From the Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, July 17, 2000, and in revised form, December 12, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The The integrin family of heterodimeric cell surface adhesion
receptors mediates not only adhesion to the extracellular matrix or
other cells but also serves to integrate signals from the outside of
the cell to the inside of the cell (1-5). Although both the To begin to explore the mechanisms underlying the distinct phenotypes
mediated by the two collagen/laminin receptors, we re-expressed either
the full-length Cell Culture and Transfection--
The murine NMuMG cell line
was maintained in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum and
insulin (5 µg/ml). The variant NMuMG subclone NMuMG-3, lacking
The X2C2- and X2C1-expressing clonal cell lines were cotransfected with
FLAG-tagged wild-type or dominant negative (DN) p38 Cell Migration on Type I Collagen--
Cell migration assays
were performed using a modification of the protocol described
previously (25). Cells were serum-starved in media containing 0.4%
serum and insulin (5 µg/ml) for 48 h and then in media
containing insulin (5 µg/ml) for 24 h. Cells were removed from
flasks with trypsin/EDTA and washed twice before being replated in
transwell chambers. Briefly, 12-mm transwell chambers (Corning Costar
Corp.) containing polycarbonate membrane with 12-µm pores were coated
overnight at 4 °C with type I collagen (25 µg/ml) (Collaborative
Biomedical Products) or fibronectin (25 µg/ml) (Sigma Diagnostics
Inc.). The filters were washed with phosphate-buffered saline and
air-dried. The bottom chamber was filled with Dulbecco's modified
Eagle's medium containing 1% bovine serum albumin (Sigma) and
Mg2+ (2 mM). Cells were placed in the top
chamber at 1.5 × 105 cells/ml in Dulbecco's modified
Eagle's medium containing 1% bovine serum albumin and
Mg2+ (2 mM) and allowed to migrate for 5 h
at 37° in a humidified CO2 incubator. In experiments in
which recombinant human EGF was included, EGF (10 ng/ml) was placed in
either the lower chamber only or in both the upper and lower chambers
as a control for chemotaxis. In experiments where inhibitors were used,
the cells were incubated with inhibitors for 15 min. Inhibitors were
included in both the upper and lower chambers of the transwell device. After the 5-h incubation, cells remaining on the upper surface of the
transwell filter were removed by mechanical scraping. Cells migrating
to the lower surface of the filter were detected by fixation and
staining with Gill's hematoxylin and eosin solution (Sigma). The
number of cells migrating to the lower surface was determined by
counting the number of cells in 10 random high power (× 40) fields.
Data presented represent the mean ± S.E. of at least three
separate experiments. Statistical analyses were carried out by unpaired
t tests using GraphPad Prism version 2.01.
Immunoblot and Immunoprecipitation Analyses--
Cells were
serum-starved and treated as described for the migration assays but
were plated onto Petri dishes coated with either type I collagen (25 µg/ml) or fibronectin (25 µg/ml) and lysed at defined time points
in lysis buffer (50 mM HEPES (pH 7.2), 250 mM
NaCl, 2 mM EDTA, 0.1% Nonidet P-40, 100 µg/ml aprotinin, 50 µg/ml leupeptin, 40 mM NaF, 0.5 mM
phenylmethylsulfonyl fluoride, 0.5 mM
o-vanadate, and 1 mM dithiothreitol). Total
protein concentration was determined by the Pierce protein assay (Fisher).
For immunoblot analyses, equivalent amounts of protein lysate were
subjected to SDS-PAGE and electroblotted onto Immobilon-P transfer
membrane (Fisher). Immunoblots were blocked in 5% bovine serum albumin
or 5% dried milk in Tris-buffered saline containing 0.5% Tween 20 and
incubated overnight with an appropriate dilution of primary antibody at
4 °C. Secondary antibody incubation was performed with horseradish
peroxidase-conjugated sheep anti-mouse IgG or anti-rabbit IgG (Amersham
Pharmacia Biotech) for 2 h at room temperature. The ECL
Chemiluminescence System (Amersham Pharmacia Biotech) was used for visualization.
For immunoprecipitation analyses, equivalent amounts of protein lysate
were pre-cleared with rabbit anti-mouse IgG (Jackson ImmunoResearch
Laboratories, Inc.) and immunoprecipitated with polyclonal anti-EGF
receptor antibody (Santa Cruz Biotechnology) and protein A-Sepharose
beads (Sigma). Immunoprecipitated protein was subjected to SDS-PAGE,
electroblotted onto Immunobilon-P transfer membrane (Fisher), and
immunoblotted with the appropriate dilution of either monoclonal
anti-PY99 (Santa Cruz Biotechnology) and subsequently with polyclonal
anti-EGF receptor (Santa Cruz Biotechnology).
Antibodies and Reagents--
Polyclonal anti-total p38
MAP kinase antibody, polyclonal anti-phospho-p38 MAP kinase antibody
(New England Biolabs), monoclonal anti-HA antibody (Roche Molecular
Biochemicals), monoclonal anti-FLAG antibody (Sigma), and polyclonal
anti-Jun kinase antibody (New England Biolabs) were used for immunoblot analysis.
We recently reported that an integrin collagen receptor containing
the 2 integrin subunit
cytoplasmic domain uniquely supported epidermal growth factor
(EGF)-stimulated migration on type I collagen. p38 MAP kinase- and
phosphatidylinositol 3-kinase-specific inhibitors, but not a
MEK-specific inhibitor, eliminated EGF-stimulated and unstimulated
2-cytoplasmic domain-dependent migration.
Following adhesion to collagenous matrices, cells expressing the
full-length
2 integrin subunit, but not cells expressing
a chimeric
2 integrin subunit in which the
2-cytoplasmic domain was replaced by the cytoplasmic
domain of the
1-subunit, exhibited sustained and robust
phosphorylation of p38 MAP kinase. Expression of dominant negative p38
MAP kinase inhibited
2-cytoplasmic
domain-dependent, EGF-stimulated migration as well as
unstimulated migration on collagen. Expression of constitutively active
Rac1(Val-12) augmented p38 MAP kinase activation and
2-cytoplasmic domain-dependent migration. It
also rescued the ability of cells expressing the
1-cytoplasmic domain to activate p38 MAPK and to
migrate. These results suggest that the
2 integrin
cytoplasmic domain uniquely stimulates the p38 MAP kinase pathway that
is required for unstimulated and EGF-stimulated migration on type I collagen.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
-subunits are composed of extracellular and transmembrane domains
and short cytoplasmic domains, only the role of the
-subunit cytoplasmic domain in interacting with cytoskeletal proteins and signaling molecules, such as focal adhesion kinase and integrin-linked kinase, has been well characterized (6-10). The role of the many different
-subunit cytoplasmic domains is not well understood. Although both the
1
1 and
2
1 integrins mediate cellular adhesion to
collagens and/or laminins (11-14), studies from our laboratory as well
as a number of other laboratories (15-22) have suggested that the
1
1 and
2
1
integrin receptors are not simply redundant receptors but serve to
mediate different downstream events.
2 integrin subunit (X2C2) or a chimeric integrin
-chain composed of the extracellular and transmembrane domains of the
2-subunit fused to the cytoplasmic domain
of the
1-subunit (X2C1) in a variant subclone of the
NMuMG cell line, which lacks endogenous expression of either the
1
1 or the
2
1 integrin. As reported earlier, the
X2C2 and X2C1 transfectants both effectively adhered, spread, and
formed focal adhesion complexes on type I collagen matrices (16).
However, the X2C2 transfectants, but not the X2C1 transfectants,
developed elongated branches and tubules in three-dimensional collagen
gels and migrated on type I collagen in response to a chemotactic
gradient of epidermal growth factor
(EGF).1 In this study we
demonstrate that
2-cytoplasmic
domain-dependent migration required activation of the p38
mitogen-activated protein kinase (MAPK) cascade. The
2-cytoplasmic domain, but not the
1-cytoplasmic domain, led to p38 MAPK activation.
Additionally, expression of constitutively active Rac1(Val-12)
rescued p38 MAPK activation and EGF-stimulated chemotactic migration in
the X2C1 transfectants.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
1 and
2
1
integrin receptors, was derived by limiting dilution techniques and has
been described in detail elsewhere (16). The full-length human
2 integrin (X2C2) cDNA, in the expression vector
pFneo, was a generous gift from Dr. Martin E. Hemler (Harvard Medical
School, Boston) (23, 24). The chimeric integrin cDNA containing the
extracellular domain of
2 and the cytoplasmic domain of
1 was constructed, as previously described (16). The
full-length and chimeric cDNA constructs were subcloned into the
expression vector pSR
(a gift from Dr. Andrey S. Shaw, Washington
University School of Medicine, St. Louis, MO) that contains a
cytomegalovirus promoter. All constructs were transfected into the
NMuMG-3 clonal cell line using calcium phosphate transfection
methodology. Clonal cell lines were selected and maintained in
geneticin (850 µg/ml) and evaluated by Southern blot analysis for
integration site determination to ensure that distinct clones were evaluated.
MAPK cDNA
clones, or DN c-Jun N-terminal kinase cDNA clones (gifts from Dr.
Aubrey R. Morrison, Washington University School of Medicine, St.
Louis, MO), or HA-tagged constitutively active Rac1(Val-12) or dominant
negative Rac1(Asn-17) cDNA clones (gifts from Dr. Margaret Chou,
University of Pennsylvania, Philadelphia) and the Selecta Vecta-Hyg
plasmid (Novagen) using Lipofectin (Life Technologies, Inc.), according
to the manufacturer's instructions. Clonal and nonclonal cell lines
were selected and maintained in media containing only geneticin (850 µg/ml) or geneticin plus hygromycin (448 µg/ml). Selected cell
lines were evaluated for the expression of the epitope tag using either
the goat polyclonal anti-FLAG tag (OctA-probe) (Santa Cruz
Biotechnology) or the mouse monoclonal anti-HA antibody (12 CA5) (Roche
Molecular Biochemicals).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 integrin cytoplasmic domain, but not the
1 integrin cytoplasmic domain, supported EGF-stimulated
chemotaxis on a matrix of type I collagen (16). In agreement with these
recent observations, NMuMG-3 subclones, lacking endogenous
1
1 and
2
1
integrins, but expressing the full-length
2 integrin
subunit cDNA construct (X2C2), migrated on type I collagen when
stimulated by a chemotactic gradient of EGF (10 ng/ml). In contrast,
NMuMG-3 transfectants expressing a chimeric integrin subunit consisting
of the extracellular and transmembrane domains of the
2
integrin subunit fused to the cytoplasmic domain of the
1 integrin subunit (X2C1) migrated poorly on type I
collagen in response to EGF (Fig.
1A), a finding also in
agreement with our previous study. Since the vector-only control
transfectants did not adhere to collagen substrates, their migration
could not be evaluated. To determine whether EGF-stimulated migration
was unique to
2
1 integrin-mediated
adhesion to collagen substrates or was a more general accompaniment of
integrin-mediated adhesion, we assessed the ability of EGF to stimulate
migration of the transfectants on fibronectin. The NMuMG-3 cell line
and the transfectants expressed similar levels of the
5
1 integrin. The level of expression of
the
5
1 integrin on these cells was comparable to the level of the
2
1
expression by the transfectants (16). Although the X2C2 and X2C1
transfectants adhered to fibronectin, both transfectants failed to
migrate on fibronectin in response to EGF (Fig. 1, B and
C). The differences in EGF-stimulated migration were not due
to differences in the expression or the extent of phosphorylation of
the EGF receptor on the two different clonal cell lines (Fig.
1D). Similar results were obtained with multiple clones (see
Ref. 16 and data not shown).
View larger version (24K):
[in a new window]
Fig. 1.
2 integrin subunit,
cytoplasmic domain-dependent, and EGF-stimulated
chemotactic migration on type I collagen. A, X2C2 and
X2C1 transfectants were serum-starved for 72 h and plated on the
upper surface of a transwell filter coated with type I collagen (25 µg/ml). Migration through the pores of the filter was either
unstimulated (Control) or stimulated with EGF (10 ng/ml)
(EGF). Cell migration proceeded for 5 h in a 5%
CO2 humidified chamber at 37 °C. The number of cells
attached to the lower surface of the transwell filter was quantitated
microscopically. Results are presented as the mean ± S.E.M. of at
least three separate experiments. B, the X2C2 and
(C) X2C1 transfectants were treated as described above and
plated on transwell filters coated with either type I collagen or with
fibronectin (25 µg/ml), and migration was either unstimulated
(Control) or stimulated with EGF (10 ng/ml)
(EGF). Results are presented as mean ± S.E.M. of at
least three separate experiments. D, the X2C2 and X2C1
transfectants expressed similar levels of the EGF receptor and
phosphorylated EGF receptor to a similar extent. The X2C2 and X2C1
transfectants were serum-starved for 72 h, either treated or not
with EGF (10 ng/ml) for 5 min, and lysed. Cell lysates were
immunoprecipitated using the polyclonal anti-EGF antibody.
Immunoprecipitated proteins were evaluated by SDS-PAGE and
immunoblotted using the anti-phosphotyrosine antibody PY99 followed by
the anti-EGF receptor antibody (EGF-R). One of three
replicate experiments is shown.
To dissect the pathways responsible for EGF-stimulated 2
integrin subunit cytoplasmic domain-dependent chemotaxis,
we determined the ability of inhibitors of different signaling pathways
to inhibit chemotactic migration. As shown in Fig.
2A, inhibition of either the
phosphatidylinositol 3-kinase (PI3K) or the p38 MAPK pathways completely inhibited
2 integrin-dependent
EGF-stimulated chemotaxis on type I collagen. Inhibition of chemotactic
migration by the p38 MAPK-specific inhibitor SB203580 was
concentration-dependent, as shown in Fig. 2B.
Surprisingly, the MEK-specific inhibitor of the MAPK/extracellular
signal-regulated kinase pathway PD98059 (50 µM) failed to
inhibit migration. Although rapamycin (5 pM), the p70 S6
kinase-specific inhibitor, has been shown to inhibit migration of
endothelial cells and leukocytes (26-28), it failed to inhibit
2-cytoplasmic domain-dependent migration.
Since the PI3K pathway is important for stimulating migration through a number of different integrins (29, 30), we focused on the role of the
p38 MAPK pathway in mediating signals downstream of the
2
1 integrin that may lead to chemotactic
migration.
|
To evaluate the role of p38 MAPK activation by the X2C2 and X2C1
transfectants, we evaluated the time course of activation and
phosphorylation of p38 MAPK resulting from adhesion to type I collagen.
The X2C2 and X2C1 transfectants were serum-starved in a manner
identical to that described for the chemotactic migration assays,
plated on type I collagen (25 µg/ml) in the absence or presence of
EGF (10 ng/ml), and lysed at defined time points. Adhesion of the X2C2
transfectants to type I collagen resulted in phosphorylation of p38
MAPK within 15 min (Fig. 3). Low level p38 MAPK phosphorylation was maintained at 1 h and then declined to base-line levels by 3 and 6 h. The presence of EGF slightly augmented p38 MAPK phosphorylation at 15 min and 1 h. Adhesion to
collagen by the X2C1 transfectants stimulated only minimal p38 MAPK
phosphorylation at 15 min; phosphorylated p38 MAPK was undetectable
after 1 h. EGF slightly augmented phosphorylation of p38 MAPK by
the X2C1 transfectant at 15 min but not at later time points (Fig. 3).
These findings demonstrate that in a cell line that expressed no
endogenous 1
1 or
2
1 integrin, so that adhesion to collagen
was entirely dependent upon the transfected collagen receptor, adhesion
to type I collagen activated p38 MAPK in a manner that was dependent
upon the presence of an
2-cytoplasmic domain. In
contrast, EGF only slightly augmented phosphorylation of p38 MAPK when
the X2C1 transfectants were adherent to type I collagen.
|
To examine further the requirement of the p38 MAPK pathway in
2-cytoplasmic domain-dependent
EGF-stimulated chemotaxis, the X2C2 transfectants were cotransfected
with either epitope-tagged wild-type or dominant negative (DN) p38 MAPK
cDNA constructs and the Selecta Vecta-Hyg plasmid. Clonal cell
lines were selected in geneticin plus hygromycin, and expression of the
epitope tag was evaluated by immunoblot analysis (Fig.
4A). Only clonal cell lines
expressing low levels of FLAG-tagged DN p38 MAPK were obtained (Fig.
4A), possibly due to toxicity at higher levels of DN p38 MAPK expression. As shown in Fig. 4B, expression of
wild-type p38 MAPK failed to augment unstimulated or EGF-stimulated
chemotactic migration. In contrast, expression of the DN p38 MAPK
cDNA markedly reduced migration on type I collagen, when cells were
either unstimulated (Control) or stimulated by a gradient of EGF (EGF)
(Fig. 4B). Unstimulated migration of the X2C1 transfectants
and the X2C2 DN p38 MAPK cotransfectants was significantly less than
the unstimulated migration of the X2C2 transfectants (p < 0.008). EGF-stimulated chemotaxis of the X2C1 transfectants and the
X2C2 DN p38 MAPK cotransfectants was significantly less than the
EGF-stimulated chemotaxis of the X2C2 transfectants (p < 0.01). These results suggest that the p38 MAPK pathway was necessary
for
2 integrin cytoplasmic domain-dependent
migration on type I collagen in the absence and presence of EGF.
However, increased levels of wild-type p38 MAPK protein alone failed to
augment migration. The failure of wild-type p38 MAPK protein to
increase migration suggests that activation of the p38 MAPK but not
simply increased protein expression was necessary to stimulate cell
migration.
|
The upstream signals required for p38 MAP kinase activation include
activation and phosphorylation of a cascade of protein kinases
including SEK (31-33). In some systems, SEK is activated by the small
G protein Rac1 (34). Since Rac1 is responsible for stimulating invasion
of malignant breast cancer and colon cancer cell lines, the role of
Rac1 in 2-cytoplasmic domain-mediated migration was
evaluated (29, 30). The X2C2 transfectants were cotransfected with
either HA-tagged constitutively active Rac1(Val-12) or dominant
negative Rac1(Asn-17) cDNA constructs and the Selecta Vecta-Hyg
plasmid, and clonal cell lines were selected in geneticin plus
hygromycin (Fig. 5A). The
unstimulated and EGF-stimulated migration of clonal cell lines
expressing constitutively active or dominant negative Rac1 was
evaluated. Constitutively active Rac1(Val-12) significantly augmented
unstimulated migration (p < 0.05) and increased
EGF-stimulated chemotaxis (although not statistically significant) of
the X2C2 transfectants on type I collagen (Fig. 5B). In
contrast, dominant negative Rac1(Asn-17) failed to inhibit unstimulated
migration but significantly decreased (p < 0.05)
chemotactic migration in response to EGF when compared with the X2C2
transfectants (Fig. 5B). Expression of dominant negative
Rac1(Asn-17) reduced p38 MAPK phosphorylation following adhesion to
type I collagen (Fig. 5C). The lack of a significant diminution in unstimulated migration is likely due to the very low
level of migration in the absence of EGF. Constitutively active Rac1(Val-12) and dominant negative Rac1(Asn-17) failed to alter migration on fibronectin (data not shown).
|
To address the ability of Rac1(Val-12) to rescue X2C1 migration, the
X2C1 transfectants were cotransfected with HA-tagged, constitutively
active Rac1(Val-12) and Selecta Vecta-Hyg plasmid, and cells expressing
high levels of HA-Rac1(Val-12) were selected in geneticin and
hygromycin (Fig. 5A). Expression of Rac1(Val-12) rescued the
ability of the X2C1 transfectants to migrate on type I collagen in the
presence or absence of EGF, as shown in Fig. 6A. Unstimulated and
EGF-stimulated migration of the X2C2 transfectants and the
X2C1Rac1(Val-12) cotransfectants was significantly greater than
unstimulated or EGF-stimulated migration of the X2C1 transfectants (p < 0.005 or p < 0.02, respectively). In fact, the X2C1 Rac1(Val-12) cotransfectants migrated
in a manner similar to the X2C2 transfectants in the presence or
absence of EGF. Migration of the X2C1 Rac1(Val-12) cotransfectants, as
well as the X2C2 transfectants was completely inhibited by addition of
SB203580, the p38 MAPK inhibitor (Fig. 6B). The X2C1
Rac1(Val-12) cotransfectants were evaluated for their ability to
phosphorylate p38 MAPK following adhesion to type I collagen.
Expression of Rac1(Val-12) augmented p38 MAPK phosphorylation by the
X2C1 transfectants (Fig. 6C). These findings suggest that
signals downstream of the 2, but not the
1, integrin cytoplasmic domain stimulated migration via
the p38 MAPK pathway. Rac1(Val-12) rescued the ability of the X2C1
transfectants to migrate in both an unstimulated and EGF-stimulated
manner and to phosphorylate p38 MAPK. Rac1(Val-12)-stimulated migration
was inhibited by the p38 MAPK-specific inhibitor.
|
Since Rac1 activates both p38 MAPK and the c-Jun N-terminal kinase
(JNK), we evaluated the short term time course of phosphorylation of
JNK resulting from adhesion to type I collagen. Following serum starvation, the X2C2 and X2C1 transfectants were plated on type I
collagen (25 µg/ml) in the absence and presence of EGF (10 ng/ml) and
lysed at defined time points. Adhesion of the X2C2 transfectants to
type I collagen resulted in rapid and robust phosphorylation of JNK
(Fig. 7A). EGF augmented the
phosphorylation of JNK at all time points. Adhesion of the X2C1
transfectants stimulated weak phosphorylation of JNK that was only
slightly augmented by EGF. To discern whether JNK activation was in
part responsible for 2-cytoplasmic
domain-dependent migration, the X2C2 transfectants were
cotransfected with the dominant negative (DN) JNK cDNA construct, and the Selecta Vecta-Hyg plasmid and cells overexpressing DN JNK were
selected in geneticin and hygromycin. X2C2 cotransfectants expressing
DN JNK at levels (Fig. 7B) comparable to the level of DN p38
MAPK kinase expressed by the X2C2 DN p38 MAPK cotransfectants migrated
at a rate similar to the X2C2 transfectants (Fig. 7C). Expression of DN JNK failed to alter significantly EGF-stimulated
2-cytoplasmic domain-dependent migration,
indicating that Rac1 activation of p38 MAPK, but not JNK, is required
for EGF-stimulated chemotaxis of the X2C2 transfectants.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Data from a number of laboratories have suggested that although
the 1
1 and
2
1 integrins are structurally similar and
both function as adhesion receptors for collagens and/or laminins, the
two receptors mediate profound differences on cell phenotype (11-22,
35-37). The results reported here demonstrate that the
2, but not the
1, integrin cytoplasmic
domain mediated signals, via the p38 MAP kinase pathway, that lead
to a migratory phenotype. Expression of a constitutively active small G
protein, Rac1, augmented p38 MAP kinase phosphorylation and migration
of the X2C2 transfectants in the absence and presence of EGF. In
addition, expression of constitutively active Rac1 by the X2C1
transfectants restored activation of the p38 MAP kinase pathway and a
migratory phenotype. Thus, the activation of the p38 MAP kinase pathway
that occurs upon adhesion to collagen is uniquely dependent on the
2 domain, but not the
1-cytoplasmic
domain, and is required for cell migration, either unstimulated or
stimulated by EGF.
Our earlier work, using complementary "gain-of-function" and
"loss-of-function" models indicated that the
2
1 integrin was required for epithelial
morphogenesis and branching in three-dimensional collagen gels (15,
38-40). The ability of epithelial cells to branch and to form
glandular structures was not supported by the
1
1 integrin. These results were the first
to suggest that these two receptors mediate distinctly different cell
phenotypes when expressed in epithelial cells. Other laboratories (17,
18) demonstrated that, when cultured in three-dimensional collagen gels, fibroblasts utilized the
1
1
integrin to down-regulate collagen gene expression but utilized the
2
1 integrin to up-regulate matrix
metalloproteinase gene expression. The
1
1
and
2
1 integrins have also been shown to
mediate distinctly different signals that promote cell cycle
progression (20, 21).2 Wary
et al. (20, 21) demonstrated that the
1
1 integrin was among a subset of
integrin receptors, including
5
1,
v
3, and
6
4,
that associated with shc to activate the Ras/MAP kinase pathway. Caveolin-1 functions as a membrane adaptor to couple this
subset of integrins via the transmembrane domain of the
integrin
subunit to the shc pathway. In the studies of Wary et al. (20, 21) and in our own unpublished
studies,3 adhesion of cells
expressing the
2
1 integrin does not
result in shc phosphorylation. Under similar conditions,
adhesion of cells expressing the
1
1
integrin produced shc
phosphorylation.3 These
results indicate that both the
1
1 and the
2
1 integrins mediate downstream signals
but do so by distinctly different mechanisms. Our results suggest that
the
2 integrin subunit utilizes the cytoplasmic domain
for signaling specificity that results in activation of the p38 MAP
kinase pathway.
The ability of different integrin - and
-subunit cytoplasmic
domains to respond to different inside-out or outside-in signaling pathways is poorly understood. Here we demonstrate that EGF-stimulated migration on type I collagen requires
2-cytoplasmic
domain-dependent p38 MAP kinase activation and not p42/44
MAP kinase activation. EGF failed to stimulate
5
1-mediated migration on fibronectin. R-Ras-stimulated migration has a number of similarities with
EGF-stimulated migration (42). The effect of R-Ras was dependent on the
2- and not the
5-cytoplasmic domain. In
addition, R-Ras-stimulated migration was independent of MEK/MAPK kinase
activation. MEK and MAPK kinase activation has been implicated in cell
migration of a number of cell types (43), and EGF stimulation of the
EGF receptor activates the p42/44 MAP kinase (44, 45). Initially, we
were somewhat surprised that EGF-stimulated
2-cytoplasmic domain-dependent migration was
independent of the p42/44 MAP kinase pathway. These results suggest the
potential cross-talk between the R-Ras pathway and the EGF-stimulated
2-cytoplasmic domain-dependent migration.
During completion of the study reported here, Ivaska et al.
(22) reported that 2
1 integrin-mediated
up-regulation of collagen gene expression in three-dimensional collagen
gels was mediated by p38 MAP kinase and required the
2-cytoplasmic domain. Our findings support an important
and unique role for the
2-cytoplasmic domain in
mediating activation of p38 MAP kinase activation. Our findings extend
the earlier studies to include a role for p38 MAP kinase in mediating
cellular migration on two-dimensional collagen substrates. Our results
demonstrate that the collagen-
2
1 integrin
interaction is sufficient to mediate p38 MAP kinase phosphorylation. Changes in cell shape are not sufficient for p38 MAP kinase
phosphorylation and activation since both the X2C2 and X2C1
transfectants spread in a similar manner.
One attractive model by which the 2-tail may activate
p38 MAPK places the
2-cytoplasmic domain upstream of the
small G proteins Cdc42 and/or Rac1. Ivaska et al. (22)
suggested that Cdc42 was responsible for
2-cytoplasmic
domain-stimulated p38 MAP kinase phosphorylation. Since both Cdc42 and
Rac1 have been shown to activate the p38 MAP kinase pathway and
stimulate carcinoma cell invasion and migration (29, 30), we evaluated
the role of both Cdc42 and Rac1 in
2
1
integrin-dependent migration on collagen. Migration studies
using a series of stable cell lines expressing either constitutively
active or dominant negative Cdc42 or Rac1 identified Rac1 as an
important mediator of cell migration. In contrast to the findings of
Ivaska et al. (22) expression of either constitutively
active or dominant negative Cdc42 failed to significantly alter
2-cytoplasmic domain-dependent unstimulated or EGF-stimulated migration (data not shown). On the other hand, expression of constitutively active Rac1 augmented migration and activation of p38 MAP kinase. Furthermore, expression of dominant negative Rac1 greatly diminished the EGF-stimulated chemotaxis. Dominant negative Rac1 had no effect on low level, unstimulated
2-cytoplasmic domain-dependent migration. In
the alternative model to that proposed by Ivaska et al.
(22), Rac1 would act, not as a downstream effector of the
2-cytoplasmic domain to p38 MAPK, but in a parallel
pathway that also activates p38 MAPK. In this case, Rac1 synergizes
with the
2-cytoplasmic domain to stimulate migration of
epithelial cells in a manner similar to that by which lysophosphatidic
acid activation of Rho synergizes with EGF and platelet-derived growth
factor to stimulate migration of fibroblasts (46).
Cooperative signals from the extracellular matrix and growth factors
are required for cells to progress through the cell cycle and to
migrate (47, 48). The pathways leading from integrins and growth factor
receptors converge at several points in the G1 phase of the
cell cycle (3-5).2 In this report, we have described a
synergy between signals initiated by EGF and signals generated by the
2-subunit cytoplasmic domain that lead to cell
migration. Unstimulated and EGF-stimulated chemotactic migration on
type I collagen are supported by the
2 integrin cytoplasmic domain. Signals initiated by EGF synergized with the
2-cytoplasmic domain-dependent
phosphorylation of p38 MAP kinase to stimulate chemotactic migration.
Cooperative synergy between specific integrin receptors and/or integrin
cytoplasmic domains and growth factors has been reported by other
investigators (41, 48-51). In cultures of primary skeletal muscle,
overexpression of the
6-subunit or the
5-extracellular/
6-cytoplasmic domain subunit (X5C6) inhibited proliferation and supported differentiation in
the presence of growth factors or serum. In contrast myoblasts expressing the
5 integrin subunit or the
6-extracellular domain/
5-cytoplasmic domain subunit (X6C5) proliferated but failed to differentiate in
response to serum or growth factors (49, 50). In the chorioallantoic membrane model, basic fibroblast growth factor or tumor necrosis factor-
-induced angiogenesis depends on the
v
3 integrin, but vascular endothelial
cell growth factor, transforming growth factor-
, or phorbol
ester-induced angiogenesis depend on the
v
5 integrin (51). Vascular endothelial
cell growth factor-induced angiogenesis requires members of the
src kinase family, whereas basic fibroblast growth factor
signals do not (41). Therefore, in angiogenesis, responses to specific
growth factors are linked to and are dependent upon the expression of
specific integrin receptors.
In summary, the results reported here demonstrate that the ability of
the 2
1 integrin to mediate a migratory
phenotype requires the cytoplasmic domain of the
2-subunit. Signals uniquely mediated by the
2-, but not the
1, cytoplasmic domain
lead to phosphorylation of p38 MAP kinase and Jun kinase. This study is
the first to demonstrate the activation of the p38 MAP kinase and not
the Jun kinase pathway is necessary for
2
1 integrin-mediated cell migration and
EGF-stimulated chemotaxis. The
1 integrin cytoplasmic
domain did not mediate these signals, but migratory activity and
phosphorylation of p38 MAPK was rescued by expression of constitutively
active Rac1 in the X2C1 transfectants. These results begin to dissect
the complex mechanisms by which the
1 and
2 integrin cytoplasmic domains, in concert with signals
from growth factors, exert profound influences on cell phenotype.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Aubrey R. Morrison, Dr. Margaret Chou, and Dr. Martin E. Hemler for providing constructs used in these studies; Bruce Linders and George Li for their technical expertise; and Mary Beth Flynn for expert secretarial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants CA70275 and CA83690.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: Dept. of Pathology and
Immunology, Box 8118, Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110. Tel.: 314-362-0108; Fax:
314-747-2040.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M006286200
2 P. A. Klekotka, S. A. Santoro, A. Ho, S. F. Dowdy, and M. M. Zutter, submitted for publication.
3 P. A. Klekotka, S. A. Santoro, and M. M. Zutter, unpublished data.
4 P. A. Klekotka and M. M. Zutter, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: EGF, epidermal growth factor; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; HA, hemagglutinin; DN, dominant negative; PAGE, polyacrylamide gel electrophoresis; JNK, c-Jun N-terminal kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Clark, E. A., and Brugge, J. S. (1995) Science 268, 233-239[Medline] [Order article via Infotrieve] |
2. | Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Annu. Rev. Cell Dev. Biol. 11, 549-599[CrossRef][Medline] [Order article via Infotrieve] |
3. | Yamada, K. M., and Miyamoto, S. (1995) Curr. Opin. Cell Biol. 7, 681-689[CrossRef][Medline] [Order article via Infotrieve] |
4. | Howe, A., Aplin, A. E., Alahari, S. K., and Juliano, R. L. (1998) Curr. Opin. Cell Biol. 10, 220-231[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Giancotti, F. G.,
and Ruoslahti, E.
(1999)
Science
285,
1028-1032 |
6. |
Renshaw, M. W.,
Price, L. S.,
and Schwartz, M. A.
(1999)
J. Cell Biol.
147,
611-618 |
7. |
Pfaff, M.,
Liu, S.,
Erle, D. J.,
and Ginsberg, M. H.
(1998)
J. Biol. Chem.
273,
6104-6109 |
8. | Pasqualini, R., and Hemler, M. E. (1994) J. Cell Biol. 125, 447-460[Abstract] |
9. |
Radeva, G.,
Petrocelli, T.,
Behrend, E.,
Leung-Hagesteijn, C.,
Filmus, J.,
Slingerland, J.,
and Dedhar, S.
(1997)
J. Biol. Chem.
272,
13937-13944 |
10. |
Wu, C.,
Keightley, S. Y.,
Leung-Hagesteijn, C.,
Radeva, G.,
Coppolino, M.,
Goicoechea, S.,
McDonald, J. A.,
and Dedhar, S.
(1998)
J. Biol. Chem.
273,
528-536 |
11. | Santoro, S. A. (1986) Cell 46, 913-920[Medline] [Order article via Infotrieve] |
12. |
Staatz, W. D.,
Fok, K. F.,
Zutter, M. M.,
Adams, S. P.,
Rodriguez, B. A.,
and Santoro, S. A.
(1991)
J. Biol. Chem.
266,
7363-7367 |
13. | Staatz, W. D., Rajpara, S. M., Wayner, E. A., Carter, W. G., and Santoro, S. A. (1989) J. Cell Biol. 108, 1917-1924[Abstract] |
14. | Hall, D. E., Reichardt, L. F., Crowley, E., Holley, B., Moezzi, H., Sonnenberg, A., and Damsky, C. H. (1990) J. Cell Biol. 110, 2175-2184[Abstract] |
15. | Zutter, M. M., Santoro, S. A., Staatz, W. D., and Tsung, Y. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7411-7415[Abstract] |
16. |
Zutter, M. M.,
Santoro, S. A.,
Wu, J. E.,
Wakatsuki, T.,
Dickeson, S. K.,
and Elson, E. L.
(1999)
Am. J. Pathol.
155,
927-940 |
17. | Langholz, O., Rockel, D., Mauch, C., Kozlowska, E., Bank, I., Krieg, T., and Eckes, B. (1995) J. Cell Biol. 131, 1903-1915[Abstract] |
18. |
Gotwals, P. J.,
Chi-Rosso, G.,
Lindner, V.,
Yang, J.,
Ling, L.,
Fawell, S. E.,
and Koteliansky, V. E.
(1996)
J. Clin. Invest.
97,
2469-2477 |
19. |
Racine-Samson, L.,
Rockey, D. C.,
and Bissell, D. M.
(1997)
J. Biol. Chem.
272,
30911-30917 |
20. | Wary, K. K., Mainiero, F., Isakoff, S. J., Marcantonio, E. E., and Giancotti, F. G. (1996) Cell 87, 733-743[Medline] [Order article via Infotrieve] |
21. | Wary, K. K., Mariotti, A., Zurzolo, C., and Giancotti, F. G. (1998) Cell 94, 625-634[Medline] [Order article via Infotrieve] |
22. |
Ivaska, J.,
Reunanen, H.,
Westermarck, J.,
Koivisto, L.,
Kahari, V. M.,
and Heino, J.
(1999)
J. Cell Biol.
147,
401-416 |
23. | Chan, B. M., Kassner, P. D., Schiro, J. A., Byers, H. R., Kupper, T. S., and Hemler, M. E. (1992) Cell 68, 1051-1060[Medline] [Order article via Infotrieve] |
24. | Takada, Y., and Hemler, M. E. (1989) J. Cell Biol. 109, 397-407[Abstract] |
25. | Santoro, S. A., Zutter, M. M., Wu, J. E., Staatz, W. D., Saelman, E. U. M., and Keely, P. J. (1995) Methods Enzymol. 245, 147-183[CrossRef] |
26. |
Poon, M.,
Marx, S. O.,
Gallo, R.,
Badimon, J. J.,
Taubman, M. B.,
and Marks, A. R.
(1996)
J. Clin. Invest.
98,
2277-2283 |
27. | Francischi, J. N., Conroy, D., Maghni, K., and Sirois, P. (1993) Agents Actions 39, 139-141 |
28. | Nogueira de Francischi, J., Conroy, D. M., Maghni, K., and Sirois, P. (1993) Br. J. Pharmacol. 110, 1381-1386[Abstract] |
29. | Keely, P. J., Westwick, J. K., Whitehead, I. P., Der, C. J., and Parise, L. V. (1997) Nature 390, 632-636[CrossRef][Medline] [Order article via Infotrieve] |
30. | Shaw, L. M., Rabinovitz, I., Wang, H. H., Toker, A., and Mercurio, A. M. (1997) Cell 91, 949-960[Medline] [Order article via Infotrieve] |
31. |
Ludwig, S.,
Hoffmeyer, A.,
Goebeler, M.,
Kilian, K.,
Hafner, H.,
Neufeld, B.,
Han, J.,
and Rapp, U. R.
(1998)
J. Biol. Chem.
273,
1917-1922 |
32. | Raingeaud, J., Whitmarsh, A. J., Barrett, T., Derijard, B., and Davis, R. J. (1996) Mol. Cell. Biol. 16, 1247-1255[Abstract] |
33. |
Guan, Z.,
Buckman, S. Y.,
Pentland, A. P.,
Templeton, D. J.,
and Morrison, A. R.
(1998)
J. Biol. Chem.
273,
12901-12908 |
34. | Zohn, I. M., Campbell, S. L., Khosravi-Far, R., Rossman, K. L., and Der, C. J. (1998) Oncogene 17, 1415-1438[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Gardner, H.,
Broberg, A.,
Pozzi, A.,
Laato, M.,
and Heino, J.
(1999)
J. Cell Sci.
112,
263-272 |
36. |
Pozzi, A.,
Wary, K. K.,
Giancotti, F. G.,
and Gardner, H. A.
(1998)
J. Cell Biol.
142,
587-594 |
37. |
Riikonen, T.,
Westermarck, J.,
Koivisto, L.,
Broberg, A.,
Kahari, V. M.,
and Heino, J.
(1995)
J. Biol. Chem.
270,
13548-13552 |
38. |
Keely, P. J.,
Fong, A. M.,
Zutter, M. M.,
and Santoro, S. A.
(1995)
J. Cell Sci.
108,
595-607 |
39. |
Saelman, E. U.,
Keely, P. J.,
and Santoro, S. A.
(1995)
J. Cell Sci.
108,
3531-3540 |
40. | Berdichevsky, F., Gilbert, C., Shearer, M., and Taylor-Papadimitriou, J. (1991) J. Cell Sci. 102, 437-446[Abstract] |
41. | Eliceiri, B. P., Paul, R., Schwartzberg, P. L., Hood, J. D., Leng, J., and Cheresh, D. A. (1999) Mol. Cell 4, 915-924[Medline] [Order article via Infotrieve] |
42. |
Keely, P. J.,
Rusyn, E. V.,
Cox, A. D.,
and Parise, L. V.
(1999)
J. Cell Biol.
145,
1077-1088 |
43. |
Klemke, R. L.,
Cai, S.,
Giannini, A. L.,
Gallagher, P. J.,
de Lanerolle, P.,
and Cheresh, D. A.
(1997)
J. Cell Biol.
137,
481-492 |
44. |
Davis, R. J.
(1993)
J. Biol. Chem.
268,
14553-14556 |
45. | Blenis, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5889-5892[Abstract] |
46. |
Sakai, T.,
de la Pena, M.,
and Mosher, D.
(1999)
J. Biol. Chem.
274,
15480-15486 |
47. |
Clark, E. A.,
King, W. G.,
Brugge, J. S.,
Symons, M.,
and Hynes, R. O.
(1998)
J. Cell Biol.
142,
573-586 |
48. | Wells, A., Gupta, K., Chang, P., Swindle, S., Glading, A., and Shiraha, H. (1998) Microsc. Res. Tech. 43, 395-411[CrossRef][Medline] [Order article via Infotrieve] |
49. | Sastry, S. K., Lakonishok, M., Thomas, D. A., Muschler, J., and Horwitz, A. F. (1996) J. Cell Biol. 133, 169-184[Abstract] |
50. |
Sastry, S. K.,
Lakonishok, M.,
Wu, S.,
Truong, T. Q.,
Huttenlocher, A.,
Turner, C. E.,
and Horwitz, A. F.
(1999)
J. Cell Biol.
144,
1295-1309 |
51. | Friedlander, M., Brooks, P. C., Shaffer, R. W., Kincaid, C. M., Varner, J. A., and Cheresh, D. A. (1995) Science 270, 1500-1502[Abstract] |