* Department of Cell and Structural Biology, Department of Pediatrics, University of Illinois at Urbana-Champaign, Urbana,
Illinois 61801; and § Lankenau Medical Research Center, Wynnewood, Pennsylvania 19096
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Abstract |
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Integrin receptors play a central role in cell
migration through their roles as adhesive receptors for
both other cells and extracellular matrix components.
In this study, we demonstrate that integrin and cadherin receptors coordinately regulate contact-mediated inhibition of cell migration. In addition to promoting proliferation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A. Horwitz. 1996. J. Cell
Biol. 133:169-184), ectopic expression of the 5 integrin in cultures of primary quail myoblasts promotes a
striking contact-mediated inhibition of cell migration.
Myoblasts ectopically expressing
5 integrin (
5 myoblasts) move normally when not in contact, but upon
contact, they show inhibition of migration and motile
activity (i.e., extension and retraction of membrane
protrusions). As a consequence, these cells tend to
grow in aggregates and do not migrate to close a
wound. This phenotype is also seen with ectopic expression of
1 integrin, paxillin, or activated FAK (CD2
FAK) and therefore appears to result from enhanced
integrin-mediated signaling. The contact inhibition observed in the
5 myoblasts is mediated by N-cadherin,
whose expression is upregulated more than fivefold.
Perturbation studies using low calcium conditions, antibody inhibition, and ectopic expression of wild-type
and mutant N-cadherins all implicate N-cadherin in the
contact inhibition of migration. Ectopic expression of
N-cadherin also produces cells that show inhibited migration upon contact; however, they do not show suppressed motile activity, suggesting that integrins and cadherins coordinately regulate motile activity. These
observations have potential importance to normal and
pathologic processes during embryonic development
and tumor metastasis.
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Introduction |
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CELL migration plays a central role in diverse processes, including embryonic development, wound
healing, inflammation, and tumor metastasis. Directional cell migration requires an integrated response to
multiple external cues and therefore is likely to require the
participation of different families of cell surface receptors
(Huttenlocher et al., 1995). However, the mechanism by
which these signals integrate to form a coordinated migratory response is poorly understood. Cell surface adhesion
receptors, including integrins and cadherins, mediate cell-
extracellular matrix (ECM)1 and cell-cell interactions that
play an important role during cell migration. Differential
expression of both integrins and cadherins has been associated with changes in the migratory phenotype of cells
during both development and other processes, including tumor invasion and metastasis (Hynes and Lander, 1992
;
Takiechi, 1993; Gumbiner, 1996
; Varner and Cheresh,
1996
).
Integrin receptors are heterodimers that recognize
and bind to components of the extracellular matrix as well
as counter-receptors on the surface of cells (Hynes, 1992
).
In addition to providing a link between the ECM and actin
cytoskeleton, integrin receptors serve as signaling receptors that transduce information from the ECM to affect
cell behavior and gene expression (Damsky and Werb, 1992
; Juliano and Haskill, 1993
; Clark and Brugge, 1995
).
They play an important role during cell migration by linking the extracellular matrix and the actin cytoskeleton and
by transmitting the forces required for migration (Lauffenburger and Horwitz, 1996
). In addition, signaling
through integrin receptors can affect migration independent of their adhesive role (Bauer et al., 1992
).
Cadherins are transmembrane glycoproteins that promote calcium-dependent homophilic cell-cell adhesion
(Takeichi, 1988, 1995
; Gumbiner, 1996
). Like integrins,
cadherins serve both a structural function, linking to the
actin cytoskeleton, and as signaling receptors that affect
cell behavior, including cell proliferation (Watabe et al.,
1994
; Caveda et al., 1996
) and differentiation (Larue et al.,
1996
; George-Weinstein et al., 1997
; Redfield et al., 1997
).
Cadherins promote strong intercellular adhesions, and
their expression is associated with decreased tumor cell invasiveness and metastasis in vivo. (Takeichi, 1993
). Studies
in vitro suggest two probable mechanisms for this inhibition: increased cell-cell adhesion and effects on cell motility (Chen and Obrink, 1991
; Chen et al., 1997
).
Since both integrins and cadherins play central roles in
regulating diverse processes such as differentiation and
cell migration, it is likely that these two families of cell surface adhesion receptors act coordinately to regulate these
processes. An example of such cross talk between cadherin and integrin receptors has been demonstrated in keratinocytes, where cadherins downregulate integrin expression during keratinocyte differentiation (Hodivala and Watt, 1994). It is likely that integrin expression also alters cadherin expression or function, although this has not
been shown previously.
In this study, we show that integrin and cadherin receptors coordinately regulate contact-mediated inhibition of
cell migration. Our previous studies have shown that ectopic expression of the 5 integrin in primary myoblasts
(
5 myoblasts) promotes cell proliferation and inhibits differentiation through enhanced adhesive signaling (Sastry,
S., and M. Lakonishok, unpublished results). Here we
show that ectopic expression of either the
5 or
1 integrin subunit or putative downstream effectors of integrin
signaling promotes a striking contact-mediated inhibition
of cell migration.
5 myoblasts, for example, move normally when not in contact, but upon contact they exhibit
inhibition of both cell migration and motile activity (membrane ruffling and lamellipodial activity). This contact-
mediated inhibition of migration is mediated by N-cadherin, which is markedly upregulated in the
5 myoblasts. Cells expressing ectopic N-cadherin also remain in contact; however, they do not show inhibited motile activity
like the
5 myoblasts. Taken together, our results with primary myoblasts suggest that contact inhibition of migration and motile activity are regulated by a synergy between integrin and cadherin receptors.
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Materials and Methods |
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Primary Cell Culture and Transfection
Myoblasts were isolated from the pectoralis muscle of 9-d-old Japanese
quail embryos as previously described (Sastry et al., 1996). Cells were dissociated from muscle tissue with 0.1% dispase (Boehringer-Mannheim
Corp., Indianapolis, IN) in PBS. The cells were filtered through a
Sweeney filter and plated on 0.1% gelatin-coated tissue culture plates.
The cells were grown in myoblast media containing DME (Sigma Chemical Co., St. Louis, MO) with 15% horse serum, 5% chicken embryo extract, 1% pen-strep, and 1.25 mg/ml fungizone (GIBCO BRL, Gaithersburg, MD). Myoblasts were used between passages 1 and 8.
Myoblasts between passages 1-3 were plated on 60-mm tissue culture
plates coated with gelatin (0.1%) in myoblast media for 12-24 h (Sastry
et al., 1996). For transfections, 8 µg of plasmid DNA and 50 µg of LipofectamineTM (GIBCO BRL) in 0.3 ml of DME (Sigma Chemical Co.) were
added to the cultures for 12-18 h as described. Transient transfection efficiency is ~40%. Cells were selected by using selection media 0.5 mg/ml of
G418 (GIBCO BRL) or 0.25 mg/ml Zeocin (Invitrogen, Carlsbad, CA).
Myoblasts were selected for 4-7 d and then maintained in myoblast medium containing either 0.2 mg/ml of G418 or 0.125 mg/ml of Zeocin. Results represent the findings from multiple separate transfections of
5 integrin, N-cadherin, and control plasmids alone, i.e., pRSVneo (Sastry et al.,
1996
) or pcDNA 3.1/zeo (Invitrogen).
Vector Construction
The eukaryotic expression vector pRSVneo and retroviral plasmid 1654 containing h5 cDNA were prepared as described previously (Sastry et al.,
1996
). The chicken N-cadherin (Hatta et al., 1988
) and mutant
390,
which lacks most of the extracellular domain (Fujimori and Takeichi,
1993
), were provided by M. Takeichi (Kyoto University, Kyoto, Japan) in
Bluescript and were subcloned into the eukaryotic expression vector
pcDNA3.1/Zeo (Invitrogen). All restriction enzymes were purchased
from GIBCO BRL. The N-cadherin and mutant
390 were both excised
using SalI and Sma. The pcDNA3.1/Zeo was cut using XbaI, converted to
a blunt end, and then cut with Xho. The 3.0-kb N-cadherin cDNA and 2.0-kb
390 cDNA were then subcloned into pcDNA 3.1/Zeo. The CH8 epitope-
tagged chicken
1 integrin in pBJ-1 vector was provided by Yoshikazu
Takada (Scripps Research Institute, La Jolla, CA) (Takada and Puzon,
1993
); the chimeric receptor, interleukin-2
1 cytoplasmic domain (IL2
1) in pCMV-IL2R/
1 cyto, was provided by S. LaFlamme (Albany Medical College, Albany, NY) (LaFlamme et al., 1992
); CD2FAK was provided by
A. Aruffo (Bristol-Meyers Squibb Pharmaceutical Research Institute, Seattle, WA) (Chan et al., 1994
), and pcDNA3 paxillin was provided by C. Turner (Albany Medical College) (Turner and Miller, 1994
). The
1 integrin and IL2
1 were both subcloned into the eukaryotic expression vector
pRSVneo (Sastry et al., 1996
). A 1-kb hindIII fragment from the CH8
1
pBJ-1 construct containing the CH8 epitope tag was subcloned into
1pRN to produce full-length CH8
1 cDNA in pRSVneo. IL2
1 was excised from pCMV at NheI and XbaI and ligated in pRSVneo at XbaI.
Flow Cytometry
Flow cytometry was performed as described previously (Sastry et al.,
1996). Briefly, cells were washed with PBS and detached using 0.02%
EDTA in calcium-magnesium-free Hepes-Hanks buffer (CMF-HH). The
cells transfected with human
5 integrin were stained with a specific human
5 antibody (VIF4) from R. Isberg (Tufts University, Boston, MA)
at a dilution of 1:5 of hybridoma supernatant in CMF-HH containing 5%
goat serum (Sastry et al., 1996
). The cells were then stained with an FITC-labeled sheep anti-mouse IgG (Cappell, Durham, NC). N-cadherin expression was determined by staining with the chicken N-cadherin mAb,
6B3, at 20 µg/ml (George-Weinstein et al., 1997
). Flow cytometry was performed using an EPICSTM cell sorter (Coulter Electronics Inc., Miami
Lakes, FL) equipped with Cyclops software for data analysis (Cytomation, Fort Collins, CO). The human
5-transfected myoblasts were sorted to enrich for a population that was >80% positive for human
5 expression as described previously (Sastry et al., 1996
). Cells expressing IL2
1
integrin and
1 integrin also were sorted to enrich a population that was
>80% positive for surface expression. Surface expression of
5 integrin,
N-cadherin,
1 integrin, CD2FAK, and IL2-
1 integrin were documented
by flow cytometry.
Antibodies and Reagents
The rat NCD-2 cadherin function perturbing antibody was purchased
from Zymed (S. San Francisco, CA) and the mAb VIF4, which recognizes
the human 5 integrin extracellular domain, was a gift of R. Isberg. The
1 integrin antibody W1B10 was purified as described previously (Hayashi et al., 1990
) and used at a concentration of 20 µg/ml. The monoclonal antibody against human
1, TS2/16, was from M. Hemler (Dana
Farber Cancer Institute, Boston, MA) and used as ascites at a dilution of
1:2000 (Hemler et al., 1984
). The N-cadherin mAb, 6B3, was prepared as
described (George-Weinstein et al., 1997
) and used at a concentration of
20 µg/ml. The
-catenin antibody IG5 and the
-catenin antibody 15B8
were prepared as described (Johnson et al., 1993
). The plakoglobin antibody PG5.1 was purchased from IBL Research Products Corp. (Cambridge, MA). A control antibody P3/X63-Ag8 was prepared as described
(Kohler and Milstein, 1976
). The fibronectin was purified from human
plasma by affinity chromatography (Ruoslahti et al., 1982
).
Immunofluorescence
Coverslips were coated with fibronectin after being acid washed and ethanol treated as described previously (Huttenlocher et al., 1996). The glass
coverslips were coated with fibronectin at 10 µg/ml, blocked with 2%
BSA, and washed with PBS before plating the cells. After 3 h, the cells
(plated at 1 × 104 cells per well in a 24-well plate) were fixed in PBS containing 3% formaldehyde (Ted Pella, Inc., Irvine, CA) for 15 min, treated with 1% Triton X-100 in PBS for 10 min, and blocked in PBS containing 5% goat serum for 30 min. For staining with 6B3, cells were quick-fixed in
cold methanol for 5 min. After incubating with the primary mAbs, N-cadherin (6B3), or
1 integrin antibody (W1B10), the cells were washed with
PBS three times and then incubated with the FITC sheep anti-mouse at
1:200 in blocking buffer (Cappell). The coverslips were mounted and observed using a fluorescence microscope at 63× (model Axioplan; Carl
Zeiss, Inc., Thornwood, NY). Pictures were taken using TMAX 400 film
(Eastman Kodak Co., Rochester, NY).
Cell Extraction and Immunoblot Analysis
Cells were prepared for extraction by plating untransfected myoblasts and
5 myoblasts at a density of 2 × 106 cells/60-mm tissue culture plate
coated with fibronectin (10 µg/ml) and culturing for 24 h in myoblast medium. The medium was aspirated, the cells were washed with cold PBS,
and then 200 µl of cold modified RIPA extraction buffer was added to the
plates. The modified RIPA buffer contained 20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% NP-40, 1.0% Triton X-100, 0.25% sodium deoxycholate, 2 mM EGTA. The following protease and phosphatase inhibitors were added before extraction: 20 µg/ml leupeptin, 0.7 µg/ml pepstatin, 1 mM
phenanthroline, 2 mM phenyl-methyl-sulfonyl-chloride, 0.05 U of aprotinin and 30 mM sodium pyrophosphate, 40 mM NaF, and 1 mM sodium
orthovanadate. Cells were scraped using a rubber spatula, incubated on
ice, and then centrifuged at 14,000 rpm at 4°C for 5 min. Lysates were frozen in liquid N2 and stored at
80°C. Protein concentration was determined using the Pierce BCA assay (Rockford, IL) with BSA as the standard.
Western blotting was performed by using 5-10 µg of protein, first
boiled in Laemmli sample buffer containing 5% -mercaptoethanol, then
separated on 10% SDS-PAGE gels, and finally transferred to nitrocellulose membranes as described (Towbin et al., 1979
). Membranes were
blocked in 1% heat-denatured BSA in TST buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween-20) overnight at 4°C. The blots were incubated with primary antibody (20 µg/ml of 6B3 or W1B10) for 30 min,
washed with TST, and then incubated with secondary anti-mouse antibody conjugated with HRP diluted 1:20,000 in blocking buffer (Jackson
ImmunoResearch Labs, West Grove, PA). Blots were visualized using
chemiluminescence (Super SignalTM; Pierce Chemical Co.). After incubating the membrane with substrate for 5 min, the membranes were exposed
to x-ray film. Expression of CD2FAK and paxillin were also documented
by Western immunoblotting. Expression of CD2FAK was at times found
to be in a cleaved rather than an intact form. The phenotype promoting
aggregate growth was only seen when the CD2FAK was expressed in its
intact form (data not shown).
Cell Migration Assays
Time lapse video microscopy was performed as previously described
(Schmidt et al., 1993). Non-tissue culture plates were coated with fibronectin (10 µg/ml) for 1 h at 37°C and blocked with 2% BSA for 1 h at
37°C. The cells were plated on a 35-mm plate at a density of 0.5 × 105 cells
for low-density cell tracking and 5 × 105 cells for high-density cell tracking. The cells were tracked and video images processed using an image
processing system (Biological Detection Systems, Inc., Pittsburgh, PA).
Transwell assays were performed as described previously (Huttenlocher
et al., 1996
). Briefly, membranes were coated with fibronectin (10 µg/ml),
and cells were plated in myoblast medium and then fixed and stained after
3 h. Wound healing assays were performed by plating cells on a fibronectin substratum (10 µg/ml). After 12-24 h, when the cells were confluent, a
wound was made using a pipet tip to scrape off the cells. The wound was
observed 24 h later by blinded observers. Number of individual cells in the
wound was quantified at 24 h from pictures of multiple fields (three to
five) for each experiment. The results of all experiments represent the average of two to five experiments from separate transfections. Transfected
cells were compared with untransfected cells and myoblasts transfected with control plasmids for all studies.
Motile activity was studied by observing cells using time lapse video microscopy 10 h after plating the cells. Cells were scored for membrane ruffling at areas of cell-cell contact by observing the cells for 5 min. Cells with visible membrane activity (protrusions and retractions in the range of 2-3 µm) at contact sites were defined as active (similar to changes demonstrated in Fig. 10). A minimum of 50 cells were studied in three separate experiments for each condition.
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Results |
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Ectopic Expression of 5 Integrin in Myoblasts
Promotes Aggregate Growth and Contact-mediated
Inhibition of Cell Migration
To address the role that integrin expression plays in cell
migration, we ectopically expressed 5 integrin subunit in
primary quail myoblasts. Myoblasts were transfected with
the expression plasmids 1654 and pRSVneo containing human
5 cDNA as previously described (Sastry et al., 1996
).
Cells expressing pRSVneo or pcDNA3.1/zeo alone were
used as controls. After selection in G418, transfected cells
were analyzed for surface expression of the human
5 integrin and then enriched by FACS®. All experiments were
performed on a population that was 80-90% positive for
5 integrin expression (Sastry et al., 1996
). Cells transfected with h
5 integrin show a three- to fivefold increase in cell surface
5 integrin expression and a twofold increase in expression of
1 integrin with little apparent change in
the expression of other integrins (Sastry et al., 1996
).
Ectopic expression of 5 integrin in primary myoblasts
was shown previously to promote proliferation and inhibit
differentiation (Sastry et al., 1996
). Myoblasts ectopically
expressing
5 integrin (
5 myoblasts) also grow in tight
aggregates (i.e., clusters) rather than dispersed like control
cells (Fig. 1, A and B). To determine if the growth in aggregates reflects a change in migration of the
5 myoblasts, wound assays were performed. Migration of cells
during wound closure was studied after scraping cells from an area in a confluent monolayer of cells. Closure is inhibited in the
5 myoblasts when assayed 24 h after wounding
(Fig. 2 A). In comparison to untransfected myoblasts or
myoblasts transfected with control plasmids, which migrate
into the wound as individual cells, the
5 myoblasts do not
migrate into the wound individually. Quantification of the
number of individual cells within the wound show that few
if any individual
5 myoblasts are found within the wound,
in contrast to the control cells (Fig. 2 B). Observations by
time lapse video microscopy demonstrate that
5 myoblasts eventually close the wound by cell proliferation
rather than migration.
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In contrast to the wound assay, which examines cells migrating away from a confluent monolayer, migration of 5
myoblasts is not significantly altered in assays that measure migration rates of individual cells. For example, using
a short-term Transwell assay (3 h), there is only a small
difference in the migration of control and
5 myoblasts
(Fig. 2 D). Furthermore, there is no significant difference
in the migration rates of
5 myoblasts and untransfected
myoblasts (or those transfected with control plasmids), as
assayed by video microscopy, when the cells are not in
contact. The average migration speed of untransfected
myoblasts on fibronectin was 17 µm/h with 70% (33/47) of
the cells defined as motile (cell speed greater than 5 µm/h),
as compared with 15 µm/h in
5 myoblasts, with 60% (16/
27) of the cells defined as motile. In contrast, time-lapse
video microscopy of higher-density cultures show that migration of
5 myoblasts is greatly inhibited when the cells
come into contact, whereas control cells are not (Fig. 3).
Thus, the
5 myoblasts show a contact-mediated inhibition of migration that results in a phenotype promoting aggregate growth and delayed wound closure.
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Enhanced Integrin-mediated Signaling Promotes Aggregate Growth and Contact-mediated Inhibition of Migration
The 5 phenotype is not specific to the
5 integrin but
rather appears to result from an increase in integrin-mediated signaling. Ectopic expression of either the
1 integrin
or putative downstream signaling molecules results in
growth in clusters as well as promoting proliferation and
inhibiting differentiation (Sastry, S., and M. Lakonishok,
unpublished) (summarized in Table I). Ectopic expression
of chicken
1 integrin, for example, promotes aggregate growth (Fig. 1 E), delays wound closure, and results in
contact-mediated inhibition of migration as measured by
time-lapse video microscopy. We find that the level of
1
integrin expressed on the surface of these cells is increased
twofold over controls and is similar to the increase in
1
integrin expression in the
5 myoblasts (data not shown).
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Downstream integrin-signaling molecules also promote
growth in aggregates (Fig. 1, summarized in Table I). Ectopic expression of the integrin-associated protein paxillin
promotes growth in aggregates similar to 5 myoblasts
(Fig. 1 C). Ectopic expression of a membrane-bound constitutively active form of FAK, CD2-FAK, also promotes
growth in aggregates when expressed at high levels (data
not shown). These data support the idea that it is enhanced integrin signaling rather than increased
5 integrin
expression per se that produces these phenotypic changes.
However, the signaling pathways regulating cell proliferation and contact inhibition of migration are separable. The
chimeric integrin, IL2
1, has previously been shown to localize to focal contacts and promote the activation of certain integrin-mediated signaling pathways (LaFlamme et al.,
1992
). We find that ectopic expression of IL2
1 in myoblasts promotes cell proliferation but not contact-inhibition of migration. Cells transfected with IL2
1 grow dispersed (Fig. 1 F), and studies by time-lapse video microscopy
show that they are not contact-inhibited in their migration
(data not shown). In contrast, cells expressing the chimeric
receptor IL2
5 cytoplasmic domain show normal differentiation and a dispersed growth pattern comparable to untransfected myoblasts (data not shown). These results support a separation of the signaling pathways regulating these two processes.
Myoblasts Ectopically Expressing the 5 or
1 Integrins
Show Increased Cadherin Expression
To characterize the mechanism of the aggregate growth
and contact-mediated inhibition of cell migration mediated by the 5 integrin, we determined whether there were
any changes in expression of cell surface adhesion receptors due to ectopic
5 integrin expression. Candidate cell-
cell adhesion receptors include integrins, cadherins, and
members of the immunoglobulin superfamily. Staining with the N-cadherin antibody 6B3 shows increased surface
staining of N-cadherin, particularly at the areas of cell-cell
contact in
5 myoblasts (Fig. 4). In contrast, staining with
neural cell adhesion molecule (N-CAM) antibodies shows
no detectable difference in staining patterns between the
5 myoblasts and untransfected cells (data not shown).
Staining with the
1 integrin antibody, W1B10, shows an
increase in
1 integrin in focal adhesions in
5 myoblasts
but no significant difference in the staining patterns at
sites of cell-cell contact (Fig. 4).
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FACS® analysis confirms the increase in surface expression of N-cadherin in 5 myoblasts when compared with
untransfected cells (Fig. 5 A). 24 h after plating,
5 myoblasts express approximately five times more N-cadherin
on their surface. Although a small increase in N-cadherin
is seen during normal differentiation (1.5-2-fold at 48 vs.
24 h), this is significantly less than that seen with ectopic
5 integrin expression. We also found a small increase (1.5-fold) in N-CAM surface expression in the
5 myoblasts in comparison to control cells (data not shown).
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Western immunoblot analyses confirm the increase in
N-cadherin expression in 5 myoblasts (Fig. 5, B and C).
Densitometry shows that
1 integrin expression increases
approximately twofold in
5 myoblasts (Fig. 5 C), whereas
N-cadherin expression increases more than fivefold. Myoblasts transfected with control plasmids show no increase
in N-cadherin by immunoblotting (data not shown). Ectopic expression of
1 integrin also upregulates N-cadherin more than fivefold in contrast to expression of IL2
1
integrin, in which there is no upregulation of N-cadherin.
The results presented in Table I demonstrate a direct correlation between upregulation of N-cadherin expression
and enhanced growth in clusters. In summary, enhanced
integrin-mediated signaling upregulates N-cadherin expression, demonstrating a cross talk between integrin and
cadherin receptor expression.
Cadherin function is mediated, at least in part, by its
association with multiple cytoplasmic proteins including
-catenin,
-catenin, and plakoglobin. Cadherin function
and signaling may be altered through changes in the levels
or functions of these cytoplasmic proteins (Gumbiner,
1995
). We therefore characterized the expression of these
proteins in myoblasts ectopically expressing the
5 integrin. Significant increases (twofold) in
-catenin and plakoglobin expression are seen in
5 myoblasts when compared with untransfected cells (Fig. 6 A). In contrast, there
is no significant change in
-catenin expression in
5 myoblasts. Similar changes in expression of the cytoplasmic
proteins are seen with ectopic expression of N-cadherin
alone (Fig. 6 B). These studies demonstrate that in addition to upregulating N-cadherin, ectopic expression of
5
integrin results in increased expression of some cadherin-associated cytoplasmic proteins.
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Aggregate Growth and Contact-mediated
Inhibition of Migration in the 5 Myoblasts Is
Mediated by N-cadherin
To implicate N-cadherin directly in contact-mediated inhibition of cell migration, we determined if perturbing
N-cadherin function disrupts the 5 phenotype. Since cadherins are calcium-dependent adhesion molecules, we
plated
5 myoblasts in a low-calcium medium that inhibits
cadherin function (growth in aggregates) but not cell-substratum adhesion.
5 myoblasts grown in low-calcium medium adhere normally to the substratum but demonstrate
a more dispersed pattern of growth than
5 myoblasts
grown in calcium-containing medium (Fig. 7). In addition,
by time-lapse video microscopy these cells do not show
the contact inhibition of migration that characterizes
5-
expressing cells grown in normal media (data not shown).
These observations suggest that the
5 integrin-mediated
contact inhibition is calcium-dependent at concentrations
consistent with cadherin function, implicating cadherin receptors in this process.
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Function-perturbing antibodies further implicate N-cadherin in the 5 integrin-mediated contact inhibition of cell
migration. Pretreatment of
5 myoblasts with NCD-2, a
function-perturbing mAb directed against N-cadherin, disrupts the
5 integrin-mediated aggregate growth at concentrations (100 µg/ml) that inhibit cadherin-mediated adhesion (Fig. 8).
5 myoblasts pretreated with this antibody demonstrate a dispersed pattern of growth, similar to that
of untransfected myoblasts.
|
Experiments using the dominant-negative cadherin
390 further support a role for N-cadherin in the contact-mediated inhibition of migration. This construct is a nonfunctional cadherin with an intact cytoplasmic domain and
a truncated extracellular domain (Fujimori and Takeichi,
1993
).
5 myoblasts were transfected with either a control
plasmid or the N-cadherin mutant
390. Unlike the control plasmid, we were unable to establish stable transfectants expressing the
390 mutant. This is consistent with a
recent study showing that
390 inhibits proliferation and
promotes differentiation in developing keratinocytes (Zhu
and Watt, 1996
). We therefore used transient transfections
and studied the effects on wound closure 48 h after transfection. The dominant-negative cadherin partially reverses
the inhibitory effect of ectopic
5 integrin expression on
wound closure (Fig. 9). The
5 cells expressing the
390
N-cadherin display wound closure with cells moving individually into the wound. The number of single cells migrating into the wound was quantified. An average of 31 ± 6.2 (SD) cells in the
390-transfected
5 myoblasts as
compared with 7 ± 3.8 in
5 myoblasts transfected with
control plasmid migrate into the wound.
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Ectopic N-cadherin expression in myoblasts also promotes contact-mediated inhibition of migration (Fig. 3).
Myoblasts were transfected with chicken N-cadherin
(N-cadherin myoblasts), and a stable population was selected. The N-cadherin myoblasts show a level of N-cadherin expression similar to that of 5 myoblasts by both
Western immunoblotting and FACS® analysis (Fig. 6 B).
The changes in expression of the cadherin-associated signaling molecules, including the catenins and plakoglobin,
are similar to the
5 myoblasts (Fig. 6 B), although there
was no significant change in the expression of
1 integrin
in the N-cadherin myoblasts (data not shown). Like the
5
myoblasts, myoblasts expressing ectopic N-cadherin grow
as aggregates (Fig. 3, I-L) and show a delay in wound closure (Fig. 2 C). Interestingly, and in contrast to
5 myoblasts, ectopic N-cadherin expression alone promotes differentiation but not proliferation (data not shown). Taken together, all of these observations provide strong evidence
for the participation of N-cadherin in contact-mediated inhibition of migration of the
5 myoblasts.
Contact-mediated Inhibition of Motile Activity in 5
Myoblasts Differs from that of Myoblasts Expressing
Ectopic N-cadherin
Two possibilities can be offered to explain the contact-
mediated inhibition of migration seen in 5 myoblasts.
One is that upregulation of N-cadherin increases cell-cell
adhesion, and as a result the cells simply cannot separate
from one another once in contact. Alternatively, the cell-
cell contact may serve a signaling function that inhibits cell
movement by negatively regulating motile activity, i.e.,
membrane protrusions and retractions. To distinguish between these possibilities, the migration of myoblasts transfected with
5 integrin or N-cadherin was observed by
time-lapse video microscopy (Fig. 3). In general, when
control myoblasts come into contact with one another,
their movement and motile activity, both locally (i.e., at
the site of contact) and globally, are not significantly inhibited. In contrast, when myoblasts expressing the
5 integrin come into contact, they show a striking inhibition of
cell migration and cessation of motile activity first locally and subsequently globally (Figs. 3, E-H, and 9). Once in
contact,
5 myoblasts generally do not separate from one
another. Video microscopic analyses show that upon initial contact (first 30-60 min), the
5 myoblasts tend to
have decreased motile activity locally (i.e., membrane protrusions and retractions), and with more prolonged contact (hours), the cells tend to demonstrate global quiescence. An exception to this is seen when cells divide. After
a division cells show increased motile activity despite being
in contact with other cells; but as these cells subsequently spread, this motile activity decreases. Furthermore, we
generally observed that the less spread cells within an aggregate have increased motile activity. Consistent with
these observations are preliminary studies showing that
the shutdown in motile activity observed in the
5 myoblasts is most marked at higher substrate concentrations where the cells are more spread. Taken together, these observations suggest that N-cadherin likely mediates a signaling process that inhibits motile activity in
5 myoblasts.
To determine if increased expression of N-cadherin
alone is sufficient to induce the cessation of motile activity,
we also studied the effects of ectopic N-cadherin expression on the contact-mediated inhibition of motile activity.
Myoblasts ectopically expressing N-cadherin display growth
in clusters and generally do not separate from one another
after making contact. In contrast to the 5 myoblasts, however, N-cadherin myoblasts do not exhibit the striking
contact-mediated cessation of motile activity, either locally or globally. The cells continue active membrane protrusions and retractions throughout prolonged contact
(Figs. 3, I-L, and 10) and frequently migrate along each
other within an aggregate. As a result, the clusters of
N-cadherin myoblasts are not as tight as those of
5 myoblasts. Therefore, the shutdown in motile activity (i.e., quiescence) seen in the
5 myoblasts seems to require an integrin-mediated event, most likely enhanced signaling
and/or cytoskeletal organization, and not just increased
cell-cell adhesion.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we show that ectopic expression of the 5
integrin upregulates N-cadherin expression on the cell surface and together with N-cadherin produces a contact-
mediated inhibition of cell migration and cessation of motile activity in contacting cells. This phenotype is not unique
to ectopic expression of
5 integrin but appears to reflect
enhanced signaling through integrin receptors. The N-cadherin specifically mediates the contact inhibition of migration. Perturbation studies using low calcium conditions, antibody inhibition, and ectopic expression of wild-type and
mutant N-cadherins all directly implicate N-cadherin. However, cessation of motile activity requires, in addition to
N-cadherin, an increase in expression of either
5 or
1 integrins, or integrin-related molecules, since cessation is
not reproduced in cells with only increased N-cadherin.
This suggests that while cadherins are necessary for the
cessation of motile activity, they are not sufficient. These
observations demonstrate that integrin signaling not only regulates cadherin expression in skeletal muscle cells but
also acts coordinately with N-cadherin to produce a highly
aggregated, nonmotile phenotype. This novel observation
has potential importance to normal and pathological phenomena including embryonic development, tumor invasion, and metastasis.
Contact inhibition of cell migration was originally described in fibroblasts by Abercrombie and Heaysman
(1953), who observed that motile activity, including ruffling and lamellipodium formation, is inhibited locally
when fibroblasts make contact. With prolonged contact,
the fibroblasts frequently separate and move away from the region of direct cell-cell contact. The contact inhibition of migration in the
5 myoblasts is more dramatic
than that described for fibroblasts. After making contact,
the
5 myoblasts do not generally separate from one another and instead exhibit an initial local cessation of motile activity followed by global cessation. Ectopic expression of the
1 integrin subunit or a downstream target of
integrin signaling, paxillin, produce a similar aggregated phenotype. Thus, the effects on cell aggregation and migration are not unique to the
5 integrin but rather appear
to reflect an increase in integrin-mediated signaling. Together, these results suggest that increased integrin-mediated signaling promotes an increase in cell-cell aggregation
and reduces migration through an increase in cadherin expression and a coordinate regulation of motile activity.
While the role of integrin-mediated adhesion in this contact inhibition of migration is seen most clearly in these ectopic expression experiments, it is likely that it reflects a
continuum. In this view, the degree of contact inhibition
would depend on the specific levels of cadherin and integrin signaling.
Although ectopic expression of both 5 integrin and
N-cadherin both produce highly aggregated cells that do
not tend to separate once in contact, they have very different effects on motile activity. Ectopic N-cadherin expression alone does not result in the contact-mediated suppression of motile activity that is seen in ectopic
5-expressing cells. When myoblasts overexpressing N-cadherin come
into contact, they do not separate from each other; however, they do continue to move relative to each other and
remain active with membrane protrusions and retractions.
This difference does not likely arise from differences in
N-cadherin, catenin, or plakoglobin expression. The levels
of these molecules in the N-cadherin-transfected cells are
similar to those in the
5-transfected cells. A more likely hypothesis is that increased integrin and cadherin signaling together promote the shutdown in motile activity, suggesting that these two signaling systems are synergistic. In
fact, the data suggest that cadherins are necessary but not
sufficient for the shutdown in motile activity. In any case,
these results show that the regulation of cadherin-mediated
adhesion and motile activity are separable. This is supported by a recent study showing a separation in the regions
of the E-cadherin cytoplasmic domain implicated in adhesion and in suppressing motile activity (Chen et al., 1997
).
The signaling pathways that promote a cessation of motile activity with cell-cell contact have not been elucidated.
Likely downstream candidates include members of the rho
family of small GTP-binding proteins, including rho, rac,
and cdc42, since they regulate the actin cytoskeleton (Hall,
1994). Rac, in particular, may play an important role since
it stimulates lamellipodial formation and ruffling (Hall,
1994
), and its expression is associated with increased invasiveness in vivo (Michiels et al., 1995
). Therefore, regulators of rac, such as GTPase-activating proteins or GAPs (Hall, 1994
), are likely candidates to be downstream effectors that may negatively regulate motile activity.
In addition to having distinct effects on migration, ectopic 5 integrin and N-cadherin expression also have different effects on proliferation and differentiation. Myoblasts expressing ectopic N-cadherin differentiate but show
inhibited proliferation. In contrast, despite comparable
levels of N-cadherin expression, the
5-transfected myoblasts continue to proliferate but do not differentiate under normal conditions (Sastry et al., 1996
). It is intriguing
that ectopic
5 integrin expression promotes contact- mediated inhibition of cell migration but does not inhibit
cell proliferation. Interestingly, despite their tight cell-cell
contacts, the
5 myoblasts show increased proliferation,
suggesting a separation in the pathways regulating contact-mediated inhibition of cell migration and proliferation.
Complex processes such as cell migration and differentiation likely require an integrated response to multiple external stimuli. Evidence for cross-talk between integrin
and growth factor receptors has been shown to play an important role in the regulation of cell proliferation, differentiation (Roskelley et al., 1995; Sastry and Horwitz,
1996
), and migration (Huttenlocher et al., 1995
). Similar
integration or cross regulation also occurs between cell surface adhesion receptors. For example, changes in cadherin expression alter integrin expression and thereby affect differentiation (Hodivala and Watt, 1994
). Our observations are the first to demonstrate that alterations in
integrin expression can result in changes in both cadherin
expression and its function (i.e., in promoting a cessation
of motile activity with cell-cell contact). We demonstrate a
fivefold increase in N-cadherin expression by ectopic
5 or
1 integrin expression in myoblasts. However, ectopic
N-cadherin expression does not result in a change in
1 integrin expression in this system (data not shown). Thus, it
appears that it is the level or alternatively the ratio of integrin and cadherin signaling that determines the phenotype
of migrating cells that come into contact.
Our observations clearly show that the level of integrins and other adhesion-related molecules must be carefully regulated since two- to threefold changes in integrin expression have dramatic effects on cadherin expression and function. Such a highly poised system, in the context of the dynamic patterns of integrin expression during development and other processes, is likely to have significant effects on cell behavior. Examples include cadherin-mediated processes in which cells need to sort out and reposition with respect to each other versus those in which stable, long-term associations form. Myogenic precursors in the somite, for example, interact via cadherins but are highly adherent and quiescent, while in contrast, myoblasts in the limb sort out, through cadherin-mediated interactions involving a more motile type of adhesion.
While our studies focus on myoblasts over-expressing
the 5 and
1 integrins, their implications are likely more
general and pertain to related phenomena in other cell
types. The increase in proliferation seen in
5 myoblasts
is also seen in myoblasts ectopically expressing some other
integrin subunits, e.g.,
1 and
3, but not
6 integrin
(Truong, T., unpublished), and
3 (Blaschuk et al., 1997)
but not
6 (Sastry et al., 1996
). Recent studies on the enhancing effects of
5 integrin and inhibiting effects of the
6 integrin on proliferation point to modulation of
1 integrin signaling as the likely origin (Sastry et al., 1996
; Sastry, S., and M. Lakonishok, manuscript in preparation). Homology in the cytoplasmic domains of the
1 and
3 integrins suggest that the phenotype of ectopic
3 arises
from similar signaling. It is interesting that expression of
the
3 subunit changes as myoblasts initiate terminal differentiation (Blaschuk et al., 1997). It is possible, therefore, that the integrin cadherin synergy functions to facilitate the sorting and alignment of myoblasts in nascent
muscle tissue. Our observations are not unique to skeletal muscle, however. It has previously been demonstrated
that increased
5 integrin expression is associated with decreased tumorigenicity and may function as a tumor suppressor (Giancotti and Ruoslahti, 1990
; Schreiner et al.,
1991
). We have preliminary evidence suggesting that ectopic
5 integrin expression in colon carcinomas promotes contact-mediated cessation of motile activity (Huttenlocher, A., unpublished), suggesting that these findings
may have direct implications for tumor invasion and metastasis.
In summary, our results provide evidence that integrin
and cadherin receptors act coordinately to regulate cell
migration through a contact-mediated inhibition of migration and motile activity. They demonstrate that enhanced
adhesive signaling through ectopic 5 or
1 integrin expression in this case leads to the upregulation of N-cadherin expression and inhibition of cell migration through
enhanced cell-cell aggregation and cessation of motile activity. These studies suggest a novel role for integrin-mediated adhesive signaling in promoting contact inhibition of
migration through a synergy with cadherin signaling. A coordination of cell-cell and cell-matrix interactions is likely
critical to cell migration and cell sorting during embryogenesis and during the invasion of tumor cells in cancer.
![]() |
Footnotes |
---|
Received for publication 11 June 1997 and in revised form 8 October 1997.
Address all correspondence to Anna Huttenlocher, Department of Cell and Structural Biology, B107 Chemistry and Life Sciences Laboratory, 601 S. Goodwin Ave., Urbana, IL 61801. E-mail: huttenlo{at}uiuc.eduThe authors thank T. Abrogast and M. Plutz for excellent technical assistance. We also thank Gary Durack and Karen Magin of the Flow Cytometry Facility at the University of Illinois for assistance in cell sorting. We thank M. Takeichi, Y. Takada, S. LaFlamme, A. Aruffo, and C. Turner for kindly providing us with constructs. We also thank S. Sastry, M. Takeichi, M. Berg, and J. Nelson for useful discussions.
This work was supported by National Institutes of Health grants GM 23244 (to A.F. Horwitz) and a grant from the Arthritis Foundation (to A. Huttenlocher).
![]() |
Abbreviations used in this paper |
---|
ECM, extracellular matrix;
FN, fibronectin;
IL21, interleukin-2
1 cytoplasmic domain;
UT, untransfected
myoblast.
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