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INTRODUCTION |
EGF1 triggers many
biological responses, including cell proliferation and differentiation
(1). In addition, EGF has been shown to induce the reorganization of
the actin cytoskeleton, and the EGF receptor has been found to be
associated with actin filaments (2-7). In this regard, EGF has been
reported to stimulate rapid cell rounding, extensive membrane ruffling,
extension of filopodia, retraction of cells from the substratum (8, 9), extensive cortical actin polymerization, and depolymerization of actin
stress fibers (10, 11). Moreover, numerous studies have shown that
activation of the EGF receptor leads to increased cell motility
(12-19) and production of ECM degrading proteases (20-23), thereby
supporting a role for the EGF receptor in normal development and
pathophysiological events such as tumor cell invasion and metastasis.
Cell migration plays a central role in a variety of biological
processes including embryonic development, angiogenesis, wound healing,
and tumor cell metastasis (24). Although the exact mechanisms of cell
migration are not well established, it is generally believed that
several coordinated events are involved, including morphological
polarization, membrane extension, formation of cell-substratum attachments, contractile force and traction, and release of attachments (24). The adhesive interactions between cells and various ECM substrates are likely to be critical in determining cell migration capacity (24, 25). Many investigations have shown that an intermediate
adhesive strength generates maximal cell migration (26-33). The
adhesion between cells and substrate are largely mediated by the
integrins, which are a family of cell surface heterodimeric receptors
that bind to ECM proteins such as laminin, fibronectin, and vitronectin
(34). Integrin expression, the affinity and specificity for their
ligands, and the integrin-cytoskeleton linkages are regulated by
various signals including those initiated by growth factors. In fact,
integrins and growth factor receptors share many common signaling
events, such as increased tyrosine phosphorylation, activation of
mitogen-activated protein (MAP) kinases, protein kinase C isoforms, and
small molecular weight GTP-binding proteins, as well as enhanced
Ca2+ fluxes (35-38). Given their common signaling
pathways, and the association of both EGF receptor and integrin systems
with the actin cytoskeleton, it is conceivable that the EGF receptor
can be involved in the modulation of integrin-mediated cellular
functions such as cell migration.
Previous analyses of the EGF receptor have revealed that the receptor
tyrosine kinase activity and the C-terminal autophosphorylation sites
are critical for EGF-stimulated signal transduction (1). The C-terminal
domain of the receptor exerts a competitive autoinhibitory restraint on
the receptor tyrosine kinase activity that can be removed by
autophosphorylation of the tyrosine residues located within this domain
(39, 40). Furthermore, the autophosphorylated tyrosine residues at the
EGF receptor C terminus provide anchoring sites for downstream Src
homology 2 (SH2) or phosphotyrosine binding (PTB) domain-containing
effectors that are involved in the transduction of EGF-stimulated
intracellular signals (1). Chen et al. (17, 18) have
reported that the receptor kinase activity and at least one C-terminal
tyrosine autophosphorylation site are required for cell movement. In
addition, EGF stimulation of phospholipase C-
and protein kinase C
has been linked to the EGF induction of cell motility (14, 18).
In the present study, we evaluated the effects of EGF on cell migration
and assessed the capacity of various EGF receptor constructs to
modulate the motility of mouse B82L-Parental fibroblasts that possess
no detectable endogenous EGF receptors. We found that EGF
synergistically stimulates integrin-mediated chemotaxis, and that the
co-positioning of EGF and the chemoattractant is critical for
EGF-stimulated migration. In particular, it is noteworthy that,
although B82L-Parental cells do not exhibit fibronectin-induced chemotaxis, they do adhere to fibronectin, but it is the introduction of an intact EGF receptor into these cells that allows for
fibronectin-induced motility. In addition, neutralizing anti-EGF
receptor antibodies inhibit cell migration toward fibronectin or
laminin alone. These findings demonstrate that the EGF receptor is
important for B82L fibroblast motility, suggesting that the EGF
receptor may act downstream of integrin activation and may directly
engage in the signal transduction events leading to cell migration.
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MATERIALS AND METHODS |
Reagents and Antibodies--
Mouse laminin was obtained from
Collaborative Research (Bedford, MA). Human fibronectin and vitronectin
were generous gifts from Dr. Deane F. Mosher (University of Wisconsin,
Madison, WI). Recombinant human EGF was purchased from Upstate
Biotechnology Inc. (UBI, Lake Placid, NY). Nucleopore membranes and the
48-well chemotaxis chamber were purchased from Nucleopore Corp.
(Pleasanton, CA). Anti-EGF receptor monoclonal antibodies were obtained
from the following sources: antibody 528 was affinity-purified from hybridoma cell line HB 8509 (ATCC, Rockville, MD), antibody C225 was
obtained from Imclone Systems, Inc. (New York, NY), and antibody LA22
was purchased from UBI. Neutralizing anti-mouse EGF and anti-human TGF-
antibodies were purchased from UBI and R&D Systems
(Minneapolis, MN), respectively.
Cell Culture Conditions--
Murine B82L-Parental fibroblasts
and those expressing the wild-type or the K721M or c'1022 EGF receptor
constructs were provided by Dr. Gordon Gill (University of California,
San Diego, CA). Mouse B82L-Parental fibroblasts were maintained in
Dulbecco's modified Eagle's medium (DMEM) containing 10% cosmic calf
serum (HyClone, Logan, UT). Cells transfected with wild-type or mutated EGF receptors were cultured in the same medium that contained 10 µM methotrexate because a mutant dihydrofolate reductase
gene was used as a selectable marker (41). The Clone B3 cells were derived from B82L-wt cells based on their ability to bind laminin (42),
and were cultured in the same medium as the B82L-wt cells. The GD25
cells are differentiated and immortalized cells derived from the
embryonic stem cell clone G201, which are deficient in the integrin
subunit
1 as the result of the introduction of a null
mutation in the
1 integrin gene via homologous
recombination (43). The stably transfected cell line
GD25-
1A was obtained by electroporating wild-type
integrin
1A cDNA into GD25 cells. In the present
study, the GD25 cells were cultured in nonselection medium consisting
of DMEM plus either 10% fetal calf serum (HyClone) or 10% cosmic calf
serum, whereas the GD25-
1A cells were continuously cultured in the same medium plus puromycin (10 µg/ml) (43). Both the
GD25 and the GD25-
1A cells were provided by Dr. Deane F. Mosher. Other cell lines used in this study, including human mammary
carcinoma cell line MDA468, human osteosarcoma cell line MG63 and human
epidermal carcinoma cell line A431, were cultured in DMEM plus 10%
fetal calf serum. 3T3-F442A fibroblasts were cultured in DMEM plus 10%
bovine calf serum (HyClone).
Cell Spreading Analysis--
Cell culture plates (24-well) were
coated with purified matrix or control proteins (laminin, 20 µg/ml;
fibronectin, 10 µg/ml; vitronectin, 10 µg/ml; polylysine, 10 µg/ml) diluted in phosphate-buffered saline using either an overnight
incubation at 4 °C or a 2-h incubation at 37 °C. Nonspecific
binding sites were blocked using a 2% (w/v) bovine serum albumin (BSA)
solution (in phosphate-buffered saline) and a 1-h incubation at
37 °C. The Clone B3 cells were detached by digestion with 0.1%
trypsin, and the cells were then resuspended in DMEM containing 0.1%
BSA. The cells were mixed with EGF (at the indicated final
concentrations), seeded at a density of 2 × 105
cells/ml, and incubated at 37 °C in a humidified incubator
containing 5% CO2. Samples were viewed using a light
microscope equipped with phase contrast (Nikon Inc., Instrument Group,
Melville, NY), and photographs were taken at different times
(magnification, ×40).
Cell Motility Assays--
The experiments assessing chemotactic
motility were performed using a 48-well migration chamber as described
previously (42). In these studies, cells were grown as described under
"Cell Culture Conditions" for 3-4 days. After being detached from
the plastic dishes using a 0.1% trypsin solution, the cells were
stabilized for 1 h at 37 °C in DMEM containing 0.1% BSA. Cells
were then counted and resuspended in DMEM plus 0.1% BSA at a final
concentration of 1 × 105 cells/50 µl. The lower
compartment of the migration chamber was filled with the indicated
proteins dissolved in DMEM + 0.1% BSA (29 µl/well), and the cells
were added to the upper compartment of the migration chamber (50 µl/well). To ascertain the effects of EGF, the growth factor was
added either to the upper or to the lower compartments at indicated
concentrations. To evaluate the effects of anti-EGF receptor, anti-EGF,
or anti-TGF-
antibodies, the antibodies were incubated with the
cells for 30 min before the cells were placed into the upper wells. In
all experiments, the two compartments of the migration chamber were
separated by a polycarbonate filter (5-µm pore size, Nucleopore
Corp.). The cells were allowed to migrate for 4 h at 37 °C in a
humidified atmosphere containing 5% CO2. The cells that
did not migrate through the membrane remained on the upper surface of
the filter and were removed mechanically by scraping; the migrant cells
on the lower surface were fixed in methanol/acetone (1:1) for 2 min,
and then stained with 1% crystal violet. The filters were
densitometrically analyzed using the OFOTO program (Light Source
Computer Images, Inc.) and quantified by Scanner Analysis (Biosoft,
Ferguson, MO).
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RESULTS |
To explore the effects of EGF on cell migration, mouse B82L
fibroblasts that lack measurable endogenous EGF receptors
(B82L-Parental) were transfected with wild-type EGF receptors (these
include two separate transfectants designated as B82L-wt and B82L-wt2)
or mutant human EGF receptors. A cell clone possessing laminin binding activity was also isolated from B82L-wt cells (labeled as Clone B3)
(42). The two EGF receptor mutants used in these studies include a
construct encoding a kinase-inactive receptor that contains a lysine to
methionine substitution at residue 721 (B82L-K721M), which is involved
in ATP binding, and a construct encoding a kinase-active receptor that
has been truncated at residue 1022 (B82L-c'1022), which lacks four
major receptor autophosphorylation sites. These cell lines were used to
explore the involvement of EGF receptor activation in cell migration
toward various ECM components.
Effect of EGF on B82L Fibroblast Spreading on ECM
Substrates--
Cell migration is a process that requires temporally
and spatially coordinated cell attachment and detachment (24, 25). Given the importance of adhesive strength between the cell and the
substrate in determining the migration speed and persistence, we tested
whether EGF could influence the adhesiveness of Clone B3 cells on
several ECM substrates. As shown in Fig.
1A, when Clone B3 cells were
plated onto fibronectin-coated wells in the presence of different
levels of EGF, the ability of these cells to spread on the fibronectin
surface was attenuated in a time- and EGF dose-dependent
manner. In the absence of EGF, nearly all the clone B3 cells were
attached to the fibronectin surface in the first 30 min. Within 1 h, the cells became flattened and exhibited the characteristic
protrusive structures of spreading cells, a process that was completed
by 2.5 h after plating. However, upon the addition of EGF, cell
spreading was impaired, i.e. the cells remained in a rounded
shape for a longer period of time and appeared to form fewer focal
contacts as assessed by immunofluorescent staining of vinculin (data
not shown). As also illustrated in Fig. 1A, the addition of
10 nM EGF delayed cell spreading until 1.5 h after
plating, whereas the presence of 50 nM EGF suppressed the
initiation of cell spreading to approximately 2.5 h after plating.

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Fig. 1.
EGF attenuates the spreading of Clone B3
fibroblasts, which contain the full-length active EGF receptor, on
various ECM substrates. A, the time- and EGF
dose-dependent spreading of Clone B3 fibroblasts on
fibronectin. Cells were collected by trypsinization and allowed to
attach and spread on wells coated with fibronectin (10 µg/ml) in the
absence or presence of the specified concentrations of EGF as described
under "Materials and Methods." The cells were photographed at the
indicated times using 40X magnification. B, EGF
dose-dependent spreading of Clone B3 fibroblasts on various
ECM components. Cells were collected by trypsinization and allowed to
attach and spread on wells coated with either fibronectin (10 µg/ml),
laminin (20 µg/ml), vitronectin (10 µg/ml), or the
non-integrin-activating protein polylysine (10 µg/ml) in the absence
or presence of the specified concentrations of EGF. At the end of a 2-h
incubation, the cells were photographed (original magnification, ×40).
For both A and B, analogous results were observed
in three other experiments.
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Because Clone B3 cells express receptors for other ECM proteins such as
laminin and vitronectin, we examined the effects of EGF on Clone B3
spreading onto laminin or vitronectin-coated wells. Similar to the
results shown in Fig. 1A, the addition of EGF was found to
dose-dependently reduce the capacity of the cells to spread
on all three substrates tested (Fig. 1B). It was also noted that the spreading of these mouse fibroblasts onto laminin- or vitronectin-coated wells was less extensive than that observed on
fibronectin-coated surfaces. Polylysine was used as a negative control
for cell spreading onto ECM components because cells attach to
polylysine without the activation of integrin-associated processes (45). Interestingly, EGF treatment had no effect on the attachment of
Clone B3 cells onto fibronectin and laminin, as measured by the number
of cells adhered to these ECM substrates (data not shown). These
results suggest that EGF does not substantially regulate the initial
interaction between the cell surface integrins, but that the later
events (e.g. focal contacts maturation and stress fiber
formation) are more likely to be EGF-sensitive.
Effect of EGF on Laminin-induced Chemotaxis of Clone B3
Fibroblasts--
Based on the ability of EGF to modulate the
adhesiveness of Clone B3 cells on various substrates, we investigated
whether EGF could regulate the chemotactic capacity of Clone B3 cells using a 48-well chemotaxis chamber. As shown in Fig.
2A, EGF synergized with
laminin in enhancing the migratory activity of these cells. This
synergistic effect of EGF on cell chemotaxis was about 2-3-fold above
that observed with laminin alone, whereas EGF alone had little or no
effect on cell migration. Interestingly, the effect of EGF on
laminin-induced chemotaxis could only be achieved when EGF was
co-present with laminin in the lower wells. When EGF was mixed with the
cells located in the upper wells, EGF was unable to augment
laminin-induced chemotaxis. Because the development of cell
polarization, such as the formation of distinct cellular leading and
trailing edges, is essential for directional cell movement (24), our
findings suggest that EGF, when co-present with laminin, may help to
enhance the cell polarization required for directional cell movement by
forming an EGF concentration gradient parallel to the laminin gradient.
Conversely, when EGF is present with the cells in the upper wells, the
cells were exposed to EGF from all directions, and thus may not be able
to enhance a polarization process. Although several studies have shown
that EGF can stimulate cell movement (14, 16-18, 46), this appears to
be the first report concerning the importance of EGF positioning in
promoting directional cell movement.

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Fig. 2.
The synergistic action of EGF in promoting
laminin-induced migration of Clone B3 fibroblasts. Cell migration
toward laminin was determined using a 48-well microchemotaxis chamber
as discussed under "Materials and Methods." In the experiments
using laminin as the chemoattractant, 200 µg/ml laminin was placed in
the lower wells. A, EGF is required to be co-positioned with
laminin to stimulate chemotaxis. EGF (0-10 nM) was mixed
either with the laminin in the lower wells or with B82L-Clone B3
fibroblasts in the upper wells. Cell motility induced by EGF alone
(placed in lower wells) was also measured. B, the direct
addition of EGF to the cells reduces the maximal migration conferred by
the EGF co-positioned with laminin. In the indicated cases
(filled circles), 1 nM EGF was added to the
lower wells, whereas the EGF concentration in the upper wells was
varied from 0 to 10 nM. The cell migration to laminin alone
(square) or in the absence of both EGF and laminin
(triangle) was also measured. In all cases, the data points
represent the mean ± S.D. of triplicate determinations. Similar
results were obtained in at least five different experiments.
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To further test the hypothesis that EGF-induced cell polarization is
important in facilitating cell migration toward laminin, EGF was added
to both the lower and upper wells, based on the rationale that the
uniform exposure of cells to EGF would disrupt the cell polarization
induced by EGF present with laminin in the lower wells. As shown in
Fig. 2B, the maximal cell migration induced by the
co-presentation of laminin and EGF (1 nM) in the lower wells was reduced in a dose-dependent fashion (33-85%)
when EGF (1-10 nM) was also mixed with cells in the upper
wells. These studies further support the concept that the stimulation
of cells with a co-gradient of laminin and EGF facilitates cell
polarization and efficient directional cell locomotion.
EGF Effects on Cell Migration with Multiple Cell Lines and ECM
Substrates--
Clone B3 cells were isolated by their ability to bind
laminin (42). To assess whether the effects of EGF on cell migration were restricted to laminin, we performed similar migration assays using
fibronectin and vitronectin as the chemoattractants. We found that EGF
addition to the upper wells had no measurable effects on fibronectin or
vitronectin-induced chemotaxis (data not shown); however, when EGF was
mixed with fibronectin or vitronectin in the lower wells, EGF
substantially elevated Clone B3 cell migration (Fig.
3, A and B).
Moreover, because Clone B3 cells were isolated from B82L fibroblasts
that were transfected with the human EGF receptor (B82L-wt) (42), we
compared the chemotactic capacity of Clone B3 and B82L-wt cells in
order to ascertain whether EGF stimulation of cell migration reflected
clonal variation. We observed that both cell types exhibited comparable
responses to EGF, fibronectin, and vitronectin (Fig.
3C).

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Fig. 3.
EGF stimulates the migration of Clone B3 and
B82L-wt cells toward multiple ECM components. Cell migration was
determined using a 48-well microchemotaxis chamber as discussed under
"Materials and Methods." Clone B3 fibroblast migration in response
to laminin (200 µg/ml), fibronectin (100 µg/ml) (A), or
vitronectin (100 µg/ml) (B) was measured in the absence or
presence of EGF in the lower wells (co-positioned with the
chemoattractant). C, the migration of B82L-wt fibroblasts
toward fibronectin (100 µg/ml) or vitronectin (100 µg/ml) was
assessed in the absence or presence of EGF in the lower wells
(co-positioned with the chemoattractant). For each experiment, the
bars represent the mean (± S.D.) of triplicate
determinations. Similar results were obtained in at least three
different experiments.
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The arginine-glycine-aspartate (RGD) sequence on many matrix proteins,
including fibronectin and vitronectin, has been shown to interact
directly with cell surface receptor integrins (34). Synthetic RGDS
peptides can block such matrix-integrin interactions. To identify
whether ECM-induced chemotaxis was mediated by integrins, Clone B3
cells were mixed with RGDS peptides before they were placed into the
chemotaxis chamber. As shown in Fig. 4,
the migration of Clone B3 cells toward fibronectin or vitronectin in
the absence or presence of EGF were almost completely blocked by RGDS
peptides but not by the control peptide SDGRG, whereas laminin-induced migration was not affected by RGDS peptides at the same concentrations (data not shown). This observation is consistent with the previous finding that, although the RGD sequence is present in the A chain of
murine laminin, it is cryptic, and therefore not accessible in native
laminin (47, 48). In addition, the laminin receptor on Clone B3 cells
mediating motility has been identified to be
6-containing integrins (42), whose recognition sites on
laminin are different from the RGD-containing region (49). These
results support the concept that fibronectin- or vitronectin-induced
chemotaxis of Clone B3 cells is mediated by integrins.

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Fig. 4.
The fibronectin- and vitronectin-induced
chemotaxis of Clone B3 fibroblasts is dependent on the broadly
selective integrin-binding sequence RGDS. The cells were mixed
with RGDS peptides at the indicated doses before they were loaded into
the microchemotaxis chamber. Cell migration toward fibronectin (100 µg/ml) (A) or vitronectin (100 µg/ml) (B) in
the absence or presence of EGF (1 nM) in the lower wells
(co-positioned with the chemoattractants) was determined as described
under "Materials and Methods." Each point represents the mean ± S.D. of triplicate determinations. Comparable results were obtained
in three different experiments.
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To verify that the EGF stimulation of chemotaxis is not limited to B82L
cells, we examined the chemotactic behavior of another mouse
fibroblastic cell line in response to EGF treatment, namely GD25 cells.
These cells were derived from the mouse embryonic stem cell clone G201,
which are deficient in the integrin subunit
1 due to the
introduction of a null mutation in the
1 integrin gene.
In addition, we evaluated a related cell line, i.e.
GD25-
1A cells, which were derived from GD25 cells that
were transfected with the integrin
1A gene and have
laminin binding ability. Both cell lines attach to fibronectin and form
focal contacts that contain
v
3 integrins
(43). As shown in Fig. 5, we observed that the migration of both GD25 and GD25-
1A cells toward
fibronectin (Fig. 5A) and vitronectin (Fig. 5B),
and of GD25-
1A cells toward laminin (Fig.
5A), was stimulated by the addition of EGF (1 nM) to the lower wells. There was no detectable difference
between the two cell lines in terms of their migration toward ECM
components and their EGF responses. Moreover, in all other cell lines
tested, as long as the cells were able to migrate toward a given ECM
component, EGF could further enhance their migration when it was
co-positioned with the ECM component, and had no effect when mixed with
the cells. Specifically, these cell lines include the 3T3-F442A murine fibroblasts, the human osteosarcoma cell line MG63, the human mammary
carcinoma cell line MDA468, and the human epidermal carcinoma cell line
A431 (data not shown). The broad spectrum of cell lines that are able
to respond to EGF in terms of their migration toward ECM components
suggests that the synergistic effect of EGF on chemotaxis is a general
phenomenon.

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Fig. 5.
EGF synergistically stimulates the migration
of GD25 and GD25- 1A murine
fibroblasts toward multiple ECM substrates. Cell migration was
determined using a 48-well microchemotaxis chamber as discussed under
"Materials and Methods." The migration of GD25 and
GD25- 1A fibroblasts toward medium alone, EGF (1 nM) alone, or laminin (100 µg/ml) (A),
fibronectin (100 µg/ml) (B), and vitronectin (100 µg/ml)
(C) in the absence or presence of EGF (1 nM), in
the lower wells (co-positioned with the chemoattractant) was measured.
Each bar represents the mean (± S.D.) of triplicate determinations.
Similar results were obtained in three different experiments.
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EGF Receptor Expression Is Critical for Fibronectin-induced Cell
Migration--
To identify EGF receptor elements that are necessary
for mediating the stimulatory effects of EGF on integrin-mediated
chemotaxis, B82L-Parental cells, which contain no detectable endogenous
EGF receptors, were used as a negative control for cells transfected with either wild-type or mutated EGF receptors. B82L-Parental cells
have been shown to adhere and spread on fibronectin-coated surfaces
(42), indicating that they express functional fibronectin receptors.
Although it was predicted that the B82L-Parental cells would migrate
toward fibronectin but not show EGF responses due to the lack of EGF
receptor expression, we instead unexpectedly observed that these cells
exhibited little detectable migration toward fibronectin (± EGF) (Fig.
6, A and B). In
addition, cell migration was not observed, even when the concentrations
of fibronectin and EGF were varied from 50 to 200 µg/ml and from 0.1 nM to 10 nM, respectively (data not shown).
However, the introduction of functional EGF receptors into these cells
clearly enabled them to migrate toward fibronectin alone (B82L-wt or
Clone B3) (Fig. 6A; also see Figs. 2 and 3). To evaluate
whether these responses were unique to the B82L-wt transfectant and
Clone B3 cells, we generated a separate population of transfected B82L
cells expressing wild-type EGF receptors, which we termed B82L-wt2. As
shown in Fig. 6B, B82L-wt2 cells were also capable of
migrating toward fibronectin, albeit at a lower level than that
observed with B82L-wt cells. These observations indicate that the
introduction of the wild-type EGF receptor into B82L fibroblasts enable
them to switch from a non-migratory cell type into a migratory one.

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Fig. 6.
Expression of wild-type EGF receptors is
required for B82L fibroblasts to migrate toward fibronectin. In
panels A and B, B82L-Parental cells
expressing no endogenous EGF receptor were transfected on two separate
occasions with the wild-type (full-length) EGF receptor designated wt
(A) and wt2 (B). Clone B3 cells were used as
positive control for fibronectin-induced chemotaxis in response to EGF.
The migration of B82L cells toward medium alone, EGF alone (1 nM), fibronectin (100 µg/ml) alone, fibronectin (100 µg/ml) and EGF (1 nM) co-positioned in the lower wells,
or fibronectin in the lower well and EGF in the upper well
(co-positioned with the cells), was measured. In panel
C, the effects of neutralizing anti-EGF or anti-TGF-
antibodies on the fibronectin-induced migration of B82L-Clone B3 cells
was determined. Clone B3 cells were incubated with anti-EGF and/or
anti-TGF- monoclonal antibodies at the indicated concentrations
(legend at right) (for 0.5 h before they were loaded to
the migration chamber. Each bar represents the mean ± S.D. of
triplicate determinations. Similar results were obtained in two
different experiments.
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In these studies, it is conceivable that the necessity of the EGF
receptor for B82L cells to migrate toward fibronectin alone may reflect
an artifact of EGF receptor transfection, e.g. EGF receptor
overexpression may lead to ligand-independent activation of the EGF
receptor. However, in our system, we found that the EGF receptor is
expressed at ~100,000 molecules/cell (42), which is within the
physiological range, suggesting that the EGF receptor in B82L cells is
not likely to be activated due to the overexpression of the receptor. A
second possibility, which may be of physiological significance, is that
the cells may secrete an EGF receptor ligand such as EGF or TGF-
,
which in turn stimulates fibronectin-induced migration in an autocrine
manner. Although this feature would still strongly support the notion
that the EGF receptor is critical for cell migration, it was important
to assess if this mechanism was occurring in our system. To examine
this possibility, antibodies that can neutralize the most common EGF
receptor ligands, EGF and TGF-
, were used to assess whether they
could reduce the migration of Clone B3 cells toward fibronectin. As
shown in Fig. 6C, neither anti-EGF nor anti-TGF-
antibodies, alone or together, at the concentrations tested, exhibited
any effects on the fibronectin-induced migration of Clone B3 cells.
These results suggest that the capacity of B82L cells expressing the
EGF receptor to migrate toward fibronectin alone does not appear to
result from an EGF or TGF-
autocrine activation of the EGF receptor.
The EGF Receptor Kinase Activity and C Terminus Are Important for
Conferring Fibronectin-induced Chemotaxis--
Upon EGF binding, EGF
receptor autophosphorylation is linked to a cascade of signaling events
leading to cellular responses such as cell proliferation and cell
motility (50). Both the receptor kinase activity and the C terminus,
which provides docking sites for SH2 and PTB domain-containing cellular
proteins, are critical for EGF-induced signal transduction (1). Chen
et al. (17, 18) have shown that EGF-elicited random cell
movement requires receptor tyrosine kinase activity and
autophosphorylation. In order to map EGF receptor regions that are
involved in the synergistic stimulation of EGF on integrin-mediated
motility, B82L-Parental cells were transfected with EGF receptors that
are kinase-inactive (B82L-K721M) or C-terminally truncated
(B82L-c'1022). As shown in Fig.
7A, B82L-K721M cells did not
migrate toward fibronectin alone, nor did they respond to EGF
stimulation. Similar results were obtained with B82L cells expressing
EGF receptors that retain only one of the five C-terminal
autophosphorylation sites (c'1022) (Fig. 7B), although
these cells exhibit increased kinase activity due to the removal of the
C-terminal autoinhibitory restraint (51, 52). The observation that the
migratory behavior of K721M or c'1022 cells are indistinguishable from
the Parental cells suggest that both the receptor tyrosine kinase
activity and the C-terminal region are important for the EGF-induced
enhancement of cell motility and for the chemotaxis induced by
fibronectin alone. In addition, Tyr-992, which has been shown to be
sufficient to confer EGF-elicited random cell movement (17), does not
appear to be sufficient to support EGF-stimulated chemotaxis,
suggesting that EGF regulation of chemokinesis and chemotaxis may
involve different signaling mechanisms.

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Fig. 7.
The EGF receptor tyrosine kinase activity and
the C-terminal region are important for conferring fibronectin-induced
chemotaxis in B82L fibroblasts. B82L-Parental cells expressing no
endogenous EGF receptor were transfected with an EGF receptor that
carries a lysine to methionine substitution at position 721, which
abolishes the receptor kinase activity (K721M) (A), or a
kinase active EGF receptor that has been C-terminally truncated from
residue 1022 through residue 1186 (c'1022) (B). This mutant
lacks four of the major EGF receptor tyrosine autophosphorylation
sites. The migration of B82L cells toward medium alone, EGF alone (1 nM),
fibronectin (100 µg/ml) alone, fibronectin (100 µg/ml) and EGF (1 nM) co-positioned in the lower wells, or toward fibronectin
placed in the lower well and EGF added to the upper well (co-positioned
with the cells), was measured. Each bar represents the mean (± S.D.)
of triplicate determinations. Similar results were obtained in three
different experiments.
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Effects of Neutralizing Anti-EGF Receptor Antibodies on Cell
Migration--
Our studies have shown that the expression of
functional EGF receptors is critical for the induction of motility in
B82L fibroblasts. These results suggest that the EGF receptor may be
used as a downstream mediator by integrins in transducing signals
leading to cell migration. Such ligand-independent activation of the
EGF receptor by other receptor systems has been reported for
G-protein-coupled-receptor in Rat-1 cells (53). Conversely, the EGF
receptor could act upstream of integrins, for example, by regulating
integrin expression, activation or coupling to key components involved
in cell motility. To further explore this system, we examined the
effect of neutralizing anti-EGF receptor antibodies on Clone B3 cell
migration. Monoclonal anti-EGF receptor antibodies (LA22, 528, and
C225) were used that recognize the EGF binding site, compete for EGF
binding, and block EGF-induced receptor autophosphorylation. As shown
in Fig. 8 (A and
B), neutralizing anti-EGF receptor antibodies inhibited not only EGF stimulation of fibronectin-induced cell migration, but also
cell migration induced by fibronectin alone. This effect appeared to be
overcome as the EGF concentration was increased. These results support
the hypothesis that the EGF receptor may participate, at least in part,
as a downstream mediator in the integrin-stimulated signaling pathways
leading to cell motility. This concept is consistent with the studies
of Klemke et al. (14), where the EGF-selective inhibitor
tyrphostin 25 blocks human carcinoma cell migration on vitronectin.

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Fig. 8.
Neutralizing anti-EGF receptor antibodies
inhibit migration of B82L-Clone B3 cells toward fibronectin
or laminin. A, cell migration toward fibronectin (50 µg/ml) alone, or fibronectin (50 µg/ml) together with different
doses of EGF (0-1 nM) was measured. Cells were incubated
with anti-EGF receptor monoclonal antibodies LA22 (10 or 100 nM) for 0.5 h before they were loaded to the migration
chamber. B, cell migration toward fibronectin alone (0-200
µg/ml) was measured. Cells were incubated with anti-EGF receptor
monoclonal antibodies LA22 (30 nM), 528 (30 nM), or C225 (30 nM) for 0.5 h before they
were loaded to the migration chamber. C, cell migration
toward laminin (200 µg/ml) alone, or laminin (200 µg/ml) together
with different doses of EGF (0-1 nM) was measured. Cells
were incubated with anti-EGF receptor monoclonal antibodies LA22 (10 or
100 nM) for 0.5 h before they were loaded to the
migration chamber. D, cell migration toward laminin (200 µg/ml) alone, or laminin (200 µg/ml) and EGF (1 nM)
together was measured. Anti-EGF receptor monoclonal antibodies 528, LA22, or nonspecific IgG at 10 nM were added either to the
cells in the upper wells or to the laminin in the lower wells. LA22,
528, and C225 can compete with EGF for the EGF receptor binding and
block EGF-induced autophosphorylation of the EGF receptor. Each point
represents the mean ± S.D. of triplicate determinations. Similar
results were obtained in two different experiments.
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When the effects of anti-EGF receptor antibodies on laminin-induced
cell migration were examined, we observed that the addition of
monoclonal anti-EGF receptor antibodies LA22 and 528 to the cells not
only reversed EGF stimulation of laminin-induced chemotaxis, but also
blocked cell migration toward laminin alone (Fig. 8, C and
D). As also shown in Fig. 8D, a relatively weaker
inhibition of cell migration was observed when the anti-EGF receptor
antibodies were put into the lower wells with laminin. This may result
from the lower effective antibody concentration available to the cells due to diffusion and dilution of the antibodies upon entry into the
upper wells. These results appear to be specific because only anti-EGF
receptor antibodies could block cell motility induced by laminin alone
whereas nonspecific IgG had no effect (Fig. 8D). Trypan blue
exclusion assays showed that all the cells treated with or without the
antibodies exhibited similar viability (data not shown), indicating
that the inhibition of laminin-induced cell motility by anti-EGF
receptor antibodies does not appear to be due to cell death.
 |
DISCUSSION |
In the present study, we demonstrate that EGF can potently
stimulate the motility of mouse B82L fibroblasts that express a full-length EGF receptor. Furthermore, these investigations reveal that
the co-positioning of EGF and the chemoattractant is critical for EGF
stimulation of cell migration, i.e. it is essential that the
cell is exposed to an EGF and substrate gradient oriented in the same
direction. This EGF-dependent stimulation of cell motility
may in part be due to the cell rounding and the reduction of cell
spreading and focal adhesion formation caused by EGF treatment. Another
major observation made in these studies is that a fully functional EGF
receptor appears critical for integrin-mediated migration, in
particular, the EGF receptor C-terminal domain and an intact kinase
domain are critical for fibronectin and fibronectin plus EGF-stimulated
cell migration. In this regard, it is noteworthy that B82L-Parental
cells that do not express the EGF receptor exhibit little
fibronectin-induced chemotaxis, whereas the introduction of the
wild-type EGF receptor enables these cells to now undergo fibronectin-induced migration. Moreover, the inhibition of fibronectin or laminin-induced chemotaxis by neutralizing anti-EGF receptor antibodies supports the concept that the EGF receptor may act downstream of integrin activation.
Many studies have shown that cell migration is a process that requires
dynamic interactions between the cell, the substrate, and the
cytoskeleton-associated motile apparatus (24, 25). Reduced cell
motility can result if cell-substratum interactions conferred by growth
factor/cytokine (32, 33) or anti-integrin antibody stimulation (28,
29), as well as by modulation of the levels of substrates (26, 27),
integrins (30, 31), or cytosketal proteins present at focal contacts
are too strong or too weak. These observations are consistent with
earlier findings that EGF can induce rapid cell rounding (9), membrane
ruffling and retraction (8), and the promotion of extensive cortical actin polymerization and depolymerization of actin stress fibers (10,
11). The present studies suggest that an EGF-induced reduction of
adhesiveness may augment the motility of B82L fibroblasts expressing
functional EGF receptors as well as the mobility of other
EGF-responsive cell types.
The synergistic action of EGF and matrix proteins to enhance fibroblast
motility suggests that an interaction exists between the signaling
pathways triggered by the activation of these two receptor systems. One
possible site of convergence could be at the level of the tyrosine
kinase Src, which has been reported to modulate EGF-induced mitogenesis
(54), actin-cytoskeleton reorganization (via the small molecular weight
G-protein Rho) (55), and integrin-initiated signal transduction. Of
note, the transformation of chicken embryo fibroblasts by the Rous
sarcoma virus is associated with a general loss of substratum adhesion, changes in cell shape, and the disorganization of the cytoskeleton (56), all of which are similar to those observed in EGF-treated cells.
Moreover, the integrin
1 subunit is
tyrosine-phosphorylated in v-Src-transformed cells, and the highly
homologous integrin
3 subunit has been reported to be
tyrosine-phosphorylated (57) and to bind the Shc PTB domain upon
platelet aggregation (58). Tyrosine-phosphorylated
1
integrin subunit exhibits a decreased ability to bind to both
fibronectin and talin (59), implying that the phosphorylated
1 subunit may be able to escape from focal contacts. The
release of
1 integrins from focal adhesions may result
in the disruption of focal contacts, and a reduction of adhesion
strength that allows the cell to enter a motile state. In fact, certain
Tyr to Phe mutations in the
1 cytoplasmic domain results
in the loss of cell motility (60). Although the
1
subunit could be a direct substrate of Src, it is also likely that the phosphorylation of
1 is mediated by other kinases such
as focal adhesion kinase (FAK), which has been shown to bind a peptide mimicking the
1 cytoplasmic domain (61). Additionally,
Src and the Src family kinase Fyn are known to form stable complexes with FAK through phosphotyrosine-SH2 domain interactions, thereby bringing these kinases into focal contacts (62-64). Src can also phosphorylate FAK at additional sites to fully activate its tyrosine kinase activity (65, 66). Therefore, as a downstream mediator of both
EGF receptor and integrin activation, Src or Src family kinases may
integrate signals from both receptor systems, thus leading to events
such as the release of phosphorylated integrins from focal contacts,
thereby reducing cell adhesiveness and enhancing cell motility.
The observation that a non-polarized addition of EGF to the cells does
not affect cell migration (Fig. 2) suggests that a generalized
EGF-induced attenuation of cell adhesion strength is not sufficient to
fully explain EGF action. EGF increases cell migration toward matrix
proteins only when EGF and the chemoattractant are co-presented to the
cells at the same time and in the same direction. The exposure of the
cell to EGF and chemoattractant gradients originating from the same
direction is likely to augment the cell polarization known to be
necessary for directional cell movement (24). When EGF is added
directly to the cells, cell polarization can be generated only by the
chemoattractant and not by an EGF gradient, and thus EGF does not
increase cell migration.
EGF-enhanced cell polarization may occur in several non-exclusive ways.
For example, a high local EGF concentration at the leading edge of a
migrating cell may facilitate the induction of various active membrane
processes. EGF is known to promote F-actin redistribution (10, 11) and
the formation of lamellipodia and filopodia (8, 9), which are processes
occurring at the leading edge of migrating cells (24). In addition, EGF
may induce integrin association/interaction with the EGF receptor at
the cell leading edge, and thus generate an asymmetric distribution of
integrins when the cells are co-exposed to an EGF gradient. Growth
factors such as PDGF (58) and insulin (67) have been reported to induce
the association of integrins (such as
v
3) with these growth factor receptors. Furthermore, as integrins engaged
in cell migration become clustered at the leading edge in the form of
macroaggregates (68, 69), EGF receptors may co-cluster with the
integrins at the cell leading edge when the cells are exposed to a
chemoattractant gradient. The idea that the clustering of EGF receptors
by activated integrins could be involved is supported by the
observation that an accumulation of EGF receptors occurs on
fibronectin-coated beads where integrin interaction with fibronectin
takes place (45). Moreover, a recent report by Moro et al.
(70) indicates that
1 integrin stimulation can enhance
EGF receptor tyrosine phosphorylation. The consequences of this EGF
receptor distribution/activation on the leading edge of the cell
surface may then be further amplified by the higher concentration of
EGF at the cell front when cells are exposed to an EGF gradient in
addition to a chemoattractant gradient. The enriched distribution of
activated EGF receptors at the leading edge may trigger asymmetric
phosphorylation of cellular proteins resulting in an augmentation of
the cell polarization induced by chemoattractant alone. This hypothesis
is consistent with the observation that tyrosine-phosphorylated
proteins are found at the tips of growth cone filopodia and that this
process is correlated with the length of the filopodia (71).
Another critical observation of this study is that B82L fibroblasts
appear to require an intact EGF receptor to efficiently migrate toward
fibronectin alone. B82L-Parental cells contain functional fibronectin
receptors in that they are able to adhere to a fibronectin-coated
surface (42), yet they are essentially non-migratory.
Interestingly, the introduction of the wild-type EGF receptor into
these cells converts them from a non-migratory cell type into a
migratory one. Although we cannot rule out the possibility that the
transfection of intact EGF receptor may alter the expression of certain
proteins that are critical for cell motility, the present
investigations support the concept that the EGF receptor can act
downstream of integrin activation and serves as a key component of the
motility machinery of B82L fibroblasts. In this regard, if the EGF
receptor is not directly involved in cell migration, the blockade of
EGF receptor function should not abolish cell migration ability, yet
anti-EGF receptor antibodies can block cell migration toward
fibronectin alone.
Altogether, these observations suggest that the EGF receptor may be
activated in an EGF-independent manner upon fibronectin engagement and
that the EGF receptor plays a direct role in the migration of these
cells. This situation may be analogous to that seen in other systems,
i.e. the EGF receptor has been reported to be activated by
G-protein-coupled receptors (53) and by UV light (72). Moreover,
Miyamoto et al. (45) and Moro et al. (70) have
shown an interaction between integrins and growth factors for
triggering the tyrosine phosphorylation of the EGF receptor under
various conditions. Although this kind of cross-talk between the EGF
receptor and integrins may be accomplished by intervening proteins such
as Src or FAK (discussed above), it may also occur directly between the
two receptors. The two NPXY motifs in the cytoplasmic domain
of the integrin
1 subunit are homologous to the tyrosine
autophosphorylation sites on the EGF receptor (1, 34), indicating that
the
1 integrin and the EGF receptor may interact
directly or may be modulated by a common system. Similarly, a physical
association between integrin
v
3 and
growth factor receptors such as the PDGF receptor and the insulin
receptor substrate (IRS-1) upon PDGF or insulin treatment (58, 67, 73)
has been reported. The present study provides evidence for an EGF
receptor-integrin system interaction, which is consistent with earlier
observations that the EGF receptor interacts directly with
-catenin, a vinculin homologous protein involved in cell-cell
adhesion (74), and that EGF receptors are accumulated and
potentially activated by integrins (45, 70).
Inactivation of the EGF receptor kinase activity by replacing Lys-721
with Met reduces EGF-induced tyrosine phosphorylation of cellular
proteins, including phospholipase C-
, and inhibits EGF-induced
receptor internalization. Although cells expressing this tyrosine
kinase negative EGF receptor still bind EGF with the same affinity, and
exhibit EGF-induced MAP kinase activation (75), EGF-stimulated cell
proliferation is blocked (76). Our study indicates that an intact
kinase domain appears essential for cell migration as well. The
inability of this kinase-inactive mutant to mediate cell motility may
reside in the transient nature of EGF-induced MAP kinase activation
(75), which has been shown to be important for focal adhesion
disassembly (77), and/or in the blockage of activation of phospholipase
C-
, which has been shown to be important for mediating random cell
movement (17, 18) and haptotaxis (14). Unlike the kinase-negative mutant, the c'1022 mutant EGF receptor mediates enhanced tyrosine phosphorylation of multiple cellular substrates (39), it undergoes normal receptor internalization and Ca2+ mobilization (78),
and it can be tyrosine-phosphorylated (39) upon EGF treatment. The
observation that c'1022-expressing cells cannot migrate suggest that
an SH2- or PTB domain-containing protein that binds to the C-terminal
autophosphorylation sites is involved in mediating signals in the
motility pathway, or that the altered substrate specificity of this
mutant may modify the activity of key effectors that regulate the
motility pathway.
One additional issue that is related to the present work is that the
inhibition of fibronectin- and laminin-induced chemotaxis by
function-blocking anti-EGF receptor antibodies could arise from EGF
contamination of the ECM preparations, i.e. EGF accounts for
part of the ECM-induced chemotaxis. Although this scenario would still
strongly support a key role for the EGF receptor system in B82L cell
migration, the argument of EGF contamination is not supported by the
observations that EGF alone, at all the concentrations tested, induced
little chemotaxis, that EGF was not detected in the laminin
preparations as assessed by immunoblotting using anti-EGF and TGF-
antibodies, and that anti-EGF or TGF-
antibodies do not alter
fibronectin-induced chemotaxis. A related issue is that because laminin
contains EGF-like motifs and it may bind EGF receptor to induce cell
migration, and that the anti-EGF receptor antibodies may block
laminin-induced chemotaxis by abolishing laminin binding to the EGF
receptor. However, this is not likely because intact laminin (79) and a
peptide encompassing the EGF-like fragment of laminin (80) cannot
compete with EGF receptor binding. Thus, the data are most indicative
of a system wherein anti-EGF receptor antibodies eliminate
laminin-induced chemotaxis by binding to the EGF receptor and
antagonizing the receptor dimerization/activation, thereby suggesting
that EGF receptor function is directly important for ECM-induced chemotaxis.
In summary, EGF can exert a synergistic effect on integrin-mediated
chemotaxis, and this process requires the co-exposure of the cell to
both an EGF and an ECM substrate gradient. We also provide evidence to
support the concept that the EGF receptor can act downstream of
integrin activation and may play a direct role in cell migration toward
matrix proteins. These events are not only likely to be critical for
both normal growth and development (e.g. embryogenesis,
organ development, and wound healing), but also may be critical for
some of the pathobiological actions of EGF, especially given that the
aberrant expression of the EGF receptor, and the other members of the
erbB receptor family, have been implicated in cancer progression. In
the latter case, whereas tumor progression may result from the strong
mitogenic effects ascribed to EGF, our study suggests that the
chemotactic effects of EGF could contribute to tumor cell metastasis.
Given that integrins appear to transmodulate the EGF receptor, abnormal
EGF receptor expression in various tumor cells may contribute to their
metastatic potential.