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INTRODUCTION |
Fibronectin is an extracellular matrix component that is also
present as a soluble protein in plasma and other body fluids. The
matrix form of fibronectin is believed to support cell adhesion and
migration during embryogenesis, tumor growth, wound healing, angiogenesis, and inflammation (1-3). Assembly of soluble fibronectin into matrix is a multistep process under cellular control (4). Among
the membrane components implicated in fibronectin matrix assembly,
integrins have been firmly demonstrated to have a central role
(5-10).
Integrins are a group of cell surface heterdimers of
- and
-glycoprotein subunits that mediate cell adhesion to extracellular matrix proteins such as fibronectin, laminin, vitronectin, and collagen
or to countereceptors on other cells (11, 12). The interactions between
integrins and their ligands influence a number of cellular processes,
including proliferation (13), differentiation (14), survival (15, 16),
and migration (17, 18). The extracellular domains of the two subunits
are noncovalently associated, forming a ligand-binding pocket, and the
cytoplasmic domains interact with cytoskeletal proteins and other
cytoplasmic components (12). In addition to mediating adherence,
ligation of integrins activates signal transduction pathways (19,
20).
The mechanisms by which integrins modulate fibronectin assembly are not
well understood. Transfection of
5-integrin and expression of
5
1-integrin by CHO1
cells results in a large increase in fibronectin assembly (5, 21). A
chimera comprising the interleukin 2 receptor and the cytoplasmic tail
of
1, working presumably in a dominant-negative manner, inhibits
assembly (22). Monoclonal antibodies to
5 or
1 inhibit binding
and assembly of fibronectin by fibroblasts and also binding of the
N-terminal fibronectin fragment to cell surfaces (23, 24). The 70-kDa
N-terminal fragment of fibronectin that mediates binding to assembly
sites colocalizes with
1-integrin in focal contacts of
cycloheximide-treated cells (25). Studies of
5 knock-out mice and
cells derived from these mice and also of
1 knock-out cells (7, 8,
26) indicate that other molecules can substitute for
5
1 in matrix
assembly. Expression of activated forms of
IIb
3 allows CHO cells
to assemble a fibronectin matrix (6, 27). In contrast, overexpression
of
v, which can pair with
1 or
3 (28), or
4, which pairs
with
1 (29), does not confer assembly competency to CHO cells.
Transfection of
3 causing overexpression of
3
1, an adhesion
receptor for entactin and other molecules, however, allows assembly of
fibronectin if CHO cells are cultured on an entactin-coated substrate;
such assembly is not blocked by antibodies to the cell adhesion domain
of fibronectin (30). Thus, expression of several different integrins
allows adherent cells to be assembly competent, and the specific
integrins specifically required seem dependent on the substrate to
which assembling cells are adherent.
We recently found that
1-integrin-null GD25 fibroblasts fail to bind
the N-terminal 70-kDa matrix assembly domain of fibronectin when the
cells are cultured on vitronectin (10). We now show that adhesion to
intact vitronectin has a dominant-negative effect on fibronectin matrix
assembly by GD25 cells and that expression of functional
1A-integrin
overcomes the suppression of fibronectin matrix assembly by the
vitronectin-coated surface. GD25 cells on vitronectin were well spread,
but GD25 cells on fibronectin or GD25-
1A cells on either vitronectin
or fibronectin were contracted and displayed numerous hairy
protrusions. These results indicate that one role of
1A-integrin is
to facilitate cytoskeleton organization and cell shape and that this
signal is missing when
1A-deficient cells are adherent to vitronectin.
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EXPERIMENTAL PROCEDURES |
Materials--
Lysophosphatidic acid (LPA) was from Avanti Polar
Lipids (Birmingham, AL). Fatty acid-free bovine albumin was from Sigma. Vitronectin was purified in its native form (31). Human plasma fibronectin and the 70-kDa N-terminal, gelatin-binding fragment of
fibronectin generated by cathepsin D were isolated, iodinated, and
reisolated as described previously (32, 33). Fibronectin was also
labeled with FITC (33). The labeled proteins were stored in portions at
70 °C in Tris-buffered saline containing 0.1% (w/v) fatty
acid-free bovine albumin. A chimeric protein comprising residues 1-53
of vitronectin and the gelatin-binding part of fibronectin (vitronectin
portion N-terminal to the fibronectin portion) was constructed and
expressed in insect cells using a published strategy (34).
Anti-fibronectin and anti-vitronectin antisera were produced in
rabbits. IgG was purified from sera by affinity chromatography on
protein A.
Recombinant modules III7-III10 of human fibronectin were expressed in
bacteria and purified (35) using the expression plasmid kindly provided
by Harold Erickson (Duke University). To introduce mutations into the
synergistic sequence DRVPHSRN in the III-9 module, in vitro
mutagenesis by two-stage polymerase chain reaction was performed using
5' or 3' oligonucleotides containing the base changes and 3' or 5'
oligonucleotides outside two convenient BamHI restriction
sites. After confirmation of the sequence, the mutated fragment was
cloned into the III7-III10-expressing vector (35), replacing the
natural BamHI-BamHI fragment. This yielded the
mutated polypeptides III7-III10 R1374A, R1374,1379A, and R1379A; in
which DRVPHSRN was changed to DAVPHSRN, DAVPHSAN, and DRVPHSAN, respectively.
Cells--
GD25 and GD10 cells were obtained by differentiation
of the
1-null stem cell clone G201, which is deficient in the
integrin
1 subunit because of disruption of the
1 gene by
homologous recombination (36). GD25-
1A cells transfected with the
1A splice variant of
1-integrin were those described by
Wennerberg et al. (7). D130A mutant
1A was generated in
the pBS
1A expression plasmid (7) by oligonucleotide-primed DNA
synthesis using the U.S.E. mutagenesis kit (Amersham Pharmacia
Biotech). Stably transformed cell lines were cloned after
electroporation of the mutant
1A cDNA into GD25 cells using
puromycin resistance for selection (7, 10). GD25 and GD10 cells were
cultured in DMEM (Life Technologies, Inc.) with 10% fetal bovine serum
(Intergen, Purchase, NY) (i.e. nonselection medium). The
1A-expressing cell lines were cultured in the same medium plus 10 µg/ml puromycin (selection medium) and placed in medium lacking
puromycin only for short-term experiments. Cell surface levels of
1A,
5, and
6 were monitored by flow cytometry, and clones were
chosen in which expression of mutant
1A was the same as GD25 cells
expressing wild-type
1A (10).
Adhesion and Migration Assays--
Cell adhesion and migration
assays were performed as described previously (10).
Binding Assay--
Confluent cell layers were usually studied
3 d after sparse seeding in DMEM plus 10% fetal bovine serum. For
2-4-h culture without serum, tissue culture plastic wells were coated
overnight with adhesive proteins, 0.02-10 µg/ml, in
phosphate-buffered saline at 4 °C, followed by blocking with 1%
bovine serum albumin at 37 °C for 1 h. In preparation for
experimentation, confluent cells were treated with trypsin and EDTA
until cells detached (usually 5 min for GD25-
1A and GD25-
1A/D130A
and 10-15 min for GD25 and GD10), followed by washing twice with DMEM
and resuspension at 4 × 105 cells/2-cm2
well in DMEM containing 0.2% bovine serum albumin and 50 µg/ml soybean trypsin inhibitor. Endogenous fibronectin accumulation in cell
layers plus medium during the 2-4-h initial incubation time was
measured by Western blot. For binding studies, freshly seeded cells on
the adhesive substrates or the confluent cell layers were washed with
Tris-buffered saline and incubated with 125I-labeled
fibronectin or 70-kDa fragment of fibronectin in DMEM containing 0.2%
fatty acid-free bovine albumin in the absence or presence of 400 nM LPA for 60 min as described (37).
Microscopy--
Cells cultured on different adhesive
substrate-coated glass coverslips were incubated with 400 nM LPA for 60 min at 37 °C. For visualization of bound
exogenous fibronectin, cells were incubated with 10 µg/ml
FITC-labeled human plasma fibronectin for 60 min at 37 °C. For
localization of endogenous fibronectin, cell layers were fixed and
incubated with 1:1000 rabbit anti-human fibronectin at 37 °C for
2 h, after the detection of the polyclonal antibody by
rhodamine-conjugated goat anti-rabbit IgG. The anti-fibronectin was
shown to be cross-reactive with mouse fibronectin at this dilution.
Coverslips were mounted with glycerol gel (Sigma), and cells were
viewed on an Olympus epifluorescence microscope or a Bio-Rad confocal
microscope at the Keck Facility of the University of Wisconsin.
For scanning electron microscopy, cells were washed, prefixed, and
post-fixed with 2.5% glutaraldehyde and then 0.1% osmium tetroxide,
both in 0.1 M Hepes, pH 7.0. Post-fixed cells were washed,
dehydrated with ethanol, and critical point-dried (38). The cells were
examined on a Hitachi S-900 high resolution, low voltage, scanning
electron microscope at the Integrated Microscopy Resource of the
University of Wisconsin.
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RESULTS |
The fibronectin matrix of confluent GD25 cells expressing
1A
integrin is more extensive than the fibronectin matrix of confluent
1-null GD25 cells (7, 10). Assembly of fibronectin matrix involves a
reversible binding step, which is mediated by the N-terminal region of
fibronectin, followed by insolubilization of the fibronectin molecules
to form SDS-insoluble multimers (4, 39). We previously found that
the N-terminal fragment of fibronectin binds to GD25 cells after
adherence to fibronectin but not after adherence to vitronectin (10).
To understand how assembly of fibronectin is modulated by vitronectin
and
1-integrins and to relate our findings to those of Wennerberg
et al. (7), we carried out additional studies with GD25
cells lacking
1-integrin, GD25-
1A cells, which are stably
transfected with the cDNA encoding wild-type murine integrin
1A,
and GD25-
1A/D130A cells, which express
1A with the inactivating
D130A mutation in the extracellular domain.
Fibronectin Assembly by Confluent versus Freshly Seeded GD25 and
GD25-
1A Cells--
Initial experiments replicated the conditions of
Wennerberg et al. (7). Cells were grown to confluence in
serum-containing medium and then incubated with the
125I-labeled N-terminal 70-kDa fragment of fibronectin in
the absence or presence of LPA, which enhances the binding and assembly
of fibronectin (37, 40, 41). In contrast to the quantitative and
qualitative decrement in fibronectin staining observed around confluent
GD25 cells compared with confluent GD25-
1A cells (7, 10), the two
cell lines bound the 70-kDa fragment equally well (Fig.
1). Similar results were also found in a
1-h assay of binding of intact fibronectin to confluent cells (data not
shown).

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Fig. 1.
Binding of the 70-kDa N-terminal fragment of
fibronectin to confluent GD25 and GD25- 1A cells. GD25 and
GD25- 1A cells were grown to confluence in nonselection medium (for
GD25) and selection medium (for GD25- 1A) for 3 days on tissue
culture plastic; the media contained 10% serum. Binding of the
125I-labeled 70-kDa fragment of fibronectin was determined
in the absence ( ) or presence (+) of 400 nM LPA as
described under "Experimental Procedures." The cell-associated
70-kDa fragment in the absence of unlabeled ligand (Total)
and in the presence of excess unlabeled 70-kDa fragment (nonspecific
binding (NSB)) are presented as mean values ± SD
(n = 3). Error bars were omitted when the value was
<0.2 ng/mg 70-kDa fragment.
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To learn whether decreased fibronectin matrix at confluence is
attributable to a deficiency in assembly as GD25 cells are growing to
confluence, we studied cells shortly after seeding onto one or the
other of the major adhesive proteins in serum; i.e. GD25 and
GD25-
1A cells were seeded densely on a substrate coated with
vitronectin or fibronectin for 2-4 h and tested for binding of
FITC-labeled fibronectin or 125I-labeled 70-kDa fragment of
fibronectin. Endogenous fibronectin accumulation in cell layers plus
medium during the 2-4-h incubation as measured by Western blotting was
similar for GD25 cells and GD25-
1A cells: 10-50
ng/2-cm2 well. GD25 and GD25-
1A cells on both
vitronectin and fibronectin substrate had small amounts of cell surface
fibronectin staining in punctate or short linear patterns when analyzed
by immunofluorescence with antibodies that recognized mouse fibronectin
(Fig. 2, a-d). The staining
was greatest on GD25-
1A cells adherent to fibronectin. GD25 cells on
vitronectin did not bind FITC-fibronectin, whereas short linear arrays
of FITC-fibronectin binding were abundant when GD25 cells on
fibronectin (Fig. 2, A and B). GD25-
1A cells adherent on either vitronectin or fibronectin had cell-associated FITC-fibronectin fibrils, especially at areas of cell-cell interaction (Fig. 2, C and D).

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Fig. 2.
Endogenous fibronectin deposition and
FITC-fibronectin binding ability of freshly seeded GD25 and GD25- 1A
cells. GD25 (a, b, A, and B) and GD25- 1A
(c, d, C, and D) cells were seeded on vitronectin
(a, c, A, and C) or fibronectin (b, d,
B, and D) in DMEM for 4 h. Cells were incubated
without (a-d) or with (A-D) FITC-fibronectin,
10 µg/ml, in the presence of 400 nM LPA for 60 min. The
endogenous fibronectin deposition (a-d) was visualized by
staining with antibodies against fibronectin, whereas FITC-fibronectin
binding (A-D) was visualized on the basis of its intrinsic
fluorescence. Bar, 10 µm.
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Binding of the 125I-labeled N-terminal 70-kDa fragment of
fibronectin to the freshly seeded monolayers correlated with results of
fluorescence microscopy. The 70-kDa fragment bound specifically to GD25
cells spread on fibronectin (Fig.
3A) and GD25-
1A cells spread on either vitronectin or fibronectin (Fig. 3B).
Binding to GD25 cells spread on fibronectin or GD25-
1A cells spread
on fibronectin or vitronectin was enhanced by LPA treatment (Fig. 3,
A and B). For LPA-treated GD25 and GD25-
1A
cells spread on fibronectin, the Kd values were 7.5 and 4.8 nM, respectively, versus 10.8 and 9.0 nM for the same cells without LPA treatment, and there were
170,000 and 190,000 binding sites per LPA-treated cell, respectively,
versus 110,000 and 130,000 sites for untreated cells.
GD25-
1A cells spread on vitronectin bound less 70-kDa fragment
compared with GD25-
1A cells spread on fibronectin (Fig. 3B); the Kd was 8.7 nM with
LPA versus 14.4 nM without LPA, and there were
80,000 binding sites per LPA-treated cell versus 59,000 sites per untreated cell. GD25 cells spread on vitronectin did not bind
the 70-kDa fragment specifically, even after stimulation with LPA (Fig.
3A).

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Fig. 3.
Binding of the 70-kDa N-terminal fragment of
fibronectin to freshly seeded GD25 and GD25- 1A cells. GD25
(A) and GD25- 1A (B) cells were seeded on
vitronectin (VN) or fibronectin (FN) in DMEM for
4 h. Increasing amounts of 125I-labeled 70-kDa
fragment were incubated with cells in the absence ( LPA) or
presence (+LPA) of 400 nM LPA for 60 min.
Specific binding was calculated by substraction of nonspecific binding
(in the presence of 500 µg/ml unlabeled fibronectin) from total
binding and expressed as ng of 70-kDa fragment bound/well. Data
represent mean ± SD (n = 3). Error bars are
missing when less than the size of symbol.
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Effect of
1A with the D130A Mutation--
The fact that
GD25-
1A cells are capable of 70-kDa fragment binding on
vitronectin-coated surfaces, whereas GD25 cells are not, indicates that
1A-integrins overcome the absent or negative input arising from
adhesion of cells to vitronectin. We previously found that expression
of
1As with inactivating mutations of cytoplasmic residues did not
restore the ability of GD25 cells on vitronectin to bind the 70-kDa
fragment (10). To test whether
1A with a wild-type cytoplasmic
domain but a nonactive extracellular domain is restorative, a
1A-integrin with a D130A mutation was generated and transfected into
GD25 cells. Mutation of the homologous residue in
3 is associated
with defective platelet
IIb
3 function and Glanzmann
thrombasthenia (42). GD25 cells expressing the D130A mutant adhered
well to vitronectin or fibronectin (Table
I) but did not adhere to laminin (data
not shown). GD25-
1A/D130A cells did not bind the 70-kDa fragment
when adhered to vitronectin, even in the presence of LPA (Fig.
4). Therefore,
1A-integrin must have
both functional intracellular and extracellular domains to support
fibronectin matrix assembly by cells cultured on vitronectin. In
contrast to GD25 cells lacking
1A (10), GD25 cells expressing
1A/D130A did not bind the 70-kDa fragment even when cultured on
fibronectin (Fig. 4). Furthermore, when D130A cells were studied by
immunofluorescence after reaching confluence, no fibronectin extracellular matrix was present (data not shown). These observations indicate that the D130A mutation in
1A has a dominant-negative effect on fibronectin matrix assembly. Adherence of GD25 cells expressing
1A/D130A to either vitronectin or fibronectin and migration of
1A/D130A cells through filters coated with vitronectin or fibronectin was not grossly impaired when compared with GD25 cells
or GD25-
1A cells (Table I). Therefore, the dominant-negative effect
of
1A/D130A on cells cultured on fibronectin is specific for
fibronectin assembly.
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Table I
Adhesion and migration of GD25, GD25- 1A, and GD25- 1A/D130A
cells
Attachment of GD25, GD25- 1A, and GD25- 1A/D130A cells to
vitronectin or fibronectin or migration through vitronectin- or
fibronectin-coated filters in response to platelet-derived growth
factor, 30 ng/ml. Results are expressed as a colorimetric assay for the
extent of cell attachment to microtiter wells coated with either
vitronectin or fibronectin or the number of cells that migrated to the
lower surface of the filter. Numbers represent mean ± S.D.
(n = 4).
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Fig. 4.
Binding of the 70-kDa N-terminal fragment of
fibronectin to freshly seeded GD25, GD25- 1A, and GD25- 1A/D130A
cells. Tissue culture plates were coated with vitronectin
(VN) or fibronectin (FN), 2 µg/ml. GD25,
GD25- 1A, and GD25- 1A/D130A cells were seeded on different
substrate in DMEM for 4 h. The 125I-labeled 70-kDa
fragment was incubated with cells for 60 min. The cell-associated
70-kDa fragment in the absence of unlabeled ligands (Total)
and in the presence of excess unlabeled 70-kDa fragment
(NSB) are presented as mean values ± SD
(n = 3).
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Effects of the Central Cell Adhesion Domain of Fibronectin and the
Vitronectin Chimera--
One explanation for the differential response
of the GD25 cells cultured on vitronectin versus fibronectin
is that substratum-bound fibronectin is used as a co-polymerizing
molecule (43, 44), in accord with the proposal that a complex of III1,
III10, and integrin mediates binding of the N-terminal fragment of
fibronectin to cells (45, 46). Therefore, responses of cells adherent to recombinant proteins constituting the central cell adhesion domain
of fibronectin were compared with the response to whole fibronectin.
GD25 cells spread on III7-III10 were capable of binding of the 70-kDa
fragment (Fig. 5). The sequence DRVPHSRN
in III9 (residues 1373-1380) has been identified as a synergistic site for integrin functions (47, 48). We mutagenized the synergy sequences
to form R1374A with an Ala in place of the Arg1374, R1379A
with an Ala replacing Arg1379, and R1374,1379A with Alas
replacing both Args. When the mutant proteins were tested, all
supported 70-kDa fragment binding to GD25 cells (Fig. 5). To further
investigate the determinants for fibronectin matrix assembly on GD25
cells, residues 1-53 of vitronectin containing the RGD sequence
(residues 42-44) were expressed as a chimera with the gelatin-binding
part of fibronectin. GD25 cells spreading on this chimeric protein
bound the 70-kDa fragment specifically (Fig. 5). Of the seven cell
adhesion proteins tested, therefore, all were adhesive for GD25 cells,
but only intact vitronectin did not support fibronectin matrix
assembly.

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Fig. 5.
Comparison of 70-kDa fragment binding to GD25
cells cultured on intact vitronectin, fibronectin, and recombinant
molecules containing the cell adhesion domains. Tissue culture
plates were coated with vitronectin (VN) or fibronectin
(FN), 2 µg/ml, recombinant fibronectin type III module
7-10 wild type (FN7-10), mutant fragments R1374A,
R1374,1379A, and R1379A, 10 µg/ml, or a chimeric protein containing
residues 1-53 of vitronectin (E53/GE2), 10 µg/ml. GD25
cells were seeded on different substrate in DMEM for 4 h. The
125I-labeled 70-kDa fragment was incubated with cells for
60 min. The cell-associated 70-kDa fragment in the absence of unlabeled
ligands (Total) and in the presence of excess unlabeled
70-kDa fragment (NSB) are presented as mean values ± SD (n = 3).
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Cell Morphology Correlation--
The LPA stimulation of
fibronectin binding and assembly by human fibroblasts correlates with
rapid changes in cytoskeleton and cell shape (37). We therefore
performed scanning electron microscopy of GD25 and GD25-
1A cells on
vitronectin or fibronectin after treatment with LPA. GD25 cells on
vitronectin were well spread (Fig.
6a). GD25 cells on fibronectin
(Fig. 6b) or GD25-
1A cells on either vitronectin (Fig.
6c) or fibronectin (Fig. 6d) were contracted and
displayed numerous hairy protrusions. Confocal microscopy demonstrated
that the GD25 cells on vitronectin were 4-5 µm high, whereas GD25
cells on fibronectin and GD25-
1A cells on fibronectin or vitronectin
were 9-11 µm high (images not shown). These results suggest that one
role of
1A integrin is to facilitate cytoskeleton organization and
cell shape change and that this signal is missing when
1A-deficient
cells are adherent to vitronectin even when LPA is present as a
costimulant.

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Fig. 6.
Cell morphology of freshly seeded GD25 and
GD25- 1A cells. GD25 (a and b) and
GD25- 1A (c and d) cells were seeded on
vitronectin (a and c) or fibronectin
(b and d) in DMEM for 4 h. Cells on
coverslips were incubated with 400 nM LPA for 60 min. The
coverslips were washed, processed for scanning electronic microscopy as
described under "Experimental Procedures," and photographed.
Bar, 10 µm.
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Studies of Mixed Substrata--
Integrin-generated signals may
depend on the concentration at which the adhesive proteins are coated
(49). Therefore, we studied the effect of coating concentration on the
effect of vitronectin on 70-kDa fragment binding. Vitronectin had a
negative effect only when its coating concentration was
2 µg/ml
(Fig. 7). Lower coating concentration of
vitronectin resulted in adherent cells able to bind the 70-kDa fragment
(Fig. 7). The other adhesive substrates tested also supported binding
of the 70-kDa fragment to GD25 cells after coating at concentrations as
low as 0.02 µg/ml (data not shown). When wells were coated with a
mixture of vitronectin and fibronectin, the 70-kDa fragment binding
ability of GD25 cells was dependent on the more abundant protein (Fig.
8). After coating with 2 µg/ml
vitronectin and increasing concentration of fibronectin, GD25 cells
bound more 70-kDa fragment in proportion to the concentration of
fibronectin in the initial coating solution. Conversely, increased concentration of vitronectin during co-coating with fibronectin, 2 µg/ml, diminished the 70-kDa fragment binding ability of GD25 cells.
The coating efficiency of iodinated fibronectin, 2 µg/ml, from the
mixed solutions was measured (data not shown), and only minor
competition (<40% decrement compared with control) for fibronectin absorption to plastic surface by vitronectin at the highest vitronectin concentration tested (10 µg/ml) could be demonstrated. When the mixed
substrate was preincubated with polyclonal antibodies against vitronectin, the down-regulation of 70-kDa binding of GD25 cells was
lost (Fig. 9). Nonimmune IgG did not show
this neutralizing effect (Fig. 9). Thus, intact vitronectin on the
surface demonstrates a dominant-negative effect on the binding of the
70-kDa fragment of fibronectin to GD25 cells when sufficient
fibronectin is present on the surface to support binding of the 70-kDa
fragment.

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Fig. 7.
Cellular phenotype as a function of
vitronectin concentration during coating. Tissue culture plates
were coated with increasing concentrations of vitronectin
(VN). GD25 cells were seeded on substrate in DMEM for 4 h, and the 125I-labeled 70-kDa fragment was incubated with
cells for 60 min. Specific binding was calculated by substraction of
nonspecific binding (in the presence of 500 µg/ml unlabeled
fibronectin) from total binding and expressed as ng of 70-kDa fragment
bound/mg of cellular protein. Data represent mean ± SD
(n = 3).
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Fig. 8.
Mixed vitronectin and fibronectin as
substrate. Tissue culture plates were coated with vitronectin, 2 µg/ml, and increasing concentrations of fibronectin (VN + FN) or vice versa (FN + VN).
GD25 cells were seeded on adhesive substrate in DMEM for 4 h, and
the 125I-labeled 70-kDa fragment was incubated with cells
for 60 min. Specific binding was calculated by substraction of
nonspecific binding (in the presence of 500 µg/ml unlabeled
fibronectin) from total binding and expressed as ng of 70-kDa fragment
bound/mg of cellular protein. Data represent mean ± SD
(n = 3).
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Fig. 9.
Antibody neutralization of the negative
effect of vitronectin in mixed substrate. Tissue culture plates
were coated with vitronectin (VN), fibronectin
(FN), or vitronectin plus fibronectin (VN + FN), 2 µg/ml for each protein, and then incubated with IgG
purified from antiserum against vitronectin (Anti-VN) or
nonimmune serum (Nonimmune), 200 µg/ml. GD25 cells were
seeded on substrate in DMEM for 4 h, and the
125I-labeled 70-kDa fragment was incubated with cells for
60 min. Specific binding was calculated by substraction of nonspecific
binding (in the presence of 500 µg/ml unlabeled fibronectin) from
total binding and expressed as ng of 70-kDa fragment bound/mg of
cellular protein. Data represent mean ± SD (n = 3). Error bars were omitted when the value was <0.2 ng/mg 70-kDa
fragment.
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Reproducibility of Results--
The presence of differential
responses of GD25 cells on vitronectin and fibronectin in assembly of
FITC-fibronectin and binding of the 70-kDa fragment were consistently
present in >10 different experiments. To exclude the possibility that
the results are unique for GD25 cells, an independent clone, GD10, was
derived from the
1A-null mouse embryonic stem cells. The
observations described above for GD25 cells held true for GD10 cells
(data not shown).
 |
DISCUSSION |
Dominant-negative Effect of Vitronectin--
Fibronectin or the
recombinant III7-III10 central adhesion domain as an adhesive
substrate supported the ability of the cell to bind the 70-kDa fragment
or fibronectin, even with GD25 cells lacking
1-integrin.
v
3-Integrin recognizes the RGD sequence in the III10 module of
fibronectin, whereas
5
1 and
IIb
3 require the presence of
the synergy sequence DRVPHSRN in III9 (47, 48, 50, 51). Both the RGD
and synergy sites are required for Xenopus gastrulation
(52). Recent studies suggest that the one role of the synergy site is
to activate
5
1 (9), such that
5
1 acquires more functions,
including support of fibronectin matrix assembly (9) and synergism with
the mitogen-activated protein kinase response to growth factor (53).
Our initial hypothesis, therefore, was that the effect of
substrate-bound fibronectin on
1-null cells was positive,
e.g. to cause the distinctive cell shape associated with the
ability of cells to assemble fibronectin (37), and that failure of GD25
cells to assemble fibronectin when adherent to vitronectin was
attributable to lack of the positive signal emanating from ligation of
fibronectin. However, when we mutagenized the DRVPHSRV sequence to
delete the guanidinium side chains critical for synergy site function,
the recombinant III7-III10 fragment retained its ability to support
fibronectin assembly. Instead, we found that the negative effect of
absorbed vitronectin was dominant, as demonstrated by loss of the
negative effect when surface adsorbed with both fibronectin and
vitronectin was treated with anti-vitronectin antibodies. The role of
fibronectin or the III7-III10 fragment is apparently simply to provide
the RGD sequence required for cell adhesion.
The negative determinant(s) of vitronectin are likely located outside
the N-terminal RGD-containing region, inasmuch as GD25 cells adherent
to a substratum coated with the chimera of the N-terminal sequence of
vitronectin and the gelatin-binding part of fibronectin were capable of
binding the 70-kDa fragment. Alternatively, the negative signal may
arise from a conformation adopted by the RGD sequence when intact
vitronectin but not the chimera is adsorbed to the substratum.
Vitronectin showed its suppression of 70-kDa fragment binding to
1-null GD25 cells only when its coating concentration was
2
µg/ml. It is not known whether the density of adsorbed vitronectin is
important for the effect or whether vitronectin adsorbed at low
concentration adopts a different conformation than vitronectin adsorbed
at higher concentration. The cell surface receptor for the negative
signal arising as a consequence of adherence to vitronectin is also
unknown but is most likely
v
3 or
v
5, which are expressed by
GD25 cells and are both vitronectin receptors (7).
Loss of the Dominant-negative Effect after Expression of
1A--
When
1A was transfected into GD25 cell, the cells
regained the ability to bind the 70-kDa fragment and assemble
fibronectin when adherent to vitronectin. Polymerization of fibronectin
is dependent on a coordinate series of events thought to involve the
actin cytoskeleton, cell surface integrin, and homophilic binding sites
within the fibronectin molecule (24, 37, 45, 46, 54-56). Enhanced
binding of the 70-kDa fragment of fibronectin induced by LPA treatment
or microtubule disruption correlates with changes in cell shape and
actin-containing cytoskeleton (37, 56, 57). Rho-dependent actin stress
fiber formation and cell contraction induce increase fibronectin
binding and represent a rapid, labile way that cells can modulate
fibronectin matrix assembly (56, 57). Cells competent for assembly of
fibronectin (GD25 on fibronectin and GD25-
1A on either vitronectin
or fibronectin) have a denser actin microfilament network compared with
the incompetent cells (GD25 on vitronectin; Ref. 10). Scanning electron
microscopy demonstrated that assembly competent cells also have many
more filopodia, which are associated with assembled fibronectin
(37).
The results with the D130A mutant indicate that
1A rescues the
matrix assembly capacity of GD25 cells on vitronectin by forming active
integrins that interact with ligands. GD25-
1A cells express three
1-containing integrins, namely
3
1,
5
1, and
6
1,
whereas
1,
2,
4,
9, and
v in complex with
1 have not
been detected (7). The most likely pair of integrin and ligand is
5
1 and cellular fibronectin. Cellular fibronectin was synthesized
in significant amounts (10-50 ng/2-4 h/4 × 105
cells) by both GD25 and GD25-
1A cells. Cycloheximide-treated human
foreskin fibroblasts fail to bind the 70-kDa fragment when cultured on
vitronectin but do so when cultured on fibronectin or an adhesive
fragment of fibronectin (25). The structural determinants in
fibronectin for interaction with
5
1 are more stringent than the
interaction of fibronectin with
v
3 (48, 58). CHO cells expressing
5
1 do not assemble fibronectin with a mutated synergy region in
III9 unless the integrin is activated by manganese (9). Such a result
supports a model in which the synergy region activates
5
1 to bind
fibronectin better, thus concentrating and conformationally altering
fibronectin molecules (9, 56). It has been suggested that
v
3 also
functions in assembly by direct interaction with assembling fibronectin
(7). One explanation for our results is that surface-adsorbed
vitronectin sequesters
v-integrins so that the integrins are
unavailable for fibronectin matrix assembly. Such a hypothesis does not
explain the differences in cell morphology. We therefore suggest an
alternative explanation, namely, that fibronectin assembly by cells on
vitronectin requires two events: 1) positive cellular signaling arising
from an adhesive interaction involving
5
1 that overcomes the
negative signaling arising from the adhesive interaction of
v-integrins with vitronectin, and 2) strong nanomolar avidity
binding of the N-terminal 70-kDa domain of fibronectin to the molecules
of large apparent molecular mass that can be identified by crosslinking techniques (41).
When GD25 cells are grown in serum-containing medium, a
fibronectin-containing network is present after reaching confluence although diminished when compared with the matrix of cells expressing
1A (7, 10). Confluent GD25 cells, however, bound the 70-kDa fragment
as well as cells expressing
1A. The major adhesive protein for cells
immediately after plating in 10% calf serum is vitronectin (59). GD25
cells, therefore, are probably defective in fibronectin matrix assembly
immediately after plating but "catch up" with
1A-expressing
cells as the cells are able to interact with a matrix deposited from
endogenous fibronectin and fibronectin present in the culture medium.
3 and
v colocalize with the fibronectin fibrils in confluent
culture of GD25 cells that are able to assemble fibronectin (7). The
signal arising from this interaction, which may involve recognition of
other components of the matrix as well as the RGD in fibronectin, is
enough to overcome the negative signal arising from the interaction
with vitronectin.
Endothelial cells and human foreskin fibroblasts bind more fibronectin
when cultured on fibronectin than when cultured on vitronectin (44,
60). In the GD25-
1A cells, 70-kDa fragment binding was consistantly
lower when the cells were cultured on vitronectin than on fibronectin.
We tested several fibroblastic cell lines and strains, including human
foreskin fibroblasts, and did not observe a reproducible decrement in
70-kDa fragment binding to those cells on substrates of vitronectin,
adsorbed either in its native form or after denaturation by urea (data not shown). The differences in our results with foreskin fibroblasts and those previously published (44) may be related to levels of
v-
or
1-integrins as well as of fibronectin produced by the cells under study.
Dominant-negative Effect of the D130A Mutation--
The
dominant-negative effect of the D130A mutation was not noted with any
of the cytoplasmic mutations of
1A that resulted in nonfunctional
integrins (10). The mechanism of the dominant-negative effect on
fibronectin matrix assembly of expression of the D130A mutant may be
similar to the mechanism by which a chimera of the extracellular domain
of the interleukin 2 receptor and an intact intracellular domain of
1A or
3 down-regulates fibronectin assembly (22) and inhibits the
activation of integrin (61). The mutant
1A subunit and the chimera
are molecules with extracellular domains unable to participate in
adhesive events but with intracellular domains potentially able to
interact with cytoplasmic effector molecules. Overexpression of CD98
has been found to rescue CHO cells expressing chimeras of interleukin 2 and cytoplasmic tails of either
1 or
3 and thus to allow
activation of integrins with wild-type
1 cytoplasmic domain (62). It
will be interesting to learn whether CD98 can also rescue the D130A cells.
Physiological and Pathophysiological Significance--
Vitronectin
is an adhesion-promoting glycoprotein that is involved in the
coagulation, fibrinolysis, and complement systems, activities that
point to a role in inflammation (59, 63). Vitronectin is present in
plasma and serum at concentrations of 200-400 µg/ml (64), and it is
also deposited in diseased tissues (59, 63). Vitronectin is synthesized
and secreted by tumor-associated fibroblast-like cells (65), suggesting
that vitronectin expression can be induced in disease states such as
cancer. The adhesion/migration-promoting properties of vitronectin, as
well the possibility that vitronectin induces collagenase expression in
the tumor stroma (66), suggest potentially important roles for
vitronectin in tumorgenesis. Down-regulation of fibronectin matrix
assembly by vitronectin and also possibly by other
v
3 ligands
could be an additional determinant of the migratory malignant phenotype
of tumor cells expressing
v
3.