Department of Surgery, University of Medicine and Dentistry-Robert Wood Johnson Medical School, CAB Room 7070, New Brunswick, NJ 08648, USA
* Author for correspondence (e-mail: fotyra{at}umdnj.edu)
Accepted 16 October 2002
![]() |
Summary |
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Much of the data elucidating the role of integrins as mediators of
cell-extracellular matrix (ECM) interactions have been generated using
conventional cell culture techniques in which cells are plated onto ECM-coated
2D surfaces. In vivo, cells are embedded in a 3D meshwork of ECM proteins. We
hypothesized that, within this meshwork, integrin-ECM interactions may impart
cohesivity to an aggregate of cells by linking adjacent cells together. To
test this hypothesis, we transfected Chinese hamster ovary (CHO-B2) cells to
express 5ß1 integrin and found that these cells formed compact,
spherical aggregates. We measured aggregate cohesivity using tissue surface
tensiometry, a novel technique that quantifies cell-cell cohesivity of
spheroids under physiological conditions. We determined that
5ß1
integrin is capable of conferring strong cohesivity (
=8.22±0.68
dynes/cm) to aggregates of
5-integrin-transfected cells. This cohesion
was found to be independent of cadherin expression and was significantly
greater than the cohesivity conferred onto CHO-B2 cells transfected with
N-cadherin (
=3.14±0.20 dynes/cm, P
0.0001), a more
traditional cell-cell cohesion system.
Fibronectin-null CHO cells that express 5ß1 integrin but do not
secrete endogenous fibronectin do not form aggregates in fibronectin-depleted
medium. Addition of increasing amounts of exogenous dimeric fibronectin to
these cells resulted in a dose-dependent compaction. However, compaction
failed to occur in the presence of fibronectin monomers. These data indicate
that fibronectin is required for
5ß1-mediated compaction and that
the dimeric structure of fibronectin is essential for this process.
Additionally, aggregate formation of the
5 integrin transfectants was
inhibited by an RGD peptide thus confirming
5ß1 integrin
specificity. Collectively, these data confirm our hypothesis that
5ß1 integrin acts through fibronectin to link adjacent cells
together, thus promoting strong intercellular cohesion in 3D cellular
aggregates.
Key words: Integrins, Cadherins, Cohesivity, Tissue surface tensiometry, 3D, Aggregates
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Introduction |
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5ß1 integrin binds to fibronectin (FN) and has a well-defined
role in cell adhesion, migration and matrix formation. FN is a ubiquitous,
multifunctional component of the ECM that exists as a dimer, with the two
chains connected by disulfide bonds at the C-terminus
(Hynes, 1992
). Structurally,
each FN chain contains a single cell-binding domain including an RGD sequence
to which
5ß1 integrin specifically binds
(Akiyama, 1996
).
Many of the assays measuring the affinity of integrins for their ligands
have relied largely upon either measurement of the binding kinetics
(Goldmann, 2000) or upon
assays in which cells, adherent to a 2D ligand-coated substrate, are subjected
to either centrifugal force (Koo et al.,
2002
) or to shear stress
(Goldstein and DiMilla, 2002
).
Although such assays are useful, they do not take into consideration the 3D
nature of tissues and, as such, do not consider the ECM as a potential
mechanism by which multicellular tissues may be crosslinked. We reasoned that
integrin-mediated adhesion to ECM proteins, when modeled in a 3D system, could
cause cells to aggregate and compact into spheroids imparting cohesivity to
these aggregates, much as cadherins have been shown to do.
To test this hypothesis, we applied a recently developed method, tissue
surface tensiometry (TST), to measure the intensity of intercellular cohesion
within tissue-like aggregates under physiological conditions. The biophysical
principles underlying TST have been previously described in detail
(Foty et al., 1994). Briefly,
TST measures intercellular binding energy by compressing spherical cellular
aggregates between parallel plates in a specially designed device. The shape
of the compressed aggregate is recorded, as is the force with which the
aggregate resists the compressive force. These parameters are then applied to
the Young-Laplace equation (Davies and
Rideal, 1963
), generating measurements of aggregate surface
tension, or cohesivity. Using TST, we have previously shown that aggregate
cohesivity specifies the spatial positioning of tissues within multicellular
aggregates (Foty et al.,
1996
), modulates the rate of tissue spreading on a substratum
(Ryan et al., 2001
) and
influences the emigration of cells from a tumor
(Foty et al., 1998
;
Foty and Steinberg, 1997
).
We generated a series of cell lines custom-designed to express
5ß1 integrin. We also generated cell lines expressing high levels
of N-cadherin in order to quantitatively compare the strength of
integrin-based cohesivity with that of a more traditional cell-cell cohesion
system. We then prepared spherical aggregates of these cells and quantified
their cohesivity by TST. We determined that
5ß1 integrin mediates
strong cohesivity in 3D tissue aggregates and that this cohesivity is greater
than that conferred by N-cadherin alone. We also determined that this
integrin-mediated cohesivity is FN dependent and results from a specific
interaction between
5ß1 integrin and the RGD site on FN.
Structurally, we found that dimeric FN is required for aggregate formation and
compaction. Therefore, using this 3D culture system and TST, we were able to
quantitatively show that
5ß1 integrin is capable of mediating
strong intercellular cohesion. These data are the first to rigorously quantify
the cohesivity imparted to tissue-like cellular aggregates by the interaction
of
5ß1 integrin with FN. These data have broad implications in
fields influenced by these processes including malignant invasion, embryonic
development and wound healing.
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Materials and Methods |
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Construction of an 5 integrin cDNA expression vector
A 1.8 kb BamHI-XhoI fragment encoding the N-terminal
portion of the human 5 cDNA in pLJ (kindly provided by Jean
Schwarzbauer, Princeton University) was cloned into pcDNA 3.1(+) (pcDNA 3.1
5-N, Invitrogen, CA). A 2.5 kb fragment containing the C-terminus of
the human
5 cDNA was then ligated into an XhoI digest of pcDNA
3.1
5-N to yield a complete human
5 cDNA in pcDNA 3.1.
Transfection
Cells were transfected by electroporation in 400 µl of transfection
medium (RPMI, 0.1 mM DTT, 10 mM dextrose) at 200 volts and 960 µF in a 0.4
cm electroporation cuvette using a BioRad Gene Pulser II apparatus. For
expression of human 5 integrin, CHO-B2 cells were transfected with 20
µg of pcDNA3 plasmid containing the coding region for human
5
integrin and resistance to G418. For expression of chicken N-cadherin, CHO-B2
cells were transfected with 20 µg of the N-cadherin expression plasmid
pMiwcN (Fujimori et al., 1990
)
(kindly provided by M. Takeichi, Kyoto University) and 5 µg of the zeocin
resistance plasmid pZeoSV (Invitrogen, CA). Cells were grown to confluence in
medium supplemented with either 800 µg/ml G418 or 500 µg/ml zeocin.
Empty vector control cells (CHO-P3; pcDNA3 only) were generated by the same
transfection and selection processes.
5-integrin-transfected (CHO-A5)
and N-cadherin-transfected (CHO-Ncad) cells were bulk sorted by FACS.
FACS and flow cytometry
CHO-A5 cells were detached with trypsin-EDTA (Gibco-BRL, NY), washed three
times with ice-cold Hanks' balanced salt solution (HBSS) and incubated with an
anti-human 5 integrin antibody (CD49e, PharMingen, CA) at 5 µg/ml on
ice for 45 minutes. Cells were again washed with cold HBSS and incubated on
ice for an additional 45 minutes with a FITC-conjugated goat-anti-mouse
secondary antibody (Zymed, CA). Cells expressing
5 integrin were FACS
sorted (EPICS ALTRA, Beckman Coulter, FL) and expanded. CHO-Ncad cells were
detached from tissue culture plates with trypsin-calcium (0.1% trypsin/5 mM
Ca2+) to preserve cadherin receptor integrity
(Hyafil et al., 1981
) and
sorted to express levels of N-cadherin similar to
5 integrin expression
by the CHO-A5 cells. CHO-Ncad cells were incubated with 10 µg/ml anti
N-cadherin primary antibody (NCD2, Zymed, CA), followed by a FITC-conjugated
goat-anti-rat secondary antibody (Zymed, CA). Cells were sorted three times to
generate pure populations. Receptor expression was confirmed monthly by flow
cytometry.
Western blot analysis
Expression of both 5 integrin and N-cadherin was confirmed by
western blot analysis using standard protocols. Cell lysates were prepared
from near-confluent 10 cm tissue culture plates. Cell monolayers were washed
twice with ice-cold HBSS then lysed by the addition of 500 µl RIPA lysis
buffer (150 mM NaCl, 50 mM TRIS pH 7.5, 1% NP40, 0.25% DOC) containing a
protease inhibitor cocktail (Calbiochem, CA), EDTA and sodium vanadate. The
lysates were transferred to microcentrifuge tubes, rotated at 4°C for 1
hour, then passed through a Qia-shredder (Qiagen, CA) and centrifuged at
14,000 g for 15 minutes at 4°C. Cell lysates from 3D
aggregates were prepared in the same manner except that aggregates were
disrupted by sonication in RIPA lysis buffer. Protein concentrations were
determined using the BCA Protein Assay Kit (Pierce, IL). 20 µg of protein
was separated on a 7% SDS-PAGE gel and electroblotted to nitrocellulose using
standard protocols. Blots were blocked overnight at 4°C in either Membrane
Blocking Solution (CHO-Ncad, Zymed, CA) or 5% nonfat dry milk in TBS-0.2%
TWEEN 20 (CHO-A5). Blots were incubated at room temperature for 1 hour in
either an N-cadherin primary antibody (3B9 at 0.5 µg/ml, Zymed, CA) or in
an
5 integrin primary antibody (AB1928 at 1 µl/ml, Chemicon, CA),
washed three times with TBS-0.2% TWEEN 20, followed by an additional 1 hour
incubation in either horseradish-peroxidase-conjugated goat anti-mouse
secondary antibody (CHO-Ncad) or goat-anti-rabbit secondary antibody (CHO-A5).
Blots were developed using SuperSignal West Pico Chemiluminescent Substrate
(Pierce, IL) and exposed to X-ray film. All blots were then stripped in 62.5
mM Tris HCl pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol for 30 minutes at
50°C and re-probed with an anti-actin antibody (Sigma, MO) to confirm
equal lane loading.
Fast aggregation assay
Aggregation assays, performed in the presence and absence of calcium,
measure rapid, calcium-dependent aggregation
(Takeichi, 1977). This
technique was used to assess cadherin function of the CHO-B2, CHO-P3, CHO-A5
and CHO-Ncad cell lines. Cells were detached from near-confluent 10 cm plates
by trypsin-calcium, washed in PBS, and stained with PKH26 (Sigma, MO) red
fluorescent membrane intercalating dye according to the manufacturer's
instructions. Stained cells were washed three times in HBSS containing either
2 mM CaCl2 (HBSS + Ca2+) or in
Ca2+/Mg2+-free HBSS (HBSS-Ca2+). Cells were
counted and resuspended at 1.0x106 cells/ml of
HBSS±Ca2+. 3 ml of each cell suspension was transferred to
individual 10 ml shaker flasks (Belco Glass, NJ) and incubated on an orbital
shaker at 120 rpm for one hour at 37°C. The degree of aggregation was
assessed by fluorescence microscopy.
Aggregate formation and hanging drop cultures
Cells were removed from near-confluent 10 cm plates with trypsin-EDTA,
washed, counted and resuspended at a concentration of 2.5x106
cells/ml in complete medium supplemented with 2 mM CaCl2. 15 and 20
µl aliquots of this suspension were deposited on the underside of a 10 cm
tissue culture dish lid. The lid was then inverted over 10 ml of 1x
phosphate-buffered saline creating hanging drops on the upper lid. Drops were
incubated under tissue culture conditions for 2-3 days, allowing the cells to
coalesce at the base of the droplets and form sheets. The sheets from hanging
drop culture were then transferred to 10 ml shaker flasks (Belco Glass, NJ) in
3 ml complete medium and placed on an orbital shaker at 110 rpm for 2-3 days.
This encouraged cell rearrangement and 3D spheroid formation. Spheroids ranged
in size from 540-800 µm in diameter.
In order to determine the molecular mechanisms of 5ß1-mediated
aggregate compaction, we performed hanging drop assays in FN-depleted medium.
Serum was depleted of FN by incubation with collagen sepharose beads as
previously described (Corbett et al.,
1997
). Depletion was confirmed by western blot analysis. Hanging
drops of CHO-A5 cells were cultured in the presence of a 100 µM cyclic
RGD-blocking peptide (FR-1, Calbiochem, CA). Hanging drops of the CHO-
5
cells were cultured in FN-depleted medium, with 3-300 µg/ml exogenous rat
plasma FN, or in the presence of a recombinant monomeric FN fragment.
Synthesis of recombinant fibronectin monomer
Construction of recombinant FN monomers was performed in the baculovirus
vector pVL1393 as previously described. Briefly, a terminal codon was
generated by adding an XbaI linker to a StuI site located at
position 6860 in the FNIII1-7 cDNA, yielding a FN molecule
structurally identical to FN
III1-7 but lacking the
dimerization site (Corbett and
Schwarzbauer, 1999
; Sechler et
al., 1996
). This fragment was inserted between the BamHI
and XbaI sites in pVL1393. The construct was confirmed by restriction
enzyme digest analysis. Recombinant protein production was performed as
previously described (Sechler et al.,
1997
).
Tissue surface tensiometry
Aggregate cohesivity was measured by TST as previously described
(Foty et al., 1994;
Foty et al., 1996
). Aggregates
ranging in size from 540-800 µm were transferred to the inner chamber of
the tensiometer and positioned on the lower compression plate (LCP,
Fig. 1A). The inner chamber
contained pre-warmed, de-gassed CO2-independent medium at 37°C.
The upper compression plate (UCP), attached to a nickel-chromium wire, was
then positioned above the aggregate and connected to a Cahn electrobalance.
The weight of the UCP was zeroed to establish a pre-compression UCP weight
baseline. In order to minimize adhesion of cell aggregates to the compression
plates, both the lower and upper plates were pre-coated with poly
2-hydroxyethylmethacrylate (poly-HEMA), a polymeric material to which cells do
not adhere (Folkman and Moscona,
1978
). Compression was initiated by raising the LCP until the
aggregate became compressed against the UCP. Adjusting the height of the LCP
controlled different degrees of compression. The force with which the
aggregates resisted compression was monitored by the Cahn recording
electrobalance. Aggregate geometry was monitored through a 25x Nikon
dissecting microscope equipped with a CCD video camera and connected to a
Macintosh Power PC computer. Images of aggregates were captured, digitized and
their geometries were analyzed using NIH Image software. Each aggregate was
subjected to two different degrees of compression, the second greater than the
first Measurements of aggregate geometry
(Fig. 1B) and the force of
resistance to the compressive force were then applied to the Young-Laplace
equation (Davies and Rideal,
1963
), producing numerical values of apparent tissue surface
tension (
) (Eqn. 1):
![]() | 1 |
|
Because R1, R2 and H can all be directly measured
with greater accuracy than R3, the latter parameter was calculated
using Eqn. 2:
![]() | 2 |
A true surface tension is one in which the measured is invariant of
the applied force, as would be expected of a true liquid surface tension. Only
those measurements of surface tension exhibiting this behavior were used to
calculate aggregate cohesivity.
The two likely material states to be considered as they apply to tissue
aggregates are liquidity and elasticity. The calculated surface tension of a
liquid aggregate, when subjected to two compressions, the second greater than
the first, will remain constant. By contrast, the calculated surface tension
of an elastic aggregate will obey Hooke's law and increase in proportion to
the applied force. For example, we have previously shown that when an elastic
poly-acrylamide sphere is subjected to two successive compressions, the
calculated surface tension increased in proportion to the applied force
(Foty et al., 1996). By
contrast, several liquid systems have been described in which surface tension
remains constant irrespective of the applied force. Such examples include
compression of aggregates of embryonic tissues
(Davis et al., 1997
;
Foty et al., 1994
;
Foty et al., 1996
), HT1080
human fibrosarcoma (Foty et al.,
1998
), Lewis lung carcinoma
(Foty and Steinberg, 1997
) and
genetically engineered cells (Duguay et
al., 2002
).
In order to confirm the validity of our TST measurements, we calibrated our
tissue surface tensiometer by compressing an air bubble in culture medium and
comparing the calculated surface tension with that obtained by the de
Noüy ring method (Davies and Rideal,
1963). Surface tension measured by TST and by the de Noüy
technique were essentially identical, 39 dynes/cm and 42 dynes/cm,
respectively (Foty et al.,
1994
). On the basis of this result, we are confident that the
measured surface tensions of our transfected aggregates represent absolute
values of aggregate cohesivity.
![]() |
Results |
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|
CHO-A5 cells formed compact spheroids
All cell lines were cultured in hanging drops and transferred to shaker
flasks as previously described. CHO-B2 and CHO-P3 cells did not form spherical
aggregates but instead remained as thick, flat sheets
(Fig. 3A). CHO-A5 cells,
however, formed compact spheroids (Fig.
3B). CHO-Ncad cells also formed spherical aggregates
(Fig. 3C). CHO-Ncad aggregates
were used as a comparison for the CHO-A5 aggregates for two reasons. First,
they represent a traditional cell-cell cohesion system, and second, they
formed spheroids and therefore their cohesivity could be quantified by
TST.
|
5ß1 integrin confers stronger aggregate cohesivity than
N-cadherin
When subjected to TST, CHO-A5 aggregates were found to have a surface
tension of 8.22±0.68 dynes/cm. These data were generated from 18
aggregates each subjected to two successive compressions, the second greater
than the first (Table 1). By
contrast, CHO-Ncad cells were found to have a surface tension of
3.14±0.20 dynes/cm (n=20), a value significantly lower than
that measured for CHO-A5 cell aggregates (P0.0001, Student's
unpaired t-test, Table
1). These data suggest that
5ß1 integrin confers
stronger cohesivity to 3D tissue aggregates than does the expression of
comparable levels of N-cadherin.
|
Assessment of liquid behavior of CHO-A5 and CHO-Ncad aggregates
To confirm that our TST measurements represented true surface tension, we
demonstrated that was size and force independent, as would be expected
of a true liquid system (Davies and Rideal,
1963
). As surface tension is an inherent physical property of a
liquid, size independence is an absolute requirement. Air bubbles should
therefore have the same surface tension irrespective of whether they are large
or small if measured under similar experimental conditions. Surface tension of
CHO-Ncad aggregates remained constant over a threefold range of volumes. A
linear regression analysis of
versus volume generated a correlation
coefficient (r2) of 0.099 for CHO-Ncad aggregates, demonstrating no
statistical correlation between the two parameters. This was confirmed by
performing a t-test to compare the confidence intervals. We found
that for an n of 20, the calculated r value (0.315) for CHO-Ncad is
below the critical value of r for testing P=0, (0.378,
=0.05),
proving the null hypothesis that the slope of the linear regression is zero.
This indicates that, for CHO-Ncad aggregates, surface tension is size
independent.
A similar analysis was generated for CHO-A5 aggregates yielding an
r2 value of 0.402, suggesting a possible weak relationship between
and volume (Fig. 4). A
t-test comparing the confidence intervals in this data set generated
an r value (0.634) greater than the critical value of r (0.400, n=18,
=0.05), thus confirming a correlation between surface tension and
volume. We have previously shown that aggregate surface tension can become
variable as a function of time in culture
(Foty et al., 1996
). To
determine if this increased variability was a possible explanation for the
correlation described above, we analyzed both CHO-Ncad and CHO-A5 TST data as
a function of time in culture. Whereas
of CHO-Ncad remained relatively
constant from 4-6 days in culture and exhibited relatively little scatter in
the data,
of CHO-A5 nearly doubled over the same time period
(Fig. 5). Linear regression
analysis of
versus volume for CHO-A5 aggregates at 5 days in culture
generated an r2 value of 0.164
(Fig. 6). The calculated r
value (0.405, n=9) is below the critical value for r (0.582,
=0.05), indicating that, for 5 days in culture, surface tension is size
independent. The 6 day data, however, were much more scattered and yielded an
r value of 0.741 (r2=0.550, n=6), which is greater than
the critical value for r (0.729), indicating size dependence. We conclude from
this analysis that, although the CHO-A5 aggregates behave as liquids, the
properties of these aggregates can, over time, become more variable, possibly
owing to matrix deposition or a transition to elasticity.
|
|
|
In true liquid systems, must also be independent of the applied
force. An air bubble, compressed in liquid, should have the same surface
tension irrespective of the degree of compression. Therefore, the ratio of the
applied forces (F2/F1) will be greater than 1, whereas
the ratio of the measured surface tensions
(
2/
1) should be equal to 1. As
Table 1 demonstrates,
of CHO-Ncad and CHO-A5 aggregates is indeed independent of the applied force
as surface tension measured after two different compressive forces was not
significantly different based on a student's t-test (CHO-A5,
P=0.47; CHO-Ncad, P=0.74;
Table 1). Moreover, the ratio
of F2/F1 relative to
2/
1 was approximately 1.3:1, indicating
that increasing the force at compression 2 had no effect on surface tension
(Table 1). This force
independence further establishes our
values as representative of true
aggregate cohesivity.
CHO-A5 aggregate cohesion is cadherin independent
Transfection of 5 integrin into chick myoblasts has previously been
shown to upregulate expression of N-cadherin
(Huttenlocher et al., 1998
).
One possible explanation for the observed cohesivity of the CHO-A5 aggregates
is upregulation of cadherin expression or function. CHO-B2 cells naturally
express low levels of N-cadherin. No upregulation of N-cadherin was detected
in the CHO-A5 cells by western blot analysis, irrespective of whether cells
were grown as 2D tissue cultures or as 3D aggregates
(Fig. 7A). Moreover, no
upregulation of N-cadherin function was noted as CHO-B2, CHO-P3 and CHO-A5
cells failed to undergo calcium-dependent aggregation
(Fig. 7B). As expected,
CHO-Ncad cells aggregated in a calcium-dependent manner. Thus, upregulation of
N-cadherin expression or function was not responsible for CHO-A5 aggregate
formation.
|
Cohesivity of CHO-A5 aggregates is fibronectin dependent
FN is the primary ligand of 5ß1 integrin. As
5ß1
integrin is not known to form strong homophilic interactions, we hypothesized
that the strong cohesivity observed in the CHO-A5 tissue aggregates was due to
5ß1 integrin's interaction with FN. To test this hypothesis, we
generated hanging drop cultures of CHO-A5 cells in FN-depleted medium. Such
cultures produced small irregularly shaped, loosely associated clusters of
cells (Fig. 8A). The normal
physiological range of plasma FN is 100-1000 µg/ml
(Ouaissi et al., 1986
;
Swisher and Rannels, 1997
).
When 300 µg/ml of plasma FN was added to the FN-depleted medium, the
ability of CHO-A5 cells to form compact spheroids was restored
(Fig. 8B). These data suggest
that FN plays a role in
5ß1-integrin-mediated aggregate formation
and compaction. As CHO cells are known to secrete small amounts of FN
(Rajaraman et al., 1980
), we
postulated that this endogenous FN production could contribute to the basal
level of aggregation of CHO-A5 cells observed in FN-depleted medium.
|
We further confirmed the role of FN in 5ß1-integrin-mediated
cohesivity by utilizing the CHO-
5 cell line that expresses
5ß1 integrin but does not produce endogenous FN
(Sechler et al., 1996
).
Hanging drop cultures of these cells did not aggregate in the absence of
exogenous FN (Fig. 9A) but
progressively formed more compact sheets as FN concentration was increased
from 3-100 µg/ml (Fig.
9B-D). The addition of 300 µg/ml of FN resulted in the
formation of spheroids (Fig.
9E). Spheroid formation was dependent purely on integrin
expression since this subclone does not express cadherin and does not
aggregate in a calcium-dependent manner (E.E.R., S.A.C. and R.A.F.,
unpublished). Moreover, de novo N-cadherin expression was not detected in 3D
spheroids (data not shown). These data demonstrate that
5ß1-integrin-mediated cohesivity of the CHO-A5 aggregates is
dependent upon FN.
|
CHO-A5 aggregate formation and compaction requires a specific
interaction between 5ß1 integrin and fibronectin
FN is a complex, dimeric ECM protein that binds both cells and other ECM
proteins including heparin, fibrin and collagen. 5ß1 integrin is
known to interact specifically with an RGD site located at type III repeat 10
in the cell-binding domain. We earlier demonstrated that the
5ß1-integrin-mediated cohesivity was dependent upon FN. This could
be due to two possible mechanisms:
5ß1 integrin specifically
binding to FN at the cell-binding domain or, alternatively, an interaction
between FN and other cell surface receptors, such as syndecans. To define the
mechanism by which FN promotes aggregate formation, we incubated the CHO-A5
cells in hanging drop culture with an RGD-blocking peptide. This peptide
competes for the ligand-binding pocket of
5ß1 integrin and
specifically interferes with its ability to bind FN
(Akiyama, 1996
). Hanging drop
cultures formed in the presence of RGD peptide produced only dispersed cell
clusters (Fig. 10). These data
support the role of a specific interaction between
5ß1 integrin
and FN in mediating cohesivity. No change was observed with the addition of
RGD peptide to hanging drop cultures of the CHO-P3 cells in FN depleted medium
(data not shown).
|
Dimeric fibronectin is required for aggregate formation and
compaction
FN exists as a dimer, each arm containing a single cell-binding site. The
dimeric structure of FN is essential for its assembly into a fibrillar matrix.
We hypothesized that this dimeric structure was a necessary component in the
linkage of adjacent cells and that monomeric FN would be unable to provide
such a linkage. To test this hypothesis, we used a recombinant FN monomer that
lacks the dimerization site but that contains all the structural elements
necessary for FN matrix assembly. CHO-5 cell hanging drops were
cultured in FN-depleted medium with 100 µg/ml of either monomeric
recombinant FN or dimeric rat plasma FN. Aggregates failed to form in the
presence of the recombinant monomer (Fig.
11). These data demonstrate that the dimeric structure of FN is
essential for
5ß1-integrin-mediated aggregate formation and is
required to link adjacent cells together.
|
![]() |
Discussion |
---|
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This observation was made possible due, in large part, to application of TST, a method that measures the strength of intercellular cohesion of multicellular aggregates under physiological conditions. The main advantage of TST is that this method measures intercellular adhesive intensity within 3D aggregates and therefore more accurately mimics in vivo cellular interactions than do conventional 2D assays. Cells in tissues establish contacts and assume shapes more reminiscent of 3D foams than of the typical `fried egg' configurations adopted when cells are placed on 2D substrate-coated surfaces. Consequently, TST is able to accurately measure the effect of both direct intercellular cohesion (as mediated by cadherins), as well as indirect intercellular contacts mediated by the ECM in which cells are embedded.
It is known that some integrins, including 1ß2,
4ß7
and
Mß2, recognize integral membrane proteins of the IgG
superfamily such as ICAM-1, ICAM-2 and VCAM-1 and are thus able to mediate
direct cell-cell adhesion (Hynes,
1992
). Others, such as integrin
IIbß3, are almost exclusively responsible
for platelet-platelet interaction through GPIb/V/IX
(Schoenwaelder et al., 2000
).
Typically, however, such interactions are not known to contribute to tissue
cohesivity. The more classically defined ECM binding heterodimers, such as
5ß1, function principally to promote the adhesive events required
for cell motility and tension-generated matrix remodeling. However their
contribution to the overall cohesivity of a tissue has never been rigorously
quantified. This work is the first to rigorously quantify the cohesivity
imparted by the interaction of
5ß1 integrin with FN in 3D
tissue-like aggregates.
5ß1 integrin is unique amongst the FN-binding integrins in that
it is the only integrin that naturally assembles FN into a matrix. Studies
have defined many of the structural requirements for FN matrix assembly. These
include FN's dimer structure, the N-terminal assembly domain and FN-binding
sites in the first two type III repeats
(Schwarzbauer, 1991
;
Sechler et al., 2001
).
Integrin binding to the RGD sequence in the cell-binding domain is also an
essential requirement for matrix assembly
(Sechler et al., 1996
).
FN-integrin interactions promote intermolecular association between the FN
dimers, leading to the formation of fibrils. The fact that FN monomers failed
to support aggregate formation supports the concept that FN matrix assembly
may contribute to aggregate cohesivity by creating a scaffold, or an organized
3D matrix, which functionally links the cells to each other and promotes force
generation. In the absence of solid structural support, the net effect of
FN-mediated force generation is tissue compaction and remodeling, processes
that are essential for a variety of biological functions.
The demonstration that the 5ß1-integrinFN interaction
confers strong intercellular cohesivity in 3D cellular aggregates
significantly impacts several areas of interest. The study of malignant
invasion, for example, represents a model system in which the forces of
adhesion and cohesion influence cellular behavior
(Behrens, 1993
;
Foty et al., 1998
;
Foty et al., 1994
;
Foty and Steinberg, 1997
;
Shiozaki et al., 1996
;
Steinberg and Foty, 1997
;
Tlsty, 1998
;
Zhou et al., 2000
). Normal
E-cadherin expression and function is considered to be important in
maintaining tumor integrity whereas overexpression of various integrins has
been associated with increased potential for invasion and migration.
5ß1 integrin is often found to be downregulated in metastatic
cancer, and overexpression has been shown to rescue a transformed phenotype
(Giancotti and Ruoslahti,
1990
). These observations diverge from the commonly held view of
integrins as metastasis-promoting molecules and suggest a possible role for
5ß1 integrin as a potential invasion suppressor molecule, much as
has been reported for E-cadherin. Being able to quantify the contribution of
integrins in regulating tumor cohesivity will provide information necessary
for the development of strategies aimed at promoting intercellular cohesivity,
thus discouraging dissemination of cancer cells.
The 4,
5 and ß1 integrin subunits have been shown to be
essential for embryonic development. The knockout of these genes in mouse
embryos always leads to embryonic lethality
(Beauvais-Jouneau and Thiery,
1997
). Davis et al. have shown that the cohesivity of the germ
layers in amphibian gastrulae correlate perfectly with their spatial position
(Davis et al., 1997
). Injection
of RGD peptide into amphibian blastulae has been shown to block gastrulation
by disrupting cell interactions with FN and preventing formation of the
meshwork of FN fibrils involved in migration
(Boucaut et al., 1984
). If
5ß1-integrin-FN interaction is indeed capable of conferring
cohesivity onto tissues, particularly very early in development when embryos
are essentially aggregate-like, then it is possible that
5ß1-integrin-FN interactions also have the capacity to specify the
relative cohesivity of cells within the gastrula. FN-mediated compaction has
also been shown to be important later in development. For instance, Downie and
Newman demonstrated that a correlation exists between FN secretion and
pre-cartilage mesenchymal condensation during wing and leg bud development in
chick embryos (Downie and Newman,
1995
), with high FN secretion correlating with compact and
spheroidal condensation. Modulation of tissue cohesivity through differential
cadherin expression during tissue development provides one possible mechanism
of tissue assembly (Gumbiner,
2000
; Steinberg and McNutt,
1999
). Modulation of
5ß1 integrin and FN provides yet
another potential cell-cell cohesion mechanism available to the embryo during
the self-assembly process. Accordingly, molecular pathways regulating either
the expression or function of
5ß1 integrin or FN have the capacity
to function as potential morphogens, modulating tissue behavior, perhaps
independently of cadherins.
During wound healing, alterations in integrin expression coincide with the
increase in cell migration required for the repair process. Cells in the wound
milieu alter their integrin expression to interact with ligands of the newly
formed provisional ECM, whose primary structural components include fibrinogen
and FN. The 5ß1-integrin-FN interaction has also been shown to be
vital for retraction of 3D FN-fibrin clot matrices
(Corbett and Schwarzbauer,
1999
), a process crucial to early wound healing and tissue
remodeling. It is thought that engagement of
5ß1 integrin with FN
generates the tractional force required for clot retraction. We propose that
5ß1-FN-mediated intercellular cohesivity also contributes to clot
retraction, in much the same way as the apparent `retraction' or compaction
observed for CHO-A5 aggregates in response to increased concentrations of FN
(Fig. 9).
The information presented in this manuscript is, to our knowledge, the
first quantitative demonstration that the interaction between 5ß1
integrin and FN can give rise to strong intercellular cohesivity of 3D
aggregates. Our data bring into question the undisputed role of cadherins as
the prime mediators of tissue cohesivity and provide an alternative or
additional mechanism by which the balance of cohesive and adhesive forces can
be used to alter tissue behavior. Further understanding of these forces is
important, for example, in such recent developments as the use of
ligand-coated microparticles to compact complex cellular aggregates for tissue
engineering and biomedical applications
(Dai et al., 1994
;
Saltzman and Olbricht, 2002
).
The balance between cell-cell cohesion and cell-substratum adhesion, the
regulatory mechanisms involved in maintaining this balance, and our ability to
quantify them are important for developing unifying principles governing such
processes as embryonic development, wound healing, malignant invasion, tissue
regeneration and tissue engineering.
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