
-receptor-expressing cells.

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Fig. 1.
PDGF-BB activates ERK and p38 but not
SAPK1/JNK in PAE cells expressing the wild-type PDGF
- or -receptors. The
cells expressing the wild-type -receptor (WT ) or
-receptor (WT ) were serum-starved for 20 h and
treated or untreated (C) with 50 ng/ml PDGF-BB at 37 °C
for 10 or 120 min or with 10 µg/ml anisomycin (An) for 10 min at 37 °C. After treatment, ERK and p38 were collected by
immunoprecipitation, and SAPK1/JNK was collected by adsorption to the
c-Jun fusion protein and subjected to an in vitro kinase
assay in the presence of the respective substrates, Elk-1 for ERK,
ATF-2 for p38, and c-Jun for SAPK1/JNK. Then the samples were separated
by SDS-polyacrylamide gel electrophoresis and transferred onto
nitrocellulose filters, and phosphorylation of the substrates was
detected by immunoblotting with the specific antibodies against the
phosphorylated substrates.
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Fig. 2.
Time course of PDGF-BB-induced p38 activation
in PAE cells expressing the wild-type PDGF -
or -receptors. The cells expressing the
wild-type -receptor (WT ) or -receptor
(WT ) were serum-starved for 20 h and treated with 50 ng/ml PDGF-BB for 0-120 min at 37 °C. After treatment, the cells
were lysed, and p38 activity of the cell lysates was determined as
described in the legend to Fig. 1. Data are expressed as relative
intensity of the phosphorylated ATF-2 bands, which was measured by
densitometric scanning of the fluorogram.
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Fig. 3.
Dose response of PDGF-BB-induced p38
activation in PAE cells expressing the wild-type PDGF
- or -receptors. The
cells expressing the wild-type -receptor (WT ) or
-receptor (WT ) were serum-starved for 20 h and
treated with the indicated concentrations of PDGF-BB for 10 min at
37 °C. After treatment, p38 activity was measured and is expressed
as described in the legend to Fig. 2.
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We next asked whether PDGF-BB induces activation of p38 in other cell
types. Human skin fibroblasts TIG103 were examined. As shown in Fig.
4, PDGF-BB activated p38 in TIG103
fibroblasts from 5 to 15 min after stimulation. The data indicate that
PDGF-BB-induced activation of p38 occurs not only in an artificial
condition where human PDGF receptors are expressed in the transfected
PAE cells but also in a natural condition where fibroblasts express
endogeneous PDGF receptors.

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Fig. 4.
Activation of p38 by PDGF-BB in TIG103
fibroblasts. Human skin fibroblasts TIG103 were serum-starved for
20 h and treated with 50 ng/ml PDGF-BB for 0-60 min at 37 °C.
After treatment, the cells were lysed, and p38 activity of the cell
lysates was determined as described in the legend to Fig. 1.
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SB203580 Inhibits PDGF-BB-induced Chemotaxis in a
Dose-dependent Manner--
It is well established that
PDGF modulates cell motility. To evaluate the role of p38 in
PDGF-induced cell migration, we examined the effect of SB203580, a
specific inhibitor of p38 MAP kinase, on PDGF-BB-induced chemotaxis
using a modified Boyden chamber method in PAE cells expressing the
wild-type PDGF
-receptor. PDGF-BB-induced chemotaxis reached the
maximal level at 20 ng/ml, a 108-fold increase as compared with control
(data not shown); therefore, the concentration of PDGF-BB was kept
constant at 20 ng/ml throughout the assays. As shown in Fig.
5A, treatment of the cells
with SB203580 resulted in a dose-dependent inhibition of
chemotaxis of PDGF-BB-stimulated cells, with a 27.1% reduction at 1 µM SB203580 and a 69.5% reduction at 10 µM
(IC50, 6.0 µM). On the other hand, PD098059,
a specific inhibitor of MEK1, did not affect PDGF-BB-induced chemotaxis
at concentrations of up to 50 µM (Fig. 5B). It
was also found that SB202474, a negative control of SB203580, did not
affect PDGF-BB-induced chemotaxis at concentrations of up to 20 µM as expected (data not shown). The data suggest that
activation of p38 but not ERK is required for PDGF-BB-induced cell
migration.

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Fig. 5.
Dose-dependent inhibition of
PDGF-BB-induced chemotaxis by SB203580 in PAE cells expressing the
wild-type PDGF -receptor. The cells that
were plated on a polycarbonate membrane (pore size, 8.0 µm) on a
96-well microchemotaxis chamber were pretreated for 15 min with the
indicated concentrations of SB203580 (A) or PD098059
(B) added to both the upper and lower chambers. The cells
were then stimulated with 20 ng/ml PDGF-BB added to the lower chambers
for 4 h at 37 °C. The membrane was then stained, and the number
of cells that migrated to the lower surface of the membrane was
counted. Data represent the percentage of cell numbers relative to
those in the absence of inhibitors and are expressed as mean ± S.E.
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SB203580 Influences PDGF-BB-induced Actin Reorganization--
An
essential feature of the chemotactic response in the cytoskeleton level
is the actin reorganization, such as the formation of lamellipodia or
membrane ruffling, extending forward the direction of movement (24). It
has been reported that PDGF stimulation of PAE cells expressing the
wild-type PDGF
-receptor leads to an induction of membrane ruffling
and a reduction of actin stress fibers (4). Therefore, next we examined
whether SB203580 affects PDGF-BB-induced actin reorganization at the
concentration that inhibits cell migration. The wild-type PDGF
-receptor-expressing cells were stimulated with 20 ng/ml PDGF-BB and
fixed, and actin filaments were stained using tetramethylrhodamine
isothiocyanate-labeled phalloidin. As shown in Fig.
6B, PDGF-BB stimulation
induced the appearance of membrane ruffling and the disappearance of
actin stress fibers. In contrast, these phenomena were not observed when the cells were pretreated with 10 µM SB203580 (Fig.
6C). Quantitative analyses of the data are shown in Fig.
7. The percentage of membrane
ruffling-forming cells to total cells was significantly (p < 0.002) reduced, whereas the percentage of stress
fiber-forming cells to total cells was significantly (p < 0.026) increased by treatment with SB203580. Both pretreatments with
10 µM SB202474 (Figs. 6D and 7) and with 20 µM PD098059 (data not shown) did not affect
PDGF-BB-induced actin reorganization as expected. These data suggest
that activation of p38 is required for PDGF-BB-induced actin
reorganization.

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Fig. 6.
Effect of SB203580 on PDGF-BB-induced actin
reorganization in PAE cells expressing the wild-type PDGF
-receptor. The cells were pretreated for
1 h with 10 µM SB203580 (C) or with 10 µM SB202474 (D) as a negative control. Then
the cells were stimulated (B, C, D) or not (A)
with 20 ng/ml PDGF-BB for 15 min at 37 °C. After stimulation, the
cells were fixed, and actin filaments were stained using
tetramethylrhodamine isothiocyanate-labeled phalloidin.
Arrows indicate membrane ruffling.
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Fig. 7.
SB203580 inhibits PDGF-BB-induced membrane
ruffling and rearrangement of actin stress fibers in PAE cells
expressing the wild-type PDGF -receptor.
The cells were pretreated with SB203580 or SB202474 and stimulated with
PDGF-BB as described in the legend to Fig. 6. Then the cells were fixed
and actin filaments were stained using tetramethylrhodamine
isothiocyanate-labeled phalloidin. The cells were viewed by
fluorescence microscopy, and 300 cells were counted in duplicate. The
percentages of membrane ruffling-forming cells to total cells as well
as that of stress fiber-forming cells to total cells were calculated.
The experiment was repeated three times with similar results.
Bars represent mean ± S.D. *,
p < 0.05.
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SB203580 Does Not Affect DNA Synthesis by PDGF-BB--
To evaluate
whether p38 is involved also in PDGF-induced mitogenesis, next we
examined the effect of SB203580 on the PDGF-BB-stimulated increase in
DNA synthesis in PAE cells expressing the wild-type PDGF
-receptor.
DNA synthesis was assessed by [3H]thymidine incorporation
assay. As shown in Fig. 8, treatment of
the cells with 20 ng/ml PDGF-BB induced a 3.9-fold increase of
[3H]thymidine incorporation as compared with the
control. Simultaneous treatment of the cells with 20 µM
SB203580, the concentration of which was shown to be enough to
considerably inhibit PDGF-induced chemotaxis (Fig. 5A),
did not affect PDGF-BB-induced [3H]thymidine
incorporation.

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Fig. 8.
Effect of SB203580 on PDGF-BB-stimulated DNA
synthesis in PAE cells expressing the wild-type PDGF
-receptor. After 48 h of serum
starvation, the cells were incubated with 20 ng/ml PDGF-BB in the
presence or absence of 20 µM SB203580 for 16 h at
37 °C. [3H]Thymidine (1 µCi/well) was then added to
each well, and the cells were incubated for an additional 8 h.
After the incubation, trichloroacetic acid precipitable radioactivity
was determined by liquid scintillation counting. The data represent the
mean ± S.D. of quadruplicate dishes.
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Activation of p38 by PDGF-BB Is Independent of PI 3-kinase and
PLC-
--
Next we examined which SH2 domain-containing proteins
mediate PDGF-induced p38 activation. PAE cells expressing the wild-type or various mutant PDGF
- or
-receptors, in which one or two tyrosine residues were replaced with phenylalanine residues, were analyzed for PDGF-BB-induced p38 activation. The cells expressing Y579F, Y740F/Y751F, Y771F, Y1009F, or Y1021F mutant
-receptors, which are unable to interact with Src family kinases, PI 3-kinase, RasGAP, SHP-2, or PLC-
, respectively, and the cells expressing Y762F
or Y1018F mutant
-receptors, which are unable to interact with Crk
or PLC-
, respectively, were used for the assay. As shown in Fig.
9, all these mutant
- or
-receptors
were found to mediate PDGF-BB-induced activation of p38 as efficiently
as the wild-type
- or
-receptors, respectively. The data indicate
that none of these SH2 proteins play a major role in transducing the
signal leading to p38 activation by PDGF-BB.

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Fig. 9.
Activation of p38 by PDGF-BB in PAE cells
expressing the wild-type or tyrosine residue-mutated PDGF
receptors. PAE cells expressing the wild-type -receptor
(WT ), Y579F, Y740F/Y751F, Y771F, Y1009F, or Y1021F mutant
-receptors, as well as PAE cells expressing the wild-type
-receptor (WT ) or Y762F or Y1018F mutant -receptors
were analyzed for PDGF-BB-induced activation of p38. The cells were
serum-starved for 20 h and then treated or untreated with 50 ng/ml
PDGF-BB for 10 min at 37 °C. After treatment, the cells were lysed,
and p38 activity of the cell lysates was determined as described in the
legend to Fig. 1.
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Among these SH2 proteins, PI 3-kinase and PLC-
have been considered
major role players in cell motility responses induced by the PDGF
-receptor (25, 26). We therefore examined whether specific
inhibitors for PI 3-kinase and PLC-
, wortmannin and U-73122,
respectively, inhibit PDGF-BB-induced p38 activation. Neither
wortmannin (1 µM) nor U-73122 (1-25 µM)
affected the activation of p38 by PDGF-BB at the concentrations
sufficient to inhibit enzymatic activation of these SH2 proteins (data
not shown). The data confirm that PI 3-kinase and PLC-
are not
involved in PDGF-induced p38 activation.
Expression of Dominant-negative Ras but Not Dominant-negative Rac
Inhibits p38 Activation by PDGF-BB--
Members of the small
GTP-binding proteins have a critical role in the activation of the MAP
kinases. Among these proteins, Ras is well known to be activated upon
ligand stimulation of the PDGF
-receptor. The activated Ras has been
shown to activate three distinct MAP kinases, ERK, SAPK1/JNK, and p38
in different conditions (27, 28). In addition, the small GTP-binding
protein Rac1 has been reported to regulate PDGF-induced membrane
ruffling (29, 30) and to play a role in mediating the p38 MAP kinase pathway in response to stimulation by interleukin-1 or tumor necrosis factor (31). To evaluate the role of these small GTP-binding proteins
in PDGF-induced activation of p38, we examined the effect of
overexpression of dominant-negative Rac1 (N17Rac1) or dominant-negative Ras (Ras57Y) on the PDGF-BB-induced activation of p38 in PAE cells expressing the wild-type PDGF
-receptor. The expression of N17Rac1 profoundly inhibited PDGF-BB-induced actin reorganization as expected (data not shown). On the other hand, as shown in Fig.
10A, the expression of
N17Rac1 did not affect the PDGF-BB-induced activation of p38. In
contrast, the expression of Ras57Y potently inhibited PDGF-BB-induced
activation of p38 as well as ERK (Fig. 10B). Ras57Y expression did not affect anisomycin-induced activation of p38 (Fig.
10B). These data suggest that Ras is a potent mediator of a
p38 pathway downstream of the PDGF
-receptor.

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Fig. 10.
Expression of dominant-negative Ras (Ras57Y)
but not dominant-negative Rac (N17Rac1) inhibits PDGF-BB-induced
activation of p38 in PAE cells expressing the wild-type PDGF
-receptor. A, the wild-type
-receptor-expressing cells were transiently transfected with
N17rac1 in pEFBOS vector or with empty pEFBOS vector. After
20 h of serum starvation, the cells were stimulated or not with 50 ng/ml PDGF-BB for 10 min 37 °C. After stimulation, the cells were
lysed, and p38 activity of the cell lysates was determined as described
in the legend to Fig. 1. B, the adenoviral vector
AdexCAHRasY57 was introduced into the wild-type -receptor-expressing
cells by incubation in F-12 medium for 2 h at 37 °C under
gentle agitation. The cells were washed twice with phosphate-buffered
saline and were then serum-starved for 20 h. The cells were
treated or untreated with 50 ng/ml PDGF-BB or with 10 µg/ml
anisomycin (An) for 10 min at 37 °C. After treatment, the
cells were lysed, and p38 and ERK activities of the cell lysates were
determined as described in the legend to Fig. 1.
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DISCUSSION |
In the present study, we show that PDGF-BB stimulation of the PDGF
- as well as
-receptors leads to activation of both ERK and p38
MAP kinase pathways but not the SAPK1/JNK pathway (Figs. 1-4).
Experiments using the specific inhibitor of p38 show that activation of
p38 is required for PDGF-BB-induced cell motility responses such as
cell migration and actin reorganization but not required for
PDGF-BB-stimulated DNA synthesis (Figs. 5-8). Our present study also
shows that SH2 domain-containing proteins including Src family kinases,
PI 3-kinase, RasGAP, SHP-2, PLC-
, and Crk do not play a major role
in mediating PDGF-BB-induced activation of p38 (Fig. 9). Instead, Ras,
but not Rac, seems to be a potent mediator in the p38 activation
pathway downstream of the PDGF
-receptor (Fig. 10).
Using fibroblast cell lines expressing a dominant-negative Ras mutant,
Kundra et al. (32) have shown that Ras inhibition suppresses
migration toward PDGF-BB. The observation is consistent with our
present findings, suggesting that Ras is involved upstream of the
PDGF-induced activation pathway of p38 leading to cell migration. It
was also reported using an inhibitor of MEK1, PD098059, that ERK
activation was involved in PDGF-directed migration of vascular smooth
muscle cells (33). However, we did not observe any inhibitory effect of
the MEK1 inhibitor on PDGF-BB-mediated chemotaxis in our assay system
(Fig. 5B). Joneson et al. (34) have reported that
membrane ruffling and ERK activation are mediated by distinct Ras
effector pathways. Our data demonstrating that PDGF-induced cell
motility response is mediated by p38 but not by ERK also suggest the
existence of a Ras/p38 pathway independent of the classical Ras/ERK pathway.
The MEKs for p38, MAP kinase kinase 3, and MAP kinase kinase 6, are
relatively specific activators of p38 (35), and it is conceivable that
they are also involved in the activation process of p38 downstream of
PDGF stimulation. Although there may be some cross-activation of p38 by
SEK1/MAP kinase kinase 4 (36), which is one of the MEKs upstream of
SAPK1/JNK, its involvement is unlikely because we did not observe
SAPK1/JNK activation after PDGF-BB stimulation (Fig. 1). MEKKs in the
p38 cascade have not been clearly identified, and the signaling link
between Ras and MAP kinase kinase 3/6 remains to be determined.
Based on a series of studies (37, 38), it is now clear that activation
of the p38 cascade results in activation of MAP kinase-activated
protein kinase-2, which in turn phosphorylates the F-actin
polymerization modulator heat shock protein 27 (HSP27). Although the
physiological significance of the phosphorylation is not fully
understood and the function of HSP27 is not yet clear, it can regulate
microfilament dynamics (39, 40) and, thereby, is likely to play an
important role in cell motility responses elicited by PDGF stimulation.
It is well established that Rac regulates growth factor-induced
membrane ruffling (29, 41). In fact, Hooshmand-Rad et al.
(30) have recently confirmed the involvement of Rac in PDGF-induced actin reorganization and chemotaxis. Therefore, it was our surprise that the expression of the dominant-negative Rac1 mutant failed to
inhibit PDGF-BB-induced activation of p38 (Fig. 10A). The
data suggest a possibility that the p38-mediated signaling pathway is
independent of the Rac-mediated pathway and both pathways function synergistically in PDGF-induced cell motility responses. Alternatively, Ras-dependent activation of p38 may be a prerequisite for
subsequent activation of Rac in the context of a PDGF-induced signaling pathway.
Zhang et al. (31) have shown that the p21-activated kinase 1 and its upstream regulators, Rac and Cdc42, couple to and regulate the
activity of p38 and are an integral part of the signaling pathway
linking cell surface proinflammatory receptors to p38 activation. On
the other hand, Tan et al. (42) have shown that fibroblast
growth factor-induced activation of p38 is mediated through an
intracellular signaling pathway that requires Ras. Our present study
shows that PDGF-induced activation of p38 is also mediated by Ras but
not by Rac, suggesting that the Ras-dependent pathway
functions as an alternative mechanism for the growth factor-mediated signal leading to p38 activation.
PI 3-kinase has been reported to be indispensable for PDGF-induced
chemotaxis, which is mediated by the PDGF
-receptor (25, 26).
Replacement of two tyrosine residues within PI 3-kinase binding sites
of the PDGF
-receptor causes a loss of chemotactic response to
PDGF-BB in cells transfected with this mutant receptor. However, Higaki
et al. (43) reported that two different PI 3-kinase inhibitors, wortmannin and LY294002, did not inhibit PDGF-induced chemotaxis and, furthermore, Chinese hamster ovary cells overexpressing a dominant-negative p85 subunit of PI 3-kinase showed a chemotactic response comparable to that of parental cells while showing a remarkable decrease in PI 3-kinase activity. The underlying mechanism of the discrepant observations concerning the functional role of PI
3-kinase in PDGF-induced chemotaxis remains to be elucidated. Our
present finding that the Y740F/Y751F mutant PDGF
-receptor, which
lacks the PI3-kinase binding sites, activated p38 as efficiently as the
wild-type
-receptor (Fig. 9) clearly rules out a possibility that PI
3-kinase is involved upstream of the p38-dependent
signaling pathway for cell motility responses.
In summary, we have demonstrated that the p38 pathway is involved in
modulating the PDGF-induced cell motility responses and obtained
evidence suggesting that the activation of p38 is mediated through
Ras-dependent mechanism. The finding that p38 may regulate cell motility is consistent with the previous report suggesting that
p38 activation by vascular endothelial growth factor mediates actin
reorganization and cell migration in human endothelial cells (44). The
report, together with our present study, supports the notion that p38
is an important mediator of cell motility responses elicited by
receptor tyrosine kinases.