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Fibroblast-mediated collagen gel contraction does not require fibronectin-╬▒5╬▓1 integrin interaction.

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THE ANATOMICAL RECORD 234153-160 (1992)
Fibroblast-Mediated Collagen Gel Contraction Does Not Require
Fibronectin-a$, lntegrin Interaction
-
JAMES J. TOMASEK AND STEVEN K. AKIYAMA
Department of Anatomical Sciences, University of Oklahoma Health Sciences
Center, Oklahomu City, Oklahoma (J.J.T.); Laboratory of Developmental Biology,
National Institute of Dental Research, National Institutes of Health, Bethesda,
Maryland 20892 (S.K.A.)
ABSTRACT
Fibroblasts cultured within free-floating collagen gels can
bind to and reorganize the surrounding collagen fibrils into a more dense
and compact arrangement. Collagen gel contraction provides an in vitro
model for studying fibroblast-collagen interactions important in wound
healing, fibrosis, scar contraction, and connective tissue morphogenesis.
We have assessed the role of fibronectin and its interaction with the aspl
“high affinity” fibronectin-specific integrin receptor in collagen gel contraction. A variety of agents, which specifically inhibit fibronectin-a,P, interactions, were tested for their abilities to inhibit fibroblast-mediatedcollagen gel contraction. These included anti-a,p, monoclonal antibodies, the
synthetic peptide GRGDSP, the cell adhesive fragment of fibronectin, and
an antibody against the cell adhesive region of fibronectin. None of these
agents inhibited collagen gel contraction. Therefore, it is concluded that
fibronectin-a,P, interactions are not necessary for collagen gel contraction.
However, collagen gel contraction is dependent on a member or members
of the PI subfamily of integrin matrix receptors. A polyclonal antiserum
and a monoclonal antibody, both directed against the P1 subunit of integrin
matrix receptors, inhibited the spreading of fibroblasts in the collagen gel
and inhibited collagen gel contraction. This study demonstrates that fibroblast-mediated collagen gel contraction is independent of fibronectin-a,&
interactions but dependent on an interaction of p1 integrin matrix receptors with collagen fibers. o 1992 Wiley-Liss, Inc.
Key words: Extracellular matrix, Wound healing, Morphogenesis
Fibroblasts cultured within a collagen gel attach and to be the result of tractional forces exerted by fibrospread on the surrounding fibrils, assuming an elon- blasts during their spreading and migration in the colgate bipolar morphology (Elsdale and Bard, 1972; lagen gel (Stopak and Harris, 1982). These actin-genTomasek et al., 1982). Over time, the fibroblasts reor- erated forces must be transmitted to the collagen
ganize the collagen fibrils and contract the collagen gel fibrils, either directly or indirectly, to result in collagen
(Bell et al., 1979). Collagen gel contraction is believed gel contraction.
The integrins, a family of extracellular matrix recepto resemble processes that are important in wound
healing, fibrosis, scar contraction, and connective tis- tors, are present on the surface of fibroblasts (Albelda
sue morphogenesis (Bell et al., 1979; Stopak and Har- and Buck, 1991; Hynes, 1987). These receptors appear
ris, 1982). The contraction of collagen gels is an active to function by linking extracellular macromolecules
cellular process. The rate of contraction is dependent with the cytoskeleton and promoting cell attachment,
on cell number, the type and concentration of the col- migration and shape changes (Wayner et al., 1988;
lagen, and serum concentration (Bell et al., 1979; But- Burridge et al., 1988; Akiyama et al., 1989; Clyman et
tle and Ehrlich, 1983; Guidry and Grinnell, 19851, but al., 1990). The integrins are heterodimers consisting of
not on collagen synthesis or degradation (Guidry and noncovalently associated a and p subunits (Albelda
Grinnell, 1985). The interaction of fibroblasts with the and Buck, 1991; Hynes, 1987). They can be divided into
surrounding collagen fibrils results in a more dense
and compact organization of the matrix. This reorganization is dependent on an organized actin cytoskeleton, as treatment with cytochalasin D or B will inhibit
Received October 30, 1991; accepted February 28, 1992.
the spreading of fibroblasts in collagen gels and collaAddress reprint requests to Dr. James J. Tomasek, Department of
gen gel contraction (Bell et al., 1979; Guidry and Grin- Anatomical
Sciences, Biomedical Sciences Bldg., Rm. 553, The Uninell, 1985; Tomasek and Hay, 1984). The mechanical versity of Oklahoma Health Sciences Center, P.O. Box 26901, Oklareorganization of the collagen fibrils has been proposed homa City, OK 73190.
0 1992 WILEY-LISS, INC
154
J.J. TOMASEK AND S.K. AKIYAMA
at least six different subfamilies, each with a different
p subunit. Integrins from the p, subfamily are important in fibroblast adhesion to fibronectin and to collagen (Wayner and Carter, 1987; Takada et al., 1988;
Wayner et al., 1988; Akiyama et al., 1989; Gullberg e t
al., 1989). Fibroblast adhesion to fibronectin involves
the integrin receptor a5p1 (Wayner and Carter, 1987;
Akiyama et al., 1989), while fibroblast adhesion to collagen may be mediated by the integrin receptors alp1,
a2p1and a3p1(Wayner and Carter, 1987; Takada et al.,
1988; Wayner et al., 1988; Gullberg et al., 1989; Clyman et al., 1990).
Fibronectin has been implicated to play a role in collagen gel contraction (Guillery et al., 1986). Fibronectin could potentially act as a ligand linking the cell
surface to surrounding collagen fibers (Kleinman et al.,
1981). However, other studies have suggested that fibronectin may not be necessary for collagen gel contraction (Guidry and Grinnell, 1985; Gullberg et al.,
1990; Asaga et al., 1991). In addition, recent studies
have demonstrated that adhesion to collagen can be
independent of fibronectin (Wayner and Carter, 1987;
Wayner et al., 1988; Takada et al., 1988; Gullberg et
al., 1989; Clyman et al., 1990). To elucidate fibronectin’s role in this process, we investigated whether the
“high affinity”
interaction of fibronectin with the
fibronectin-specific receptor is necessary for collagen
gel contraction. Antibodies, synthetic peptides or fibronectin fragments, which can either compete with or
block the fibronectin-a& integrin interaction, were
used in collagen gel contraction assays. In addition, the
role of p, integrins in collagen gel contraction was investigated using a n antiserum and a monoclonal antibody, which can inhibit the function of p1 integrins.
Antibodies and Fibronectin Fragment
Monoclonal anti-p, antibody (mAb 13) and monoclonal anti-a, antibody (mAb 16) were produced and
characterized as previously described (Akiyama et al.,
1989). Fab fragments of mAb 13 were prepared a s previously described (Akiyama et al., 1989). Monoclonal
antibody against the fibronectin cell adhesive fragment was produced and characterized as previously described (McDonald et al., 1987). The 75 kD cell adhesive fragment of fibronectin was produced and
characterized as previously described (Hayashi and
Yamada, 1983; Yamada and Kennedy, 1984). Polyclonal anti-p, antiserum was obtained from Dr. Martin
Hemler (Dana-Farber Cancer Inst., Boston, MA) and
has been previously characterized (Takada et al.,
1987). Monoclonal anti-a, antibody (PlD6) was obtained from Telios Pharmaceuticals and has been previously characterized (Wayner et al., 1988).
Collagen Gels
Collagen gels were manufactured by rapidly mixing
together 0.125 ml of M-199 media with or without antibodies, synthetic peptides or fibronectin fragment
with 0.125 ml of cells (4 x lo5 cells/ml) and 0.25 ml of
collagen solution (1.5 mg/ml) in the well of a 24 well
bacteriologic plate (Corning, Corning, NY). Plates
were immediately incubated at 37°C in 5% C 0 2 and
95% air to promote collagen fibrillogenesis. Cells were
harvested as described above except that they were
washed and suspended in M-199 supplemented with
10% fibronectin-depleted FBS. Collagen solution was
prepared as described previously (Tomasek et al., 1982;
Tomasek and Hay, 19841, except that fibronectin-depleted FBS was used. In some experiments, complete
MATERIALS AND METHODS
FBS was used in place of the fibronectin-depleted FBS.
The final cell concentration was 1 x lo5 cells/ml and
Reagents
Type I collagen (rat tail tendon, acetic acid extracted) final collagen concentration was 0.75 mg/ml.
Gel contraction was determined by measuring the
was obtained commercially (Collaborative Research,
Bedford, MA). GRGDSP- and GRGESP-peptides were diameter of the gel to the nearest 0.25 mm using a
obtained from Telios Pharmaceuticals (San Diego, CA). Nikon SMZ-1 stereoscope. Fibroblast morphology in
M-199 medium was supplemented with 2 mM glu- the gel was documented with a n Olympus IMT2 intamine and 1%antibiotic-antimycotic (GIBCO, Grand verted microscope with Hoffman modulation contrast
Island, NY). Fetal bovine serum (FBS) was purchased optics.
from Irvine Scientific (Santa Ana, CA). FibronectinAssay for Cell Spreading
depleted FBS was obtained by passing the FBS over a
gelatin-sepharose affinity column (Sigma, Chemical
Quantitation of fibronectin-mediated cell spreading
Co., St. Louis, MO) as described (Engvall and Ruo- of human palmar fibroblasts was performed by a modification of previously described methods (Yamada and
slahti, 1977).
Kennedy, 1984; Akiyama et al., 1986, 1989). Multiwell
Cells
tissue culture dishes (96 wells, Falcon) were incubated
Monolayer cultures of adult human palmar fibro- with 100 ~1 of 20 Fg/ml human plasma fibronectin
blasts were established from explant cultures of pal- (Collaborative Research, Inc.) in Dulbecco’s phosphate
mar aponeurosis. Normal-appearing palmar aponeu- buffered saline (D-PBS) overnight at 4°C and blocked
rosis was obtained a s surgical discard tissue from for 30 min with 100 ~1 of 10 mg/ml heat-denatured
patients undergoing carpal tunnel release. Pieces of (SOOC for 3 min) bovine serum albumin in D-PBS withtissue were placed onto 60 mm tissue culture dishes out C a + + or M g + + . The wells were washed seven
(Falcon, Oxnard, CA), allowed to attach, and cultured times with D-PBS and the prepared substrates were
in supplemented M-199 containing 10% FBS. Cells covered with serum-free M-199 which was removed
were subcultured by treatment with 0.05% trypsin- just prior to use. All subsequent steps of the assay were
0.02% EDTA in Ca/Mg-free Hanks BSS (GIBCO) for 2 performed at 37°C. Subconfluent palmar fibroblasts
min. Cells were washed three times with supplemented were washed with D-PBS and incubated for 2 min in
media containing 10% FBS and cultured in 75 cm2 tis- 100 Fg/ml TPCK trypsin (Worthington Biochemical
sue culture flasks (Falcon). Fibroblasts used in these Corp.) in D-PBS. The cells were then dislodged by
experiments were between cell passages 4 and 10.
shaking, suspended in a n equal volume of regular cul-
FIBRONECTIN IN COLLAGEN GEL CONTRACTION
155
ture media, centrifuged, resuspended in regular culture media, allowed to recover from the trypsinization
for 20 min a t 37"C, and counted. The cells were then
centrifuged and resuspended in serum-free-M-199 at a
concentration of 2 x lo5 celldml. Aliquots (50 p1) of
cells were mixed with 50 pl aliquots of various agents
to be tested in serum-free M-199, added to fibronectin
prepared substrates, and incubated for 1hr at 37°C in
a 95% air, 5% CO,, humidified atmosphere. Cells were
then fixed by adding 100 ~1of glutaraldehyde in D-PBS
directly to the wells. Cell spreading was quantitated by
phase contrast microscopy as previously described
(Grinnell et al., 1977; Yamada and Kennedy, 1984; Akiyama et al., 1986). The percentage of cells that spread
was determined by counting four random microscopic
fields of about 100 cells per field and calculating the
average percent of cells spread per well. Duplicate
wells were then averaged together.
RESULTS
Removal of Serum Fibronectin From FBS Has no Effect on
Collagen Gel Contraction
Normal palmar fibroblasts, when incorporated into a
free-floating collagen gel, will reorganize the collagen
fibrils resulting in contraction of the gel (Fig. 1). Contraction was quantitated by measuring the diameter of
the gel at different time points. The majority of the
contraction of the collagen gel occurred within the first
10 hr under the conditions used in this study (Fig. 2).
Contraction occurred in the presence of fibronectin-depleted fetal bovine serum (Fig. 2). The plasma fibronectin concentration was reduced by at least 95% as
determined by immunoblot analysis (data not illustrated). No differences were observed in collagen gel
contraction in complete FBS or in fibronectin-depleted
FBS, suggesting that exogenous plasma fibronectin is
not necessary for collagen lattice contraction (Fig. 2).
Fibronectin-a,p, interaction is not Necessary for Collagen
Gel Contraction
To determine whether fibronectin-a5Pl integrin matrix receptor interactions play a role in collagen gel
contraction, a variety of agents which compete with or
block this interaction were added to collagen gels. No
inhibition of collagen gel contraction was observed
with either the GRGDSP peptide (Fig. 3) or the 75 kd
fibronectin cell adhesive fragment (Fig. 41, both of
which compete with fibronectin-a5P1 interactions
(Pytela et al., 1985a; Hayashi and Yamada, 1983; Yamada and Kennedy, 1984). In addition, no inhibition
was observed with either a monoclonal antibody that
binds to the cell adhesive domain of fibronectin (Fig. 5)
or two different monoclonal anti-a, integrin antibodies, mAb 16 (Fig. 6) or P1D6 (data not shown), all of
which block fibronectin-a,P, interactions (McDonald et
al., 1987; Akiyama et al., 1989; Wayner et al., 1988). In
addition, none of the agents tested affected the spreading of cells in collagen gels (data not shown).
These agents were tested for their ability to specifically block the spreading of human Palmar fibroblasts
on fibronectin. Palmar fibroblasts spread on substrates
prepared with fibronectin (Fig. 7).
shown in Figure
7, all these agents significantly inhibited cell spreading on fibronectin ( p < 0.01) at the Same concentrations as those used in the collagen gels.
Fig. I . Darkfield photomicrographs of free-floating collagen gels
after 0 (a),2 (b), and 24 hr ( c ) of incubation. Human palmar fibroblasts and type I collagen were mixed to yield 0.75 mg/ml and 1 X lo6
cells/ml. The collagen gels were cultured in M-199 supplemented with
10%fibronectin-depletedFBS as described in Materials and Methods.
Contraction resulted in a symmetrical reduction in the diameter of
the lattice. Bar = 3.5 mm.
156
J.J. TOMASEK AND S.K. AKIYAMA
n
N
150
E
E
W
0
100
2
6
50
"
0
0
5
10
15
20
25
0
5
10
Fig. 2.Effect of exogenous plasma fibronectin on contraction of collagen gels. The collagen gels were prepared as described in Materials
and Methods and cultured in M-199 supplemented with either 10%
normal FBS (-1
or 10% fibronectin-depleted FBS (&A). Similar
contraction occurred under both conditions. Averages of duplicate collagen gels are shown and the total ranges are indicated by the vertical
bars.
0
'
'
5
.
'
10
'
'
15
.
20
25
Time (hrs)
Time (hrs)
01
15
'
20
.
'
25
'
Fig. 4. Effect of the 75 kD fibronectin cell adhesive fragment on
contraction of collagen gels. The collagen gels were prepared as described in Materials and Methods and cultured in M-199 supplemented with fibronectin-depleted FBS. Contraction occurred in the
presence (&A) or absence (0-0) of 1mglml of the fragment. Averages
of duplicate collagen gels are shown and the total ranges are indicated
by the vertical bars.
0
5
10
15
20
25
Time (hrs)
Time (hrs)
Fig. 3.Effect of a soluble RGD-containing peptide on contraction of
collagen gels. The collagen gels were prepared as described in Materials and Methods and cultured in M-199 supplemented with 10%
fibronectin-depleted FBS. Contraction occurred in the presence of 1
mg/ml GRGDSP (&A), 1mglml GRGESP (U),
and no peptide (-1.
Averages of duplicate collagen gels are shown and the total ranges are
indicated by the vertical bars.
Fig. 5. Effect of a monoclonal antibody that binds to fibronectin's
cell adhesive domain (mAb 333) on contraction of collagen gels. The
collagen gels were prepared as described in Materials and Methods
and cultured in M-199
10% fibronectin-depleted FBS.Contraction
occurred in the presence (&A) or absence (c-0) of 100 pgiml of the
antibody. Averages of duplicate collagen gels are shown and the total
ranges are indicated by the vertical bars.
Necessity of the p, Subfamily of lntegrin Matrix Receptors
in Collagen Gel Contraction
plete FBS (data not shown). This inhibition of
contraction was dose-dependent, with the highest concentration resulting in almost total inhibition of contraction. Collagen gel contraction was not inhibited by
either control nonimmune rabbit antiserum added a t
the same dilution as the anti+, antiserum (Fig. 8A) or
by 1 mg/ml of a control rat IgG,, monoclonal antibody,
which is the same antibody subclass as mAb 13 (Fig.
8B). Also, monoclonal antibodies against the a,p? integrin matrix receptor, at much higher concentrations,
did not affect collagen gel contraction (see above). In
To determine the role of PI integrins, in general, in
collagen gel contraction, antibodies were used which
functionally block the interaction of all PI integrins
with their extracellular matrix ligands. The addition of
either a polyclonal anti-p, antiserum or a monoclonal
anti+, antibody (mAb 13) inhibited collagen gel contraction (Fig. 8A,B). Inhibition occurred in the presence of fibronectin-depleted FBS (Fig. 8A,B) or com-
+
FIBRONECTIN IN COLLAGEN GEL CONTRACTION
0
5
10
15
20
157
25
Time (hrs)
Fig. 6. Effect of anti-a, integrin antibody (mAb 16) on contraction of
collagen gels. The collagen gels were prepared as described in Materials and Methods and cultured in M-199 + 10% fibronectin-depleted
, pg/ml
FBS. Contraction occurred in the presence of 1m g / m l ( ~ )200
(V-01,and 100 pg/ml (A-A) of anti-a, integrin antibody (mAb 16).
Also shown is contraction in the presence of 1 mg/ml of a control rat
IgG,, monoclonal antibody (0-0).Averages of duplicate collagen gels
are shown and the total ranges are indicated by the vertical bars.
I
.
1
.
I
.
I
.
I
.
1
I
0
loo
0
5
10
15
20
25
Time (hrs)
Fig. 8. Effects of different anti-p, antibodies on the contraction of
collagen gels. The collagen gels were prepared as described in Materials and Methods and cultured in M-199 + 10% fibronectin-depleted
FBS containing different concentrations of anti-p, antibody or control
antibody. A Contraction in the presence of 1/10 (C.)
and 1/50 (MI
polyclonal anti+, antiserum. Also shown is contraction in the presence of 1/10 (u
and
)1/50 (A-A) pre-immune rabbit serum. B Contraction in the presence of 250 pg/ml(-),
100 pg/ml(V-V), 50 pgiml
(A-A), and 25 pg/ml (M)
anti-p, antibody (mAb 13). Also shown is
contraction in the presence of 1 mg/ml of a control rat IgG,, monoclonal antibody (-).
Averages of duplicate collagen gels are shown
and the total ranges are indicated by the vertical bars.
C
G
F
16 13
P 333 B
Fig. 7. Effect of agents on palmar fibroblast spreading on fibronectin
substrates. Palmar fibroblasts were added alone (C) or in the presence
of GRGDSP (1mg/ml) (GI, 75 kD fibronectin cell adhesive fragment (1
mg/ml) (F), mAb 16 anti-a, integrin antibody (1mg/ml) (16), mAb 13
anti-p, integrin antibody (250 pg/ml) (131, P1D6 anti-a, integrin antibody (1:1500 dilution) (P),and mAb 333 anti-cell adhesive domain of
fibronectin (100 pg/ml) (333). There was no spreading if the substrate
was not coated with fibronectin (B). Each bar is the average of eight
determinations from random fields of cells ( 2SEM).
the control cells assumed an elongate bipolar configuration in the collagen gel (Fig. 9b).
The inhibition of cell spreading and collagen gel
contraction by anti-p, antibodies was reversible. Replacement of the antibody solution with media lacking
antibody, after 24 hr in culture, resulted in cell elongation and collagen gel contraction (data not shown).
DISCUSSION
another control experiment, Fab fragments of mAb 13
also inhibited collagen gel contraction (data not
shown).
Cell spreading within the collagen gel was also inhibited by the anti+, antibodies a t concentrations
which inhibited collagen gel contraction (Fig. 9). After
24 hours in the presence of the antibody, most of the
cell bodies were still round with only short pseudopodia
projecting into the collagen gel (Fig. 9a). In contrast,
It has been previously demonstrated that fibroblasts
will spread on collagen fibrils in hydrated collagen lattices and assume an elongate bipolar configuration
(Elsdale and Bard, 1972; Tomasek et al., 1982). In addition, fibroblasts can exert force upon the collagen
fibrils, resulting in reorganization and contraction of
the collagen gel (Bell et al., 1979). Collagen gels provide a simple and very sensitive model for examining
the functional interaction of cell surface receptors with
their extracellular ligands. In this study, we demon-
158
J.J. TOMASEK AND S.K. AKIYAMA
Fig. 9. Effect of anti-p, antibody (mAb 13) on cell spreading in
collagen gels. The collagen gels were prepared as described in Materials and Methods and cultured for 24 hr in M-199 + 10% fibronectindepleted FBS containing 250 kg/ml of anti-p, antibody (a)or 1mg/ml
of a control rat IgG,, monoclonal antibody (b). Cells were photographed with Hoffman modulation contrast optics. Bar = 75 pm.
strate that fibroblasts can contract collagen gels independent of an interaction between fibronectin and the
a&
“high affinity” fibronectin-specific receptor. The
ability of fibroblasts to spread on collagen fibrils and to
contract the collagen gel is dependent upon the p1 subfamily of integrin matrix receptors. These results suggest that collagen gel contraction can occur by a direct
interaction of integrin matrix receptors for collagen
with surrounding collagen fibers.
The role of P,-integrin matrix receptors in the contraction of collagen gels was examined with the use of
antibodies against p1 integrins. Two different anti+,
antibodies were used: (1)a polyclonal antiserum previously demonstrated to inhibit fibroblast attachment
to and spreading on a collagen substratum (Takada et
al., 1988); and (2) a monoclonal antibody that will inhibit the spreading of human palmar fibroblasts on a
fibronectin substratum. The inhibition of collagen gel
contraction with these antibodies demonstrates that
this process is dependent upon P,-integrin matrix receptors. The effects of these antibodies on spreading
and contraction were reversible, demonstrating that
their inhibition was not due to toxicity. In addition, the
inhibition was not due to effects of cross-linking the
P,-integrin receptors on the cell surface with the antibodies, since Fab fragments of mAb 13were effective in
inhibiting contraction.
Previously, Gullberg et al. (1990)have demonstrated
that a monospecific polyclonal anti-rat p1 antibody
could inhibit collagen gel contraction by rat heart fibroblasts but could only partially inhibit contraction
by human fibroblasts. In this study, we have demonstrated that a monoclonal antibody raised against the
human p1 integrin subunit can inhibit collagen gel
contraction by human fibroblasts. These results demonstrate that collagen gel contraction by different cell
types from different species is dependent upon the P1
subfamily of integrin matrix receptors.
In the present study, we determined that fibroblastmediated collagen gel contraction is not affected by a
variety of inhibitors of cell-fibronectin interactions.
The GRGDSP peptide has been previously shown to
inhibit a variety of fibronectin-dependent processes
(Hemler et al., 1987; Hynes, 1987; Albelda and Buck,
1991).In this paper, we demonstrate that it can inhibit
fibronectin-mediated spreading of human palmar fibroblasts. This peptide does not inhibit fibroblast-mediated collagen gel contraction at similar concentrations,
in agreement with a previous report by Gullberg et al.
(1990). Although this peptide can inhibit numerous fibronectin-dependent processes, its affinity for the integrin receptor asplis about 100-fold lower than the intact fibronectin molecule (Akiyama and Yamada,
1985). The 75 kD cell-binding fragment of fibronectin
interacts with cells with a similar affinity as the intact
molecule (Akiyama et al., 1985). The addition of the 75
kD fragment to collagen gels does not inhibit contraction, similar to that observed for GRGDSP. This result
demonstrates that even agents that can compete with
intact fibronectin for binding to its receptor do not inhibit collagen gel contraction. consistent with these
results, we have also observed that a monoclonal antibody which binds to the cell adhesive region of fibronectin and sterically hinders interaction with the aqpl
integrin receptor (McDonald et al., 1987) does not inhibit collagen gel contraction.
This report also demonstrates that directly blocking
the interaction of the a5p1integrin receptor with fibronectin does not inhibit collagen gel contraction. mAb
16 binds specifically to the a5p1 integrin receptor and
blocks its binding to fibronectin (Akiyama et al., 1989).
As demonstrated here, mAb 16 will bind to human
palmar fibroblasts and inhibit fibronectin-mediated
spreading; however, mAbl6 does not inhibit collagen
gel contraction. These results demonstrate that fibronectin-a5pl integrin interaction is not necessary for
collagen gel contraction.
Recently, it has been proposed that the “promiscuous” integrin receptor a3p1 also binds to the RGD
cell-binding region of fibronectin (Elices et al., 1991).
The binding of this receptor to fibronectin can be
blocked by RGD peptides (Elices et al., 1991). The lack
FIBRONECTIN IN COLLAGEN GEL CONTRACTION
of inhibition of collagen gel contraction with GRGDSP
would suggest that the interaction of a3p1 integrin
with fibronectin is not necessary for this process.
It should be emphasized that this study does not rule
out the possibility that fibronectin may function as a
ligand linking the cell surface with collagen fibrils under certain circumstances. In fact, other studies have
suggested that fibronectin can play a role in collagen
gel contraction (Guillery et al., 1986; Asaga et al.,
1991). However, in those studies, the contraction occurred over a greater period of time compared to our
study and that of Gullberg et al. (1990). We purposely
set conditions which would allow for collagen gel contraction to occur rapidly for two reasons: (1) the less
time in culture, the less cellular fibronectin synthesized, and (2) the forces required t o reorganize collagen
fibrils should be the least in a lattice that undergoes
the most rapid contraction (Nishiyama et al., 1988). In
lattices undergoing slower contraction, fibronectin
may augment the binding to collagen to increase the
force exerted by cells on collagen. Studies are currently
underway to test this possibility.
The p1 integrins that have been implicated in cell
attachment to type I collagen include the alp1,a2p1,
and a3p1 integrin matrix receptors (Santoro, 1986;
Wayner and Carter, 1987; Wayner et al., 1988; Takada
et al., 1988; Gullberg et al., 1989; Clyman et al., 1990).
Consistent with our study, fibroblast and platelet binding to collagen is RGD-independent (Gullberg et al.,
1989; Santoro 1986). Multiple integrin receptors on the
same cell may participate in attachment to collagen.
Antibodies against either the azpl or the a3p1integrin
matrix receptor can inhibit fibroblast adhesion to collagen (Wayner and Carter, 1987). The integrin matrix
receptors alp1and a2p1 both function on rat aortic
smooth muscle cells in attachment to type I collagen
(Clyman et al., 1990). Whether the direct binding of
fibroblasts to collagen by one, two, or all three of these
collagen-binding integrins is sufficient for collagen gel
contracting is presently unknown.
ACKNOWLEDGMENTS
The authors thank Mr. Melville Vaughan for his
technical assistance with this project. We also thank
Dr. Robert Buchanan of the Department of Plastic Surgery and Dr. Gaza Rayan of the Department of Orthopedic Surgery, University of Oklahoma-Health Sciences Center, for their contributions of tissue, and Dr.
Martin Hemler for generously providing the anti-p,
antiserum used in this study. This research was supported by grants from the Presbyterian Health Foundation (PHF 101 and PHF 85) to Dr. James Tomasek.
NOTE ADDED IN PROOF
After the submission of this manuscript, two papers
have appeared examining the role of the integrin receptor azpl in collagen lattice contraction: Schiro et al.
(1991) and Klein et al. (1991).
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interactions, contractile, fibronectin, gel, integrins, required, collagen, mediated, fibroblasts
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