Synergism among Lysophosphatidic Acid,
1A
Integrins, and Epidermal Growth Factor or Platelet-derived Growth
Factor in Mediation of Cell Migration*
Takao
Sakai
§,
J. Manuel
de la Pena
, and
Deane F.
Mosher
¶
From the
Departments of Medicine and Biomolecular
Chemistry, University of Wisconsin, Madison, Wisconsin 53706 and
§ Department of Experimental Pathology, Lund University, 221 85 Lund, Sweden
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ABSTRACT |
GD25 cells lacking the
1
integrin subunit or expressing
1A with certain
cytoplasmic mutations have poor directed cell migration to
platelet-derived growth factor (PDGF) or epidermal growth factor (EGF),
ligands of receptor tyrosine kinases, or to lysophosphatidic acid
(LPA), a ligand of G-protein-coupled receptors (Sakai, T., Zhang, Q.,
Fässler, R., and Mosher, D. F. (1998) J. Cell
Biol. 141, 527-538 and Sakai, T., Peyruchaud, O., Fässler,
R., and Mosher, D. F. (1998) J. Biol. Chem. 273, 19378-19382). We demonstrate here that LPA synergizes with signals
induced by
1A integrins and ligated EGF or PDGF
receptors to modulate migration. When LPA was mixed with EGF or PDGF,
migration was greater than with EGF or PDGF alone. The enhancement was
greater for
1A-expressing cells than for
1-null cells. Cells expressing
1A with
mutations of prolines or tyrosines in conserved cytoplasmic
NPXY motifs had blunted migratory responses to mixtures of
LPA and EGF or PDGF. The major effects on
1A-expressing
cells of LPA when combined with EGF or PDGF were to sensitize cells so
that maximal responses were obtained with >10-fold lower
concentrations of growth factor and increase the chemokinetic component
of migration. Sensitization by LPA was lost when cells were
preincubated with pertussis toxin or C3 exotransferase. There was no
evidence for transactivation or sensitization of receptors for EGF or
PDGF by LPA. EGF or PDGF and LPA caused activation of mitogen-activated
protein kinase by pertussis toxin-insensitive and -sensitive pathways
respectively, but activation was not additive. These findings indicate
that signaling pathways initiated by the cytoplasmic domains of ligated
1A integrins and tyrosine kinase receptors interact with
signaling pathways initiated by LPA to facilitate directed cell migration.
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INTRODUCTION |
Directed cell migration is of obvious importance in diverse
physiological and pathological processes, including development, immunity, wound healing, and cancer metastasis (1-3). Cell migration requires fine control of cellular association with and release from the
extracellular matrix (4-6). Migration in a concentration gradient
(chemotaxis) is accomplished by dynamic coordination among receptors
for chemotactic agents, cellular adhesion receptors, and the
actin-containing cytoskeleton. At any one time, a given cell likely
encounters a variety of chemotactic substances and adhesive substrates.
The mechanisms by which these signals are integrated to form a
coordinated migratory response are poorly understood.
Integrins are transmembrane heterodimeric cell surface adhesion
receptors composed of noncovalently associated
and
subunits. The fact that ligation of integrins by adhesive ligands can induce intracellular signaling events ("outside-in" signaling) and
intracellular signaling pathways can control binding avidity of
integrins for extracellular ligands ("inside-out" signaling)
(7-11), makes integrins good candidates to mediate the
adhesion-deadhesion events required for migration. In addition,
integrins are linked to the cytoskeleton in a dynamic fashion by the
molecules recruited to focal contacts (7-12). Finally, cell migration
may be facilitated by the cycling of integrins between cytoplasmic
compartments and the cell surface (13, 14).
Lysophosphatidic acid (LPA)1
is a product of activated platelets and cells and has been shown to
mediate multiple cellular responses (15, 16). LPA is the serum
enhancement factor of fibronectin matrix assembly; enhancement of
assembly closely correlates with LPA-induced actin stress fiber
formation and cell contraction (17-19). LPA is a mitogen for a number
of cells (16, 20, 21) and induces in vitro invasion across
host cell monolayers by several types of tumor cells (22, 23) and also
stimulates random nondirectional migration (chemokinesis) of Rat-1
fibroblasts (24) or both chemokinesis and directional migration
(chemotaxis) of mouse GD25 fibroblastic cells (25). Recently, a
specific G-protein-coupled receptor (GPCR) for LPA, ventricular zone
gene-1 (vzg-1) (26) or endothelial differentiation gene-2 (edg-2) (27),
was identified. Edg-1, a homologous protein, is a receptor for
sphingosine-1-phosphate (S1P), another lysophospholipid generated
during platelet activation, and has a low affinity to LPA (28, 29).
Three other Edg GPCRs have also been identified, Edg-3 and Edg-5 as
receptors for S1P and Edg-4 as a receptor for LPA, (16, 30, 31). LPA
and S1P stimulate several transduction cascades through GPCRs,
including activation of the botulinum C3
exotransferase-sensitive small GTP-binding protein p21Rho
(Rho) through G
12/13 and the Ras/mitogen-activated
protein kinase (MAPK) pathway through pertussis toxin-sensitive
Gi (15, 16). Expression of a dominant-negative Ras mutant
inhibited migration of NIH(M17) cells in response to LPA as well as to
other chemoattractants such as platelet-derived growth factor (PDGF) (32). The latter results suggest that Ras plays a key role in regulation of both LPA- and growth factor-induced cell migration.
Growth factors such as PDGF and epidermal growth factor (EGF), acting
via tyrosine kinase receptors (RTKs), stimulate cell migration in
concert with integrin-extracellular matrix interactions (33). In NR6
fibroblasts expressing EGF receptor, EGF alters migration speed and
directional persistence in a matrix-dependent manner (34).
Mouse B82L-B3 fibroblasts expressing EGF receptor do not migrate in
response to EGF alone but when co-presented with laminin or
fibronectin, EGF mediates EGF receptor-mediated migration, suggesting
an interaction between EGF receptor and integrins ligated by laminin or
fibronectin (35). PDGF enhances migration of NIH 3T3 cells when plated
on vitronectin, a ligand for
v
3; other
v integrins but not
1 integrins associate
with activated PDGF receptor (36-38).
We recently expressed a series of
1A integrin subunits
in GD25 mouse fibroblasts derived from
1-null stem cells
and demonstrated that restoration of
1 integrin function
is required for efficient migration of GD25 cells across matrix-coated
filters in response to LPA, PDGF, or EGF (25, 39). Because LPA, PDGF or
EGF, and integrin ligands stimulate cells via three distinct but
overlapping signaling systems as described above, we questioned whether
engagement of two of the three systems is sufficient for maximum cell
migration. In the present study, we demonstrate that LPA greatly
increases the migratory activity of EGF or PDGF and that a functional
1A integrin is required for this enhancement.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The GD25 fibroblast line, which was established
after differentiation of
1-null stem cells and
immortalization with SV40 large T antigen, and its derivatives
transfected with
1A or
1As with mutations
of the cytoplasmic domain were as described previously (39-41). PDGF
from porcine platelets (R&D Systems, Minneapolis, MN), recombinant
human EGF (Upstate Biotechnology, Lake Placid, NY), 1-oleoyl-LPA and
S1P (Avanti Polar Lipids, Birmingham, AL, and LC Laboratories, Woburn,
MA, respectively), and pertussis toxin (List Biological Laboratories,
Campbell, CA) were purchased. Also purchased were rabbit polyclonal
antibodies against active MAPK (Promega, Madison, WI), rabbit
polyclonal antibodies that recognized mouse EGF or PDGF receptors
(Santa Cruz Biotechnology, Santa Cruz, CA), and mouse monoclonal
antibody (mAb 4G10) against phosphotyrosine (Upstate Biotechnology).
Recombinant C3 exotransferase was a generous gift from Dr. Tracee
Panetti, University of Wisconsin (Madison, WI).
Cell Migration--
Cell migration assays were performed in
modified Boyden chambers containing Nucleopore polycarbonate membranes
(5-µm pore size; Costar Corp., Cambridge, MA) as described (25,
39).
Cell Lysis, Immunoprecipitation, and Immunoblotting--
For
immunoprecipitation of EGF or PDGF receptors, cells grown to 80-90%
confluency were starved in medium without serum for 16 h, released
from the substrate, reseeded onto plates coated with 100 µg/ml
gelatin, incubated for 3 h in medium without serum, and then
stimulated with agonists for 5 min or left unstimulated. For Western
blotting of phosphorylated Erk1/2, cells were similarly seeded onto
gelatin-coated plates, starved in serum-free medium for 3 h, and
then stimulated with agonist for various time perioids. Cells were
lysed on ice in buffer containing 1% (v/v) Triton X-100, 150 mM NaCl, 5 mM EDTA, 100 mM sodium
fluoride,1 mM sodium orthovanadate, 0.5 mM
sodium molybdate, 2 mM PMSF, 5 µg/ml leupeptin, 0.1 µg/ml pepstatin A, 0.4 mM pefabloc SC, and 20 mM Tris-HCl, pH 7.4. The same amounts of protein from
different experimental samples were used for analyses, as determined
using a BCA protein assay (Pierce). The proteins were run on
SDS-polyacrylamide gel electrophoresis under reducing conditions.
Immunoprecipitation analysis was performed as described previously
elsewhere (42), with a slight modification. Briefly, the supernatants
were precleaned with protein A-Sepharose 4 Fast-Flow (Pharmacia LKB
Biotech, Sweden) and subsequently incubated with antibody. The
complexes were precipitated with protein A-Sepharose 4 Fast-Flow, and
the proteins were eluted from the resins by incubation with SDS-sample
buffer. Samples were then subjected to SDS-polyacrylamide gel
electrophoresis and transferred onto polyvinylidene difluoride membranes. For immunoblotting, the blots were probed with the primary
antibody, then with a horseradish peroxidase-conjugated secondary
antibody (Organon Teknika Corp., Westchester, PA; Promega, Madison,
WI). Immunoreactive bands were developed using the enhanced chemiluminescence (ECL) substrate system (NEN Life Science Products).
Immunoprecipitation of EGF or PDGF receptors followed by immunoblotting
for phosphotyrosine or
1 integrin was also carried out
on cells lysed 60 min after plating on gelatin (100 µg/ml), vitronectin, fibronectin, or laminin-1 (10 µg/ml) coated substratum, or left in suspension.
 |
RESULTS |
LPA Sensitizes Cells to Growth Factors and Increases
1 Integrin-mediated Cell Migration
The effect of LPA in combination with EGF or PDGF on directed cell
migration through gelatin-coated filters was analyzed with GD25 cells
expressing wild type
1A (Fig.
1). The cells did not migrate in response
to EGF alone. PDGF alone stimulated modest migration at a concentration
>1 ng/ml. 500 nM LPA alone stimulated robust migration.
When EGF or PDGF were mixed together with LPA, cell migration was
enhanced further by EGF or PDGF concentrations as low as 0.03 ng/ml
(Fig. 1). The concentration of PDGF that supported maximal migration in
the presence of both PDGF and LPA was 3 ng/ml as compared with >30
ng/ml in the absence of LPA.

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Fig. 1.
Cell migration through gelatin-coated filters
of GD25 cells expressing wild type
1A in response to LPA and/or EGF or
PDGF. Migration was quantified for EGF alone ( ), PDGF alone
( ), LPA and EGF ( ), and LPA and PDGF ( ). LPA (500 nM) and/or EGF or PDGF in concentrations from 0.003 to 30 ng/ml was added in the lower chamber, and the dose dependence of the
growth factor was analyzed. Each symbol represents the mean
of cell number/0.16-mm2 field. Brackets indicate
mean ± S.D. of quadruplicate determinations.
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The requirement for
1A for migration of GD25 cells
through gelatin-coated filters is more stringent when the
chemoattractant is LPA than when the chemoattractant is PDGF (25). The
effects of
1A integrin on cell migration induced by LPA
in combination with EGF or PDGF were therefore investigated. When EGF
and LPA were mixed together, cell migration was 2-3-fold greater for
cells expressing
1A than for cells lacking
1A (Fig. 2A).
When PDGF and LPA were mixed together, the number of migrating cells
was 1.2-1.5-fold greater for cells expressing wild type
1A than for cells lacking
1A (Fig.
2B). S1P, a phospholipid that, like LPA, reacts with Edg
receptors (28, 29), is unlike LPA in that it inhibits cell migration
(43). S1P did not induce migration through gelatin-coated filters by
either cell type and inhibited LPA-induced migration in cells
expressing
1A (Fig. 2C). These findings
indicate that
1A-dependent migration in
response to LPA is enhanced by EGF or PDGF and attenuated by S1P.

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Fig. 2.
Cell migration through gelatin-coated filters
of 1-deficient GD25 cells and GD25
cells expressing wild type 1A in
response to the mixtures of LPA and EGF (A), LPA and
PDGF (B), or LPA and S1P (C).
LPA (0-500 nM) and EGF (0-30 ng/ml) or PDGF (0-30 ng/ml)
or S1P (0-200 nM) were added in the lower chamber. Results
are expressed as the mean of duplicate experiments with duplicate
determinations in each experiment (n = 4). Standard
deviations were <10% of the means in all cases. GD25,
1-deficient cells; 1GD25, GD25 cells
expressing wild type 1A.
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1A-dependent migration in response to LPA
(25) or PDGF or EGF (39) is lacking in GD25 cells expressing
1As with certain mutations in the cytoplasmic domain.
These mutants fell into two groups, active (Y783F, Y795F, and
Y783/795F) and inactive (T788P, P781A, P793A, and P781/793A), based
upon the reactivity with anti-
1 (9EG7) antibody,
6
1-dependent adhesion to
laminin-1, and ability to support fibronectin assembly (25). LPA and
EGF stimulated the same minimal migration of GD25 cells lacking
1A and of GD25 cells expressing
1A with
the T788P, P781/793A, Y783/795F, Y783F, or Y795F mutations (Fig.
3). LPA and PDGF were less effective in
causing migration of T788P, P781/793A, Y83/795F, Y783F, or Y795F cells
than of GD25 cells lacking
1A (Fig. 3). When LPA and EGF
or PDGF were tested together on cells expressing
1A with the activating D759A mutation, migration was similar to cells expressing wild type
1A (Fig. 3).

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Fig. 3.
Cell migration through gelatin-coated filters
in response to LPA and EGF or PDGF of
1-deficient GD25 cells, GD25 cells
expressing wild type 1A, or
1A with the indicated mutations.
LPA (500 nM) and EGF or PDGF (3 ng/ml) were in the lower
chamber. Bars represent the means of cell
number/0.16-mm2 field. Brackets indicate
mean ± S.D. of quadruplicate determinations. In the presence of
EGF or PDGF alone, less than 10 cells/0.16 mm2 moved across
filters coated with gelatin.
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We previously demonstrated that the magnitudes of the chemotactic
response to EGF or PDGF of GD25 cells expressing wild type
1A were 2-3-fold greater than the chemokinetic response
(39), as opposed to the 1.2-1.5-fold difference seen with LPA (25). To
learn if the enhancing effect of LPA on EGF- or PDGF-induced migration
is due to increased chemotaxis or chemokinesis in cells expressing wild
type
1A, we studied the effect of adding LPA with cells
in the upper chamber or in both the upper and lower chambers (Fig.
4). When LPA was present at equal
concentrations in both chambers, the synergistic effect of LPA on EGF-
or PDGF-induced migration was 90-95% of that observed when LPA and
EGF or PDGF were present in just the lower chamber. When LPA was in the
upper chamber and EGF or PDGF was in the lower chamber, more cells
migrated toward the lower chamber than in the complete absence of LPA
but less than when LPA alone was in the lower chamber. The same results were observed with filters coated with gelatin, vitronectin, or fibronectin (Fig. 4). These results indicate that LPA causes increased migration when mixed with EGF or PDGF mainly by a chemokinetic effect
with some contribution of chemotaxis.

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Fig. 4.
Trans-filter migration of GD25 cells
expressing wild type 1A through
gelatin-, vitronectin-, or fibronectin-coated filters in response to
various combinations of LPA and EGF or PDGF in the upper and lower
chambers. LPA (500 nM) and/or PDGF or EGF (3 ng/ml)
were added in the following combinations: No add./LPA, No add./EGF, and
No add./PDGF: No addition in the upper chamber, and LPA, EGF, or PDGF
alone added in the lower chamber; No add./LPA+EGF, and No
add./LPA+PDGF: No addition in the upper chamber, and LPA and EGF or
PDGF added together in the lower chamber; LPA/EGF, and LPA/PDGF, LPA
added in the upper chamber and EGF or PDGF added in lower chamber;
LPA/LPA+EGF, and LPA/LPA+PDGF, LPA added in both chambers and EGF or
PDGF added in the lower chamber. Bars represent the means of
cell number/0.16-mm2 field. Brackets indicate
mean ± S.D. of quadruplicate determinations normalized as percent
of cells migrating to LPA in the lower chamber (No add./LPA). The mean
numbers of migrated cells in the presence of LPA (500 nM)
in the lower chamber were: 155 on gelatin, 254 on vitronectin, or 276 on fibronectin.
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Signaling Pathways Induced by LPA and/or EGF or PDGF
Lack of Effect of LPA or
1A-mediated Adhesion on
Phosphorylation of EGF or PDGF Receptors--
Treatment of Rat-1
fibroblasts, HaCaT keratinocytes, or COS-7 cells with high
concentration of LPA (10-25 µM) causes phosphorylation of EGF receptors (44, 45). GD25 cells lacking
1A or
expressing
1A expressed comparable amounts of EGF and
PDGF receptors when analyzed by SDS-polyacrylamide gel electrophoresis
and immunoblotting, and both receptors in both cells were activated
appropriately by concentrations of 3 ng/ml EGF or PDGF but not by 500 nM LPA when analyzed by anti-phosphotyrosine immunoblotting
of immunoprecipitated receptors (25). We tested a wider range of growth
factor concentrations to learn whether LPA sensitizes EGF or PDGF
receptors to activation by low concentrations of EGF or PDGF. The
amount of EGF or PDGF receptor phosphorylation in cells expressing wild
type
1A as assessed by anti-phosphotyrosine
immunoblotting of immunoprecipitated receptors was increased in
response to EGF or PDGF in a concentration-dependent manner
(Fig. 5). When EGF or PDGF was mixed with
500 nM LPA together, there was no enhancement of EGF or
PDGF receptor phosphorylation when compared with the stimulation by EGF
or PDGF alone (Fig. 5). When 20 µM LPA alone was added,
no tyrosine phosphorylation of EGF or PDGF receptors was demonstrated
(not shown). These experiments, therefore, indicate that GD25 cells do
not respond to LPA with transactivation of EGF receptors like Rat-1,
HaCaT, or COS-7 cells. We also compared the phosphorylation states of
EGF and PDGF receptors in
1GD25 cells adherent for 60 min to vitronectin, fibronectin, or laminin-1-coated surfaces. No
phosphorylation was noted over and above the baseline phosphorylation
present in suspended cells. In addition, no
1A was
co-immunoprecipitated with EGF or PDGF receptors (not shown).

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Fig. 5.
Dose-response analysis of tyrosine
phosphorylation of EGF or PDGF receptor stimulated by EGF or PDGF
and/or LPA in GD25 cells expressing wild type
1A. Serum-starved cells were
seeded onto plates coated with 100 µg/ml gelatin, incubated for
3 h in medium without serum, and then stimulated with agonists for
5 min as indicated or left unstimulated ( ). The EGF or PDGF receptor
was immunoprecipitated from cell lysates and analyzed by immunoblotting
with anti-phosphotyrosine antibody 4G10 (upper panels). The
same samples were analyzed by immunoblotting with anti-EGF or anti-PDGF
antibody (lower panels).
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Activation of Erk1/2 Stimulated by LPA and/or EGF or PDGF--
LPA
signaling is mediated through Ras and known to result in activation of
MAPK (15, 16). We therefore studied whether cell migration in response
to LPA and EGF or PDGF is associated with the hyperactivation of MAPK
in GD25 cells wild type
1A. To replicate conditions of
the cell migration assay, cells were incubated for 3 h without
serum on a gelatin-coated substratum and then stimulated with agonists
for various time periods. Doubly phosphorylated Erk1 and Erk2, which
represent the activated forms of these proteins, were analyzed using
anti-active MAPK antibodies. Basal activation of Erk1/2 was minimal
(Fig. 6). In response to the stimulation
of LPA, EGF, or PDGF alone, activation of Erk1/2 was increased in a
concentration-dependent manner (Fig. 6). When LPA and EGF
or PDGF were added together, only modest additive effects of Erk1/2
activation were demonstrated when compared with LPA, EGF, or PDGF alone
(Fig. 6). This finding was true after both 5 and 30 min of
stimulation.

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Fig. 6.
Dose-response analysis of phosphorylation of
Erk1/2 stimulated by EGF or PDGF and/or LPA in GD25 cells expressing
wild type 1A. Cells were
seeded onto plates coated with 100 µg/ml gelatin and starved in
medium without serum for 3 h and then stimulated with agonists for
various time periods or left unstimulated ( ). Cell lysates were
analyzed by immunoblotting with anti-active MAPK polyclonal
antibody.
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Correlation Between Cell Migration and Ras- or Rho-mediated
Signaling Pathway--
Treatment of GD25 cells expressing
1A with pertussis toxin, which modifies Gi
and blocks stimulation of Ras induced by LPA (20), caused nearly
complete loss of migration through gelatin-coated filters in response
to LPA alone at a toxin dose of 3 ng/ml (Fig. 7A). The 50% inhibitory dose
of pertussis toxin, determined in a separate experiment, was 0.01-0.03
ng/ml (not shown). When LPA and EGF or PDGF were added together to
cells treated with pertussis toxin, enhancement by LPA of growth
factor-induced migration was completely lost at 3 ng/ml toxin, such
that the number of migrated cells were the same as in response to PDGF
or EGF alone (Fig. 7A). Treatment of pertussis toxin caused
down-regulation of Erk1/2 activation in response to LPA but did not
significantly affect Erk1/2 activation in response to EGF or PDGF alone
or to a combination of LPA and EGF or PDGF (Fig.
8).

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Fig. 7.
Effect of pertussis toxin
(A) or C3 exotransferase (B) on LPA-
and/or EGF- or PDGF-induced cell migration through gelatin-coated
filters of GD25 cells expressing wild type
1A. Cells were pretreated
overnight with pertussis toxin or C3 exotransferase at the
concentrations of 0, 3, 10, 30, and 100 ng/ml pertussis toxin, or 0, 3, 10, 30, and 100 µg/ml (C3 exotransferase). LPA (500 nM)
and/or EGF or PDGF (3 ng/ml) were in the lower chamber. Migration was
then quantified in response to LPA alone ( ), EGF alone ( ), PDGF
alone ( ), LPA and EGF ( ), and LPA and PDGF ( ). Each
symbol represents the mean of cell
number/0.16-mm2 field. Brackets indicate
mean ± S.D. of quadruplicate determinations.
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Fig. 8.
Effect of pertussis toxin on LPA- and/or EGF-
or PDGF-induced phosphorylation of Erk1/2 in GD25 cells expressing wild
type 1A. Cells were
pretreated overnight with pertussis toxin at concentrations of 0, 3, 10, 30, and 100 ng/ml, seeded onto plates coated with 100 µg/ml
gelatin, starved in medium without serum for 3 h, and then
stimulated with agonists (LPA (500 nM) and/or EGF or PDGF
(3 ng/ml)) for 5 min or left unstimulated ( ). Cell lysates were
analyzed by immunoblotting with anti-active MAPK polyclonal
antibody.
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LPA signaling through G
12/13 causes direct activation of
Rho (46). Migration of cells expressing wild type
1A in
response to LPA alone or PDGF alone was decreased by the treatment of
cells with C3 exotransferase, which inactivates Rho in a
concentration-dependent manner up to exotransferase doses
of 100 µg/ml (Fig. 7B). When LPA and EGF or PDGF were
added together to cells treated with C3 exotransferase, the enhanced
effect of EGF or PDGF on LPA-induced migration was lost at an
exotransferase dose as low as 10 µg/ml (Fig. 7B). The
treatment of C3 exotransferase did not block LPA-stimulated Erk1/2
activation at any of the toxin concentrations tested (not shown).
 |
DISCUSSION |
1A with an intact cytoplasmic tail, including
intact NPXY motifs, is important for chemotaxis of GD25
fibroblasts in response to EGF or PDGF (39). The requirement for
1A is more stringent when the chemotactic agent is LPA
than when the chemotactic agent is EGF or PDGF (25). In this study, we
describe a tripartite interaction among ligated
1A
integrins, LPA GPCRs, and EGF or PDGF RTKs. Although signaling induced
by two of the three receptor systems supports chemotaxis, engagement of
all three receptor systems leads to greater migration. The major
effects of LPA when combined with EGF or PDGF were to sensitize cells
so that maximal responses were obtained with >10-fold lower
concentrations of growth factor and to increase the chemokinetic
component of migration. The synergism between LPA and EGF or PDGF was
lost when cells were preincubated with pertussis toxin or C3 exotransferase.
S1P mimics some of the effects of LPA (47-50), and both lipids signal
via Edg receptors. GD25 cells lacking
1A and expressing wild type
1A have been shown to express edg-2 and edg-1
receptors by reverse transcription-polymerase chain reaction (25). In contrast to the effect of LPA, neither cell type was induced to migrate
by S1P. S1P inhibits invasion and motility of melanoma cells and
PDGF-induced chemotaxis of human smooth muscle cells, whereas LPA
induces invasion of rat hepatoma cells and migration of NIH 3T3 or
human skin fibroblasts (22, 32, 43, 51, 52). Similarily S1P inhibited
LPA-induced migration of
1A-GD25 cells. Presumably, the
two bioactive lipids activate a different balance of signaling
pathways, thus leading to different effects on cellular motility.
The results indicate that the
1A integrin must not only
be active but have intact NPXY motifs in the cytoplasmic
domain. The latter requirement differentiates the LPA effect on
migration from its effect on fibronectin matrix assembly, which is
up-regulated by LPA in GD25 cells expressing
1A with
conservative Tyr to Phe substitutions (39). An unexpected finding
of the present experiments was the deleterious effect of mutations
of the NPXY motifs on migration due to LPA and PDGF when
compared with migration of cells lacking
1A completely.
This result suggests that the mutant
1A tails depress
migration mediated by other cell surface receptors.
Recent evidence suggests at least three mechanisms of "cross-talk"
among signaling pathways. First, there is evidence of cross-talk between GPCRs and RTKs. LPA and other GPCR agonists can induce activation of EGF or PDGF receptors in the absence of EGF or PDGF with
subsequent activation of the Ras-MAPK cascade (44, 45, 53-55).
G
13-mediated Rho activation induced by LPA has also been
suggested to stimulate EGF RTK activity in Swiss 3T3 cells (56).
Second, there is evidence of cross-talk between RTKs and
1 integrins. Stimulation of
1 integrin on
human AG1518 fibroblasts by interaction with extracellular matrix
induces ligand-independent tyrosine phosphorylation of PDGF receptors,
but not of EGF receptors (38). Similarly, adhesion of human skin
fibroblasts, endothelial cells to matrix proteins, or antibody to
1 integrin stimulates ligand-independent tyrosine
phosphorylation of EGF receptors in the absence of receptor ligands and
association of
1 integrins with EGF receptors (57).
Evidence of cross-talk between GPCRs and EGF or PDGF receptors appears
to be cell type-specific and dependent, e.g. on the presence
or absence of cell surface EGF or PDGF receptors (55). We found no
LPA-induced phosphorylation of EGF and PDGF receptors in GD25 cells
despite good expression of both receptors and thus have no evidence for
transactivation of receptor tyrosine kinases through GPCRs in these
cells. Further, we found no evidence for adhesion-dependent
phosphorylation of EGF or PDGF receptors. However, we cannot exclude
the possibility that LPA treatment or
1-dependent adhesion caused activation of an
undetectably small but still functionally significant subpopulation of receptors.
A third type of cross-talk is between GPCRs and adhesion receptors.
Activation of Rho by LPA stimulation results in reorganization of actin
stress fibers, recruitment or phosphorylation of different focal
contact proteins including integrins, and formation of focal contacts
(58). PDGF or EGF initially activate Rac, which stimulates membrane
ruffling, and also leads to Rho-dependent responses (59). Stimulation of multiple coordinated pathways of actin remodeling and
coupling of remodeled actin to cell surface complexes would explain the
increased migration that we observed. Pretreatment of GD25 cells
expressing wild type
1A with pertussis toxin ablated both LPA-stimulated Erk1/2 activation and LPA-induced cell migration, whereas C3 exotransferase pretreatment partially inhibited LPA-induced cell migration but did not influence MAPK activation. These results indicate that MAPK activation by LPA in GD25 cells is a
Gi-Ras-Raf-mediated pathway, but that the
Gi-Ras-Raf pathway alone is not sufficient for cell
migration induced by LPA. Importantly, LPA did not sensitize cells
pretreated with pertussis toxin or C3 exotransferase to growth factors.
This finding suggests the potential participation of Rho in pathways
leading to Ras and Erk activation (60). Thus, both
Gi-Ras-Raf and G
12/13-Rho mediated pathways
seem critical for the sensitization induced by LPA.
The phenomenon described herein may be of considerable importance for
the migration of one group of cells in response to a growth factor
diffusing from a second distant group of cells. The sensitivity and
specificity of such communication would be enhanced considerably if the
first group of cells was sensitized to the product of the second group.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Julie Nowlen for
valuable technical help and Marcy Salmon for manuscript preparation.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL21644, HL56396, T32 GM07215 and fellowship funds from the Cell
Science Research Foundation (to T. S.).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: Depts. of Medicine
and Biomolecular Chemistry, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-1576; Fax: 608-263-4969; E-mail:
dfmosher{at}facstaff.wisc.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
LPA, lysophosphatidic acid;
EGF, epidermal growth factor;
Erk, extracellular
signal-regulated kinase;
GPCR, G-protein-coupled receptor;
MAPK, mitogen-activated protein kinase;
PDGF, platelet-derived growth factor;
RTK, receptor tyrosine kinase;
S1P, sphingosine-1-phosphate.
 |
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