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Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611
Address all correspondence and requests for reprints to: Dr. Laurie G. Hudson, Department of Molecular Pharmacology and Biological Chemistry, Searle 8–565, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611.
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Abstract |
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5-fold)
EGF receptor levels. Within a single SCC line, submaximal
concentrations of EGF or reduction of EGF receptor activity by an
anti-EGF receptor neutralizing antibody resulted in delayed kinetics of
in vitro reepithelialization. Thus, suppression of EGF
receptor activity in an overexpressing SCC line restores a migratory
response that more closely resembles that of normal keratinocytes.
Conversely, ligand-induced colony dispersion was augmented in stable
clonal cell lines in which EGF receptor expression was elevated after
introduction of an EGF receptor complementary DNA construct.
Collectively, these findings suggest that the migratory potential of
keratinocytes is modulated at the level of both receptor expression and
ligand concentration, with a positive correlation between EGF receptor
levels and ligand-induced cell motility. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TGF), in transgenic mice promotes hyperplasia of
many epithelial tissues (2, 6, 7). In addition to the well characterized mitogenic actions of EGF, the EGF receptor mediates chemotaxis and migration in a number of different cell types (8, 9, 10, 11). Cell migration is an important component of embryogenesis and wound healing, and a role for EGF receptor activity is suggested by modulation of ligand expression or the EGF receptor itself during these processes (12, 13, 14). For example, in keratinocytes, ligands for the EGF receptor augment reepithelialization in vitro (15) and wound healing in vivo (8, 16, 17). Interestingly, the EGF receptor concentration is transiently up-regulated approximately 5-fold at the borders of tape-stripped wounds, and this transient increase in receptor levels is hypothesized to play a functional role in wound healing (14).
Constitutive overexpression of the EGF receptor is observed in many tumors, particularly squamous cell carcinoma (SCC) (18). In certain tumors, elevated EGF receptor levels have been associated with tumor progression and increased invasive or metastatic potential (18). Development of an invasive phenotype involves alterations in cell:cell contacts, cell:substrate adhesion, and proteolytic degradation of the extracellular matrix in addition to tumor cell migration (19). There is considerable evidence that the EGF receptor signaling pathway is involved in each of these cellular processes, which are required for invasion and metastasis (20, 21).
Based on evidence for transient and sustained changes in EGF receptor expression in epidermal wound healing and SCC, respectively, we wanted to determine the role of EGF receptor abundance or activity on modulation of keratinocyte migration. In our studies, we observe a positive correlation between EGF receptor levels and subsequent ligand-induced motility in several human SCC lines. Reduction of EGF receptor activity in an overexpressing SCC line suppressed ligand-dependent migration to more closely resemble the response observed for normal keratinocytes. Conversely, elevation of EGF receptor levels through stable introduction of an EGF receptor expression vector markedly enhanced ligand-induced motility. These results suggest a direct relationship between EGF receptor expression and ligand-mediated cell locomotion. Thus, we propose that either transient or sustained elevation of EGF receptor levels may contribute to the migratory potential of keratinocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was purchased from Life
Technologies. Neutralizing anti-EGF receptor antibody (LA1) was
obtained from Upstate Biotechnology (Lake Placid, NY). Mitomycin C was
purchased from Sigma Chemical Co. (St. Louis, MO). For all experiments
involving growth factor addition, normal keratinocytes were transferred
to keratinocyte SFM without EGF or bovine pituitary extract, and SCC
cells were placed into DME:F12 containing 0.1% BSA for 48 h
before growth factor addition. SCC lines 9 and 25 were obtained from
the American Type Culture Collection (Rockville, MD), SCC line 13 was
provided by Dr. Kathleen Green (Northwestern University, Chicago, IL),
SCC line 12F were provided by W. A. Toscano, Jr. (Tulane University,
New Orleans, LA), and A431 cells were obtained from Gordon N. Gill
(University of California-San Diego).
Transfection and isolation of stable cell lines
Subconfluent SCC 13 cells grown on 6-cm plastic tissue culture
plates were transfected with an expression vector containing the
neomycin resistance gene (pRSV-neo) or a vector containing the EGF
receptor complementary DNA (pRcCMV-EGFR, generously provided by Dr.
Gordon N. Gill, University of California-San Diego) (23) using a
modification of a lipid-mediated protocol (24). Cells were washed twice
with serum-free medium followed by the addition of 1.5 ml serum-free
medium containing 15 µg expression vector and 45 µl of a lipid
solution containing 300 µg/ml
L--phosphatidylethanolamine dioleoyl (C18:1,
cis-9), and 700 µg/ml dimethyldioctadecyl ammonium
bromide. Cells were incubated for 4 h at 37 C with intermittent
gentle agitation to facilitate DNA distribution. After this incubation,
2 ml DME:F12 containing 5% iron-supplemented defined calf serum
(complete DME:F12) were added to each plate, and the cells were
incubated for an additional 2 h. Cells were then subjected to
glycerol shock (10% glycerol in medium) for 3 min at room temperature,
rinsed twice with medium, and placed in complete DME:F12. Stable clones
were selected in 600 µg/ml G418 (Life Technologies), and at least 10
independent cell lines from 2 separate transfections were isolated for
each vector.
Photography
Photographs of cell cultures were taken at a magnification of
x10 or x25 using a Nikon N2000 camera mounted upon a Nikon
Diaphot-TMD inverted phase contrast microscope (Nikon Corp., Melville,
NY). Results shown are representative of at least three independent
experiments.
Measurements of cell motility
Evaluation of colony dispersion (cell scattering) and in
vitro reepithelialization were performed as previously described
(15). Briefly, cells were subcultured and maintained in growth medium
until colonies of greater than 16 cells were established. Cultures were
deprived of growth factors and serum for 24 h before treatment
with or without EGF at the concentrations and times indicated in the
figure legends. Colony dispersion was documented by photography. For
evaluation of in vitro reepithelialization, confluent cell
monolayers were deprived of serum and growth factors for 24 h, and
a cell-free area was introduced by scraping the monolayer with a
standard dimension blue pipette tip (Research Products International
Corp., Mount Prospect, IL) followed by extensive washing to remove
cellular debris. In vitro reepithelialization was monitored
by repopulation of the cleared area (wound width typically between
200–300 µm) with cells over time. To assess the contribution of cell
migration to in vitro reepithelialization in the absence of
proliferation, experiments were conducted in cells pretreated with
mitomycin C (10 µg/ml for 2 h). This treatment with mitomycin C
has been previously shown to inhibit EGF-induced mitogenesis (15).
Western blot analysis
Cells were washed with ice-cold 1 x Hanks’ Balanced Salt
Solution containing 1 nM NaVO3 and lysed in
2 x sample buffer [20 mM Tris-HCl (pH 8.0), 2
mM EDTA, 2% SDS, 1% ß-mercaptoethanol, 0.002%
bromophenol blue, 20% glycerol, and 1 mM
NaVO3]. The samples were boiled for 5 min, and the total
protein concentration was quantitated using the Lowry protein assay as
modified by Peterson (25). Equal protein for each sample was separated
by electrophoresis through a 7.5% SDS-polyacrylamide gel. The protein
was transferred onto polyvinyl difluoride membranes (Millipore Corp.,
Bedford, MA) and blocked with 0.25% gelatin in 10 mM
Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween-20 (TBST)
for 2 h at room temperature, then incubated with an anti-EGF
receptor antibody (Sigma or Upstate Biotechnology as indicated in the
figure legends) at a dilution of 1:1000 for 2 h at room
temperature. Membranes were then washed in TBST containing 0.25%
gelatin for 15 min at room temperature and incubated with a sheep
antimouse conjugated horseradish peroxidase secondary antibody (Sigma)
at a dilution of 1:5000 for 1 h at room temperature, washed with
TBST for 1 h at room temperature, and developed using the
SuperSignal chemiluminescent detection system (Pierce Chemical Co.,
Rockford, IL). EGF-stimulated and unstimulated cells were collected,
blotted, and probed with the antiphosphotyrosine antibody PY-20
(Transduction Laboratories, Lexington, KY) at a 1:1000 dilution
using the procedure described above.
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Results |
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(26, 27). We evaluated the SCC lines used in these studies for differential
expression of the EGF receptor and EGF-dependent receptor activity.
Each SCC line displayed elevated EGF receptor messenger RNA (data not
shown) and protein (Fig. 1
) compared to normal human
keratinocytes. The degree of EGF receptor overexpression in these SCC
lines is modest compared to that in A431 cells. SCC 12F cells exhibit
200,000 receptors/cell (28), which represents approximately 5-fold
overexpression relative to normal keratinocytes (29). In contrast, A431
cells typically display more than 1 x 106
receptors/cell (30). EGF receptor function was retained in each SCC
line, as determined by ligand-dependent receptor autophosphorylation
and EGF-dependent stimulation of cell proliferation (data not shown).
We found no evidence for elevated TGF messenger RNA expression, as
determined by Northern analysis, in these SCC lines relative to that in
normal keratinocytes.
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); however, the response
varied with cell type. SCC lines 9, 12F, and 25 significantly
overexpressed the EGF receptor (Fig. 1
), and EGF induced full colony
dispersion within 24 h (Fig. 2A). In SCC 12F cells, cell spreading
and colony disruption were apparent within 4–6 h after treatment with
10 nM EGF (data not shown). EGF-induced colony dispersion
was mediated through the EGF receptor and required receptor tyrosine
kinase activity. The response was completely abolished by coincubation
with an anti-EGF receptor-neutralizing antibody (LA1; 10 µg/ml) and
the tyrosine kinase inhibitors genistein (10 µg/ml) and tyrphostin
B46 (10 µM), but not by the inactive tyrphostin A1 (30
µM).
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, SCC 13 cells only modestly (2-fold)
overexpressed the EGF receptor compared to the other SCC lines.
Interestingly, for both normal keratinocytes and SCC 13 cells,
scattering was essentially absent after exposure to EGF (Fig. 2A). As
colony dispersion is a composite response that involves disruption of
cell:cell adhesive contacts in addition to induction of cell migration,
we also evaluated normal keratinocyte and SCC 13 colony dispersion
under less stringent conditions. Cell:cell contacts mediated by
adherens junctions and desmosomes are disrupted by reduction of
extracellular calcium concentrations of less than 100 µM
(31, 32), and even under these conditions, substantial dispersion of NK
or SCC 13 colonies required more than 48-h exposure to EGF (Fig. 2B)
compared to less than 24 h in the other SCC lines (data not
shown). The reduced responsiveness of SCC 13 cells and normal
keratinocytes relative to the other SCC lines suggested that the
observed differences in EGF-dependent motility may be related to the
level of EGF receptor expression rather than the transformed phenotype
per se.
In vitro reepithelialization kinetics vary with EGF receptor
activation
We have previously reported that for normal keratinocytes,
in vitro wound closure requires more than 48 h of EGF
treatment (15) as was observed for cell scattering (Fig. 2), whereas an
EGF receptor-overexpressing cell line (SCC 12F) displays a full
response within 24 h (15) (Fig. 2). We wanted to determine whether
partial EGF receptor occupancy using submaximal EGF concentrations
would produce an in vitro reepithelialization response in
SCC 12F cells that more closely resembles that in normal keratinocytes.
To more clearly evaluate the migratory component of in vitro
reepithelialization, cells were pretreated with mitomycin C to abolish
growth factor-induced proliferation. Under these conditions, a
concentration-dependent increase in EGF receptor phosphorylation and
cell migration into an in vitro wound were observed, with a
maximal response observed at an EGF concentration of 10 nM
(Fig. 3, A and B). Continued exposure to EGF at
submaximal concentrations (0.1 nM) for an additional
24 h promoted full in vitro wound closure. Thus,
partial occupancy and activation of the EGF receptor in SCC 12F cells
elicited migratory response kinetics similar to those observed in
normal keratinocytes.
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Titration of active EGF receptor in SCC 12F cells using anti-EGF
receptor-neutralizing antibody (LA1) resulted in a
concentration-dependent decrease in EGF receptor tyrosine kinase
activity and cell motility (Fig. 4). At LA1
concentrations of 10 µg/ml, EGF-dependent receptor
autophosphorylation was abolished (Fig. 4A) as was EGF-stimulated DNA
synthesis (data not shown) and in vitro reepithelialization
(Fig. 4B). Control IgG did not alter EGF-dependent responses (data not
shown). At an antibody concentration (0.1 µg/ml) that partially
inhibited EGF-dependent phosphorylation (Fig. 4A), in vitro
reepithelialization was incomplete after 24 h (Fig. 4B).
Additional incubation for 48 h resulted in complete
reepithelialization at an antibody concentration of 0.1 µg/ml,
although the antibody at higher concentrations (10 µg/ml) was still
fully effective at preventing EGF-dependent in vitro wound
closure at this time point. Readdition of antibody after 24 h did
not alter the response, so migration attained at 48 h is unlikely
to reflect inactivation of the neutralizing antibody. Thus, partial
elimination of EGF receptor activity in SCC 12F cells resulted in a
ligand-regulated motile response that more closely resembled that of
normal keratinocytes (15).
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) and display kinetics and magnitude of
EGF-dependent motility similar to those of normal cells (Fig. 2).
EGF receptor expression and ligand-stimulated autophosphorylation were
compared in selected clonal cell lines, and the results are shown in
Fig. 5. Modest overexpression of the EGF receptor
similar to that observed in SCC 12F cells was detected in the EGF
receptor-transfected SCC 13 cell lines compared to the control
transfectants (Fig. 5A). Clonal isolates derived from both control and
EGF-R transfections retained ligand-dependent enzymatic activity, as
determined by EGF receptor autophosphorylation (Fig. 5B).
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. EGF-stimulated colony dispersion of SCC 13 clonal
isolates that expressed elevated EGF receptor levels (Fig. 6, EGF-R C6
and EGF-R C7) was markedly enhanced relative to that in the control
clones (Fig. 6, Control B5 and Control C6) or the parental SCC 13 line
(Fig. 6). Notably, this increased response was observed under the
stringent conditions (>1 mM Ca2+) in which
intercellular calcium-dependent adhesive junctions were retained. In
contrast to the parental cells (Fig. 2), dramatic colony dispersion was
detected within 24 h of ligand treatment. This suggests that both
cell migration and modulation of cell:cell adhesive contacts are
augmented upon elevation of EGF receptor levels. Interestingly, one of
the clonal isolates derived from the control transfections (B6) that
displayed elevated EGF receptor expression (Fig. 5A) also exhibited an
increase in EGF-dependent cell migration relative to that in the
parental SCC 13 cells (data not shown). This may reflect isolation of
an EGF receptor-overexpressing colony based on a growth advantage while
under selective pressure and provides additional support for the
relationship between increased EGF receptor expression and
ligand-mediated motility.
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Discussion |
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Migration of cells from a colony is a complex response that requires dynamic reorganization of the actin cytoskeleton and modulation of cell:cell and cell:matrix adhesive properties (34, 35). The underlying basis for growth factor receptor-mediated cell motility is not well defined, although many proteins associated with the actin cytoskeleton, including components of focal adhesions and adherens junctions, are substrates for tyrosine kinases such as the EGF receptor (1, 35). Interestingly, the EGF receptor associates with the actin cytoskeleton through a distinct intracellular domain (36), and cytoskeletal association is increased upon the addition of ligand (37, 38).
Potential consequences of increased EGF receptor expression include
enhanced stimulation of signal transduction pathways required for cell
motility. Two enzymes implicated in the generation of phospholipid
products important for actin remodeling and cell motility are
phospholipase C-
and phosphoinositide-3-kinase (34, 35, 39). In
addition to their proposed roles in chemotactic and migratory responses
induced by various receptor tyrosine kinases, both proteins have been
shown to associate with actin microfilaments and are activated by EGF
(1, 40). Phospholipase C has been reported to play a role in
EGF-stimulated murine fibroblast motility (41), and there is recent
evidence directly linking the EGF receptor to phosphoinositide-3-kinase
through erbB3 (42, 43). Another class of signaling molecules
involved in actin cytoskeleton remodeling are the Ras-like small G
proteins Rac and Rho. This family of proteins acts as mediators of
growth factor-regulated membrane ruffling, formation of actin stress
fibers, and formation of focal adhesion contacts (reviewed in Ref.35).
Although the transient membrane ruffling response may not be directly
related to cell motility, at least one of these functions, the
regulated formation and disassembly of focal adhesions, is an important
determinant of migratory rate (34).
In addition to modulation of cell:substratum attachments mediated by
focal adhesions, alterations in cell:cell contacts are observed in
motile cells. Although EGF-dependent colony dispersion in the parental
SCC 13 cells was apparent only under conditions of disrupted cell
contacts (<100 µM Ca2+), cell migration was
readily apparent in the EGF receptor-transfected SCC 13 clonal cell
lines even under conditions of intact calcium-dependent junctions.
Recent studies suggest that tyrosine kinases may be important in
modulating the adhesive function of adherens junctions and desmosomes
(44, 45). It has been observed that the function of adherens junctions
is compromised by tyrosine phosphorylation of its associated proteins,
including the catenins (46, 47). EGF stimulation leads to increased
tyrosine phosphorylation of ß-catenin, and ß-catenin appears to
mediate the interaction between the cadherin-catenin complex and the
EGF receptor (47). EGF receptor activation also results in tyrosine
phosphorylation of plakoglobin/-catenin, which is a component of
both adherens junctions and desmosomes (46, 48). Interestingly, there
is a strong correlation between the catalytic activity of
v-src and subsequent disruption of adherens junctions (45, 49). This suggests that there may be a parallel link between the
magnitude of EGF receptor activation and subsequent alterations in
cadherin-mediated adhesive function.
There is considerable evidence in support of EGF receptor involvement in cellular functions associated with cell migration, including actin remodeling and modulation of both cell:substrate and cell:cell adhesive contacts (1, 18, 19, 20, 21). Our findings directly demonstrate increased ligand-dependent colony dispersion as a consequence of elevated EGF receptor levels in a SCC line. Importantly, our findings suggest that full ligand-dependent migratory potential is not attained at basal EGF receptor levels in normal keratinocytes. Therefore, up-regulation of EGF receptor expression may augment ligand-stimulated cell motility under circumstances where receptor levels are transiently increased, such as during wound healing and embryogenesis (12, 13, 14), or with constitutive overexpression, as observed in many tumor cells (18). Thus, we suggest that the migratory capacity of keratinocytes may be modulated at the level of receptor density in addition to ligand concentration, and that in certain cell types, dynamic regulation of EGF receptor expression offers an additional level of control over the cell migratory potential.
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Acknowledgments |
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Footnotes |
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Received July 1, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the epidermis of transgenic mice elicits hyperplasia, hyperkeratosis
and spontaneous squamous papillomas. Cell Growth Diff 4:1071–1082[Abstract]
in wound healing and repair. J Cell Biochem 45:346–352[CrossRef][Medline]
(TGF-) relative to epidermal growth
factor (EGF). J Invest Dermatol 106:590–597[CrossRef][Medline]
and epidermal growth factor receptor
messenger RNA are early markers of carcinogenesis in head and neck
cancer. Cancer Res 53:3579–3584This article has been cited by other articles:
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