(Received for publication, October 21, 1996, and in revised form, March 19, 1997)
From the Department of Stomatology, School of Dentistry, University of California, San Francisco, California 94143-0650
Fibronectin is an extracellular matrix molecule
composed of repeating subunits that create functional domains. These
domains contain multiple binding sites for heparin and for various
cell-surface receptors that modulate cell function. To examine the role
that the high affinity heparin-binding region and the alternatively spliced V region of fibronectin play in tumor invasion, we expressed and purified four complementary recombinant fibronectin proteins. These
proteins either included or excluded the alternatively spliced V region
and contained either a mutated, non-functional high affinity heparin-binding domain (Hep) or an unmutated
heparin-binding domain (Hep+). Cultured oral squamous cell
carcinoma cells were assayed for invasion into a Matrigel/collagen
matrix supplemented with these four purified recombinant proteins, and
for spreading and motility on plastic. Increased invasion was observed
in gels supplemented with the V
Hep+ protein
when compared with the V
Hep
protein.
Inclusion of the V region in the proteins enhanced the invasion and
migration associated with both Hep+ and Hep
proteins, whereas cell spreading was enhanced with the Hep+
recombinant proteins. These data demonstrate that both the high affinity heparin-binding domain and the V region of fibronectin play
important roles in invasion, motility, and spreading of oral squamous
cell carcinoma cells.
Squamous cell carcinomas are the most common type of malignant oral neoplasm and account for a major portion of deaths related to oral cancer. These tumors represent 4% of all cancers of males in the United States, but in some Asian countries they are the most common malignant tumor. At present the survival rate approximates 50% and has not improved significantly in patients treated over the past several decades (1). Much of the morbidity and mortality associated with these tumors is related to their invasive characteristics. Studies relating to mechanisms used by squamous cell carcinomas for adhesion, motility, and invasion are, therefore, an important aspect of cancer therapy.
Previous studies have implicated cell-surface receptors belonging to the integrin and the proteoglycan families in the invasion and metastasis of tumors (2-4). Furthermore, coordinate interactions among these receptors have been implicated in tumor cell adhesion to extracellular matrix (ECM)1 components (5, 6). Since some of these adhesive interactions can be blocked by small synthetic peptides derived from ECM molecules, peptide blocking experiments have also identified specific molecules that are potentially useful for therapy (7).
One such molecule which has been studied for its potential therapeutic
use is fibronectin (FN), an ECM adhesion molecule composed of multiple
functional domains that interact with multiple cell-surface receptors.
The domains are composed of repeating structural units, as evidenced by
sequence homology, and are designated as type I, type II, and type III
repeats. Repeats are numbered from the amino terminus of the molecule.
The functional regions include a cell-binding domain, which contains an
arginine-glycine-aspartic acid (RGD) adhesive sequence, and a
carboxyl-terminal heparin-binding domain (8, 9). The translated protein
also includes additional regions that arise through alternative
splicing. In rat FN these domains are designated EIIIB, EIIIA, and
the V region (see Fig. 1).
Many cell-surface receptors interact with specific portions of the FN
molecule. The RGD site in the 10th type III repeat (III-10) has been
shown to interact with both the 5
1 (10)
and
v
1 integrins (11). The
4
1 integrin binds to the V region as well as to sequences in the adjacent 14th type III repeat (III-14) (12). FN
also contains numerous heparin-binding sites within the
carboxyl-terminal portion of the molecule, including a high affinity
binding site in the 13th type III (III-13) repeat and multiple low
affinity binding sites within III-14 (5, 6). Many of these FN domains
retain biological activity when isolated as purified proteolytic or
recombinant protein fragments (13). Fragments from the RGD
cell-binding, heparin-binding, and alternatively spliced V regions of
FN have been used extensively to examine interactions between cells and
FN in general (14) and to better understand tumor cell adhesion,
motility, and invasion (5, 6, 15-20).
Although many domains of FN have been implicated in mediating tumor cell functions, the contributions of each domain and the relative importance of each family of receptors to these processes are difficult to assess. However, these evaluations are important since they may suggest potential therapeutic interventions by targeting the portions of the FN molecule that play the greatest role in the invasion process. These inquiries are often best made using recombinant proteins that exhibit altered function because of specific point mutations rather than deletions of large protein segments, which may alter protein function nonspecifically. In the present study, we used four different purified recombinant FN proteins, with or without function-perturbing point mutations in the high affinity heparin-binding region and with or without the alternatively spliced V region, to evaluate the relative contributions of these domains to the process of invasion by oral squamous cell carcinoma cells. Our results showed that the high affinity heparin-binding domain and the alternatively spliced V region of FN both contribute to the invasive behavior of these cells.
Growth factor reduced Matrigel basement membrane matrix was purchased from Collaborative Biochemical Products, Bedford, MA. Vitrogen 100-purified collagen was purchased from the Collagen Corp., Palo Alto, CA. Nutridoma-HU, a serum-free medium supplement, was purchased from Boehringer Mannheim. Transwell porous cell culture inserts, polycarbonate membranes in 24-well plates (6.5 mm diameter, 8.0 mm pore size), were purchased from Costar, Cambridge, MA.
Cells and Cell CultureHuman oral squamous cell carcinoma
cells (HSC-3 and HOC313) were a gift from Dr. Randall Kramer
(University of California, San Francisco) and have been described
previously (21). These cells were maintained in -minimum essential
medium containing 10% fetal bovine serum, 1% penicillin, and 1%
streptomycin.
Rat fibronectin cDNA
was engineered between type III repeats 10 and 15 (Fig. 1A)
to form four constructs, which were expressed as recombinant FN
proteins and subsequently purified. The proteins spanned repeats III-10
to III-15, and all contained the RGD cell-binding region in III-10, the
alternatively spliced EIIIA type III repeat, and the low affinity
heparin-binding sequences in the carboxyl-terminal heparin-binding
domain. However, the proteins either included (V+) or
excluded (V) the alternatively spliced V region and
differed in the high affinity heparin-binding region of III-13 (22). In
the latter region, two point mutations were introduced into the
cDNA to replace adjacent arginine residues with threonines (Fig.
1B). Since the threonine mutations have subsequently been
shown to virtually abrogate heparin-binding function, these recombinant
proteins have been designated as either Hep+ for the
unmutated protein or Hep
for the mutated protein. The
four proteins are therefore identified as
V
Hep+, V
Hep
,
V+Hep+, and
V+Hep
.
The cDNAs used to generate the four recombinant proteins were
engineered in two parts as follows. The cDNAs corresponding to
repeats III-13, -14, and -15 between the 5 ApaI and 3
HincII restriction sites in the FN cDNA were cloned into
the pECE expression plasmid (23). Two cDNAs that either included or
excluded the V region were used. The 3
HincII portion was
ligated into the blunted XbaI site of the pECE polylinker to
attach an engineered three-frame stop within the vector to the 3
end
of the FN cDNA fragment. The ApaI restriction site was
used to insert synthetic oligonucleotides into the 5
end of III-13.
These nucleotides introduced an XbaI restriction site into
the cDNA corresponding to the hinge region between III-13 and
III-12 in the FN protein (Fig. 1B, shown in bold
type). The new restriction site was engineered by altering a
single nucleotide in the FN cDNA at the third position of a codon.
This mutation preserved the protein sequence of the cDNA but
inserted a unique restriction site, the XbaI recognition sequence, into the gene. The FN sequence between XbaI and
ApaI could then be altered by inserting synthetic
oligonucleotides between these two restriction sites. As indicated in
Fig. 1B, one such pair of oligonucleotides was inserted
between these restriction sites to mutate adjacent arginine residues to
threonines. This mutation was subsequently shown to virtually abrogate
heparin-binding function in the expressed recombinant protein.
The region engineered between III-13 and III-15 in the pECE expression vector was removed from the plasmid by digesting with XbaI and EcoRI. EcoRI digests within the plasmid vector downstream from the three-frame stop and polyadenylation sequence. The cDNA fragment removed from pECE therefore contained repeats III-13 to III-15, an engineered translation termination site and a polyadenylation sequence. This cDNA fragment was bounded by restriction sites for XbaI and EcoRI. Thus, fragments containing the adjacent arginines as well as fragments with adjacent arginines mutated to threonines were isolated. Each of these fragments also contained cDNA in which the V region was either present or removed by alternative splicing.
The upstream portion of the FN cDNA between type III repeats 10 and
13 was engineered by adding restriction sites, using primers in a
polymerase chain reaction. These primers introduced the unique restriction site for XbaI at the 3 end (in the hinge region
between III-12 and III-13) and a second site for XhoI at the
5
end (in the cDNA corresponding to the hinge region between III-9
and III-10 in the FN protein). Sense primers (5
-AA TCT CGA GTT TCC GAT
GTC GGC TCT-3
) provided the recognition sequence (CTCGAG) for
XhoI. Antisense primers (5
-TT CTC TAG AGT CGT GAC GAC TCC
CTG AGC-3
) provided the recognition sequence (TCTAGA) for
XbaI. The product of the polymerase chain reaction was
therefore bounded by restriction sites for XhoI and
XbaI. Products were subcloned into TA subcloning vectors
(Invitrogen, San Diego, CA), and inserts were removed by digestion with
these two restriction enzymes.
Two cDNA fragments (III-10 to III-13 and III-13 to III-15) were
then ligated into the pTrcHis-C bacterial expression plasmid (Invitrogen) in a three-part ligation as follows. The cDNA inserts encoding repeats III-10 to III-13 were bounded by XbaI and
XhoI. The cDNA inserts encoding repeats III-13 to III-15
plus terminator were bounded by XbaI and EcoRI.
These two cDNA fragments were ligated into the pTrcHis-C expression
plasmid vector (in the appropriate reading frame) following digestion
with XhoI and EcoRI. The two resulting sets of
expression plasmids contained type III repeats 10-15, with the FN
sequences stopping and starting in the hinge regions of the FN molecule
adjacent to these repeats. The V region was either included or excluded
in each set, as was the Hep+ or Hep sequence.
Initiation and termination signals were provided by the pTrcHis-C
expression plasmid and the three-frame stop from the pECE plasmid.
All expressed proteins also contained the six-histidine (His6) purification sequence on the amino terminus, which was used for purification with metal-binding columns (Invitrogen). Proteins were eluted from the columns under denaturing conditions (pH 5.0) and were separated from bacterial proteins, as described by the manufacturer. After purification, elution buffer was removed by passage over G25 Sepharose columns.
All of the recombinant FN proteins contained the 10th type III repeat
(with the RGD cell-binding region), the alternatively spliced EIIIA
domain, and the 11th to 15th type III repeats, which include the low
affinity heparin-binding sequences in the carboxyl-terminal heparin-binding domain. These four proteins also either included or
excluded the V region, and they differed in III-13 such that it either
contained the adjacent arginines or the substituted threonines. The
predicted molecular masses of the four recombinant proteins are 69.3 kDa for the V proteins and 82.5 kDa for the
V+ proteins.
The four recombinant proteins were analyzed by standard sodium dodecyl
sulfate-polyacrylamide gel electrophoresis using 10% polyacrylamide
gels stained with 0.5% Coomassie Brilliant Blue R250 and destained in
a solution of 50% methanol and 5% glacial acetic acid. The proteins
were further positively identified by standard Western immunoblots
probed with a mouse monoclonal antibody to rat FN (24), used at a
dilution of 1:200. Primary antibody was then exposed to an alkaline
phosphatase-conjugated goat anti-mouse secondary antibody (Life
Technologies, Inc.) used at a dilution of 1:5000. Bound antibody was
detected using nitro blue tetrazolium chloride and
5-bromo-4-chloro-3-indolyl phosphate, toluidine (Promega, Madison, WI).
The concentration of the four recombinant proteins was determined by
comparison to a standard curve of sequential dilutions of bovine serum
albumin. Molecular mass standards (Life Technologies, Inc.) for
electrophoresis included the following prestained proteins with
apparent molecular mass as follows: myosin (214 kDa), phosphorylase
b (111 kDa), bovine serum albumin (74 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), -lactoglobulin (18 kDa), and
lysozyme (15 kDa).
To determine the presence or
absence of heparin-binding function, the V recombinant
proteins were analyzed for binding to heparin-Sepharose. Heparin-Sepharose columns were prepared by packing Poly-Prep
chromatography columns (Bio-Rad) with heparin-Sepharose CL-6B
(Pharmacia Biotech Inc.) according to the manufacturer's instructions.
The column was then equilibrated with 10 mM Tris, pH 8.0, and the sample was applied to the column and allowed to adhere to the
heparin-Sepharose matrix for 6 h at 4 °C. Then, the sample was
eluted from the column, using a continuous salt gradient of 0-0.5
M NaCl in 10 mM Tris, pH 8.0. Fractions of 3 ml
were collected for each of the assays and analyzed using standard
Western immunoblots as described above.
In vitro assays for invasion
were conducted as described previously (25). The gel solution was
prepared by mixing 300 µl of a Matrigel solution and 300 µl of a
collagen solution (Vitrogen, final concentration of collagen 0.8 mg/ml). The Matrigel solution was prepared by mixing 200 µl of
Matrigel with 100 µl of serum-free medium, which contained
-minimum essential medium supplemented with 0.66 × Nutridoma
solution, 0.036 mg/ml sodium pyruvate, and 3.3 mM Hepes, pH
7.4. The collagen solution contained 1.6 mg/ml collagen, 25 µl of a
10 × phosphate-buffered saline that was calcium- and
magnesium-free (PBS-CMF), 125 µl of a 1 × PBS-CMF solution, and
6.6 mM NaOH. The test proteins were subsequently added to this combined gel solution to a final concentration of 0.1 mM. The control gels contained the gel solution without the
test protein. The gel solution was coated onto polycarbonate filters
inside the Costar inserts in 24-well plates and allowed to set for
1 h at 37 °C before cells were added to the wells. The
transwell membranes were coated with 10 µl of the gel solution.
Following preparation and setting of the gels, human squamous cell
carcinoma cells were trypsinized, pelleted, rinsed twice with
serum-free medium, and counted. A 200-µl aliquot from a suspension of
1 × 106 cells/ml was added to each well. Cells were
allowed to invade the gel on the membrane filter for approximately
18 h. At the end of the incubation time, transwell filters were
washed three times with PBS, fixed in a 1:3 glacial acetic
acid:methanol solution for 30 min, washed three times with double
distilled water, and stained for 15 min with 0.05 mg/ml of Hoechst
stain (bis-benzimide 2-[hydroxyphenyl]-5-[4-methyl
1-1-piperazinyl]-2,5
-bis-1H-benzimidazole; Sigma). Filters were
washed another three times with double distilled water, placed on a
glass slide, covered, and sealed to a glass coverslip using mounting
medium (Vectashield, Vector, Burlingame, CA). The cells on the
underside of the membrane that invaded the gel and passed through the
pores of the membrane appeared bright against a dark background and
were visualized for counting with an Axiophot Photomicroscope equipped
with a filter for Hoechst stain detection (Zeiss, Oberkochen, Germany).
The number of cells from five high power fields (400 ×) from each
membrane was counted. Each experimental condition was tested in
triplicate chambers, with cell counts averaged between the triplicate
samples for each experiment.
HSC-3 cells were trypsinized,
pelleted under centrifugation, washed twice with PBS, and suspended in
control medium (serum-free -minimum essential medium supplemented
with 0.2% lactalbumin hydrolysate (Life Technologies, Inc.) and 1%
penicillin/streptomycin) at a density of 3 × 105
cells/ml. 100 µl of this cell suspension was aliquoted per well into
96-well plates and subsequently incubated at 37 °C in a humidified 5% CO2 incubator. The four recombinant FN proteins were
added to the wells immediately before plating cells, yielding a final protein concentration of 100 µM/well. Cells were
photographed after 2 h of incubation with the recombinant proteins
at 200 × magnification (26).
Cells were prepared as described for the cell adhesion assay, except that they were suspended at a density of 1 × 106 cells/ml; 100 µl of this cell suspension was aliquoted per well into a 96-well plate. After 2 h of incubation, a scratch was made down the center of the well with a sterile needle, removing cells from this area and creating a clear zone into which cells could migrate (27). Photographs of the cell-free scratched area were taken, and then the 96-well plates were incubated for another 7 h. After this time, photographs were again taken to determine if cells had migrated into the scratched area in the presence of the recombinant test proteins.
The diagrams in Fig. 1A depict the
III-10 to III-15 repeats of FN, which were expressed as fusion proteins
in bacteria. These complementary proteins either included
(V+) or excluded (V) the V region and
contained either the wild-type high affinity heparin-binding region
(Hep+) or the mutated sequence (Hep
). The
high affinity heparin-binding consensus sequence (22) was mutated in
III-13 to create the Hep
proteins (Fig. 1B).
Point mutations were incorporated into the FN cDNA such that in the
resultant proteins, adjacent arginines were replaced with threonines.
These mutations effectively abrogated heparin-binding function.
Electrophoresis of the four purified recombinant FN proteins produced
two bands at approximately 70 kDa, which correspond to the
VHep+ and V
Hep
proteins (Fig. 2A, lanes 1 and
2, respectively), and two bands at approximately 80 kDa,
which correspond to the V+Hep+ and
V+Hep
proteins (Fig. 2A,
lanes 3 and 4, respectively). Proteins were detected by staining with Coomassie Blue to demonstrate efficacy of
purification. The four recombinant proteins all reacted with a mouse
monoclonal antibody to rat FN (24) (Fig. 2B).
We examined the functional significance of the heparin-binding mutation
by determining the ability of the V proteins to bind to
columns of heparin-Sepharose. The recombinant proteins were applied to
the column and eluted in 3-ml fractions with continuous salt gradients
from 0 to 0.5 M NaCl. Recombinant protein was detected in
the 3-ml fractions by Western immunoblots. The mutated
V
Hep
protein bound to heparin-Sepharose but
eluted from the column in 0.07-0.08 M NaCl (Fig.
3A). In contrast, the native protein, V
Hep+ (Fig. 3B), required
approximately 0.5 M NaCl to elute. Since physiologic salt
concentration is 0.14 M NaCl, the mutated protein would not
bind heparin under physiologic conditions.
Having purified and functionally characterized the recombinant FN
proteins, we next examined the invasive properties of HSC-3 squamous
carcinoma cells in response to these FN proteins in a collagen/Matrigel
matrix. Initial assays performed with only the VHep+ and V
Hep
proteins demonstrated that both stimulated cell invasion, but that the
Hep+ protein was more effective than the Hep
protein in facilitating this process (Fig. 4,
B and C). There was at least a 3-fold increase in
invasion in gels supplemented with the Hep+ protein as
compared with gels supplemented with the Hep
protein
(Table I). Additional experiments were performed with all four recombinant proteins (Table II). Both
V+ proteins induced significantly elevated levels of
invasion when compared with gels containing either the
V
Hep
protein or Matrigel alone. The
V
Hep+ protein was also more effective than
the V
Hep
protein in facilitating invasion.
The V
Hep
protein was marginally better at
inducing invasion than was Matrigel alone.
|
|
These results were confirmed in assays with HOC313, a second oral
squamous cell carcinoma cell isolate. These cells invaded the gels with
the same order of efficacy as the HSC-3 cells (Fig. 5,
Table III). The relative magnitude of invasion induced
by the FN proteins was as follows: VHep
< V
Hep+ < V+Hep
< V+Hep+.
|
Since adhesion and migration are components of invasion, these
processes were evaluated separately for HSC-3 cells in the presence of
the recombinant FN proteins. Migration assays were conducted by
observing cell movement into a denuded "scratched" area in the
culture dishes. As was seen with the invasion assays, the V region
played the dominant role in this process (Fig. 6). At
7 h of migration, differences in migration between HSC-3 cells exposed to V+ and V recombinant proteins were
easily apparent, with substantially increased motility in cells exposed
to the V+ proteins.
The importance of the heparin binding function was more readily
demonstrated in assays measuring cell spreading. Cell spreading was
maximized in wells supplemented with the Hep+ FN proteins
but not the Hep proteins (Fig. 7). The
V+Hep
protein, which was highly effective in
inducing invasion, was not effective in mediating cell spreading. These
observations demonstrate that properties of the V region and the
heparin-binding domain are both important factors influencing the
behavior of the squamous cell carcinoma cells in culture, but that
these domains may modulate functions that are separable, depending on
the assay.
In this study, invading HSC-3 squamous cell carcinoma cells showed
a 3-fold increase in invasion in gels supplemented with the
V+Hep recombinant FN protein when compared
with gels containing the V
Hep
protein.
Inclusion of the V region of FN enhanced the invasion associated with
both Hep+ and Hep
proteins. Similar results
were obtained with HOC313, another oral squamous cell carcinoma
isolate. Assays for cell migration demonstrated that inclusion of the V
region substantially increased motility of HSC-3 cells. However, cell
spreading was most enhanced when HSC-3 cells were incubated with
recombinant FN proteins in which the high affinity heparin-binding
domain was functional. These data demonstrate that both the high
affinity heparin-binding domain and the V region of FN mediate invasion
by human oral squamous cell carcinoma cells. However, cell spreading is
more associated with the heparin-binding function.
The FN constructs used in this study contain all known low affinity
heparin-binding sequences within the carboxyl-terminal type III
repeats, yet these sequences did not impart significant functional
heparin binding or in vitro invasion. Inclusion of functional high affinity heparin-binding sites did, however, increase invasion. These experiments therefore reveal a structure-function correlation between high affinity heparin binding and important cellular responses. We demonstrated that the
VHep+ protein induced an increase in cell
invasion of HSC-3 cells when compared with the
V
Hep
protein. The invasion induced by the
V
Hep+ protein may be mediated in part through
heparan-sulfate and chondroitin-sulfate proteoglycan receptors (5, 6).
However, since the V region, which contains the principal binding site
for the
4
1 integrin, is not present,
these responses are probably not mediated by this integrin (28). Since
the constructs do contain the III-10 repeat and the RGD cell-binding
sequence of FN, any interactions with known members of the integrin
family probably involve the
5
1 integrin (13).
The V region of FN enhanced the invasive phenotype of both HSC-3 and HOC313 cells. In fact, its presence may predict a change toward a more invasive phenotype. The increase in invasion may be assisted by the high affinity heparin-binding region, but the V region appears to play the dominant role in this process. Other portions of the FN molecule such as low affinity heparin-binding sequences may also be important factors in the malignant process but were not examined in this study.
The high affinity heparin-binding sequence likely mediates interactions
with other FN domains and receptors over large spans of FN. This
sequence is separated from the binding sites for the 4
1 integrin in the V region by
approximately two type III repeats, and from the RGD binding site for
the
5
1 integrin by either two or three
type III repeats (depending on whether the alternatively spliced EIIIA
domain is excluded or included). Analysis of HSC-3 cells using
fluorescence-activated cell sorting has in fact shown that these cells
express
1,
4, and
5
integrin subunits.2 Interactions between
cell-surface receptors such as proteoglycans, which may bind the high
affinity heparin-binding domain, and either of these integrins must,
therefore, occur between separated portions of the FN molecule. In
contrast, virtually all other known heparin-binding sequences in the
type III repeats of FN are clustered in repeat III-14 and are
immediately adjacent to the leucine-aspartic acid-valine (LDV) binding
site for the
4
1 integrin in the
alternatively spliced V region (14). In fact, all three of these low
affinity heparin-binding sites and the LDV sequence are located within a stretch of 91 amino acids that encompasses the carboxyl-terminal portion of III-14, the amino portion of the V region, and the intervening hinge region between these repeats. This entire span is the
approximate size of one type III repeat. Thus, it seems likely that the
low affinity heparin-binding sites mediate cellular interactions
coordinately with the
4
1 integrin over a
short distance.
Other studies have investigated cellular responses to heparin-binding regions of FN by using either of two approaches. In one series of multiple studies (7, 26, 29, 30), synthetic peptides were constructed from short heparin-binding sequences, and cellular responses to these peptides were evaluated in vitro and in vivo. The results demonstrated that some of the peptides were highly effective in modulating functions such as migration and invasion in many different cell types. Most of these peptides were clustered in the 91-amino acid sequence within III-14 and the V region. Interestingly, the synthetic peptide corresponding to sequences within the high affinity heparin-binding domain was often ineffective in modulating cellular functions (7).
The second approach was to express and purify recombinant FN type III repeats (12, 15). The intervening sequences between the repeats of interest were often deleted in an attempt to eliminate amino acids believed to be nonessential to the biologic responses being measured. These studies have shown that repeats containing either the RGD cell-binding sequence or the high affinity heparin-binding sequence were marginally effective at promoting invasion of HT1080 fibrosarcoma cells (14). Mixtures of these two individual repeats were not appreciably better. The recombinant proteins were, however, highly effective in promoting invasion when repeats containing the RGD cell-binding site were linked in tandem with repeats containing the high affinity heparin-binding segment. Taken together, these two sets of approaches suggest that the high affinity heparin-binding domain needs to be presented to cells as a contiguous unit, in tandem with other functional domains, such as the RGD site. In addition, these data suggest that for maximal cell function, the high affinity heparin-binding region may indeed mediate interactions over large stretches of the FN molecule.
On the basis of these studies, the carboxyl-terminal heparin-binding
sites of FN can be classified into two groups. One group is the low
affinity heparin-binding sequences located on repeat III-14 immediately
adjacent to the V region, which contains the LDV binding site for the
4
1 integrin. Because of their proximity, the low affinity heparin-binding sequences and the V region may therefore be primarily responsible for modulating interactions between
heparin sulfate proteoglycans and the
4
1
integrin. Since these interactions are constrained to a small portion
of FN, any agent such as an antibody or a synthetic peptide would be
highly effective in altering functions associated with this complex and may interfere with binding of this integrin. Also, since the
heparin-binding synthetic peptides with blocking functions are
contained within the 91-amino acid segment that also contains the LDV
binding site, it is possible that the main effect of these peptides is
in blocking receptor complexes that are formed with the
4
1 integrin.
The second group is the high affinity heparin-binding site located on repeat III-13, which may require other, nonadjacent cell adhesion sequences to modulate cell function. Previous studies have indicated that small synthetic peptides corresponding to sequences in the high affinity heparin-binding domain are often ineffective in blocking biological activity in vivo (7). This region, in isolation, may also be ineffective in inhibiting tumor metastasis (15). However, our results would indicate that the high affinity heparin-binding domain plays an important role in tumor invasion. Other studies in which type III repeats containing cell-binding sequences were joined to and expressed with repeat III-13 have also indicated that these larger fragments are effective in inhibiting tumor metastasis (16). Interactions of other sequences with the high affinity heparin-binding domain might also be sensitive to differential splicing of either the EIIIA or the V region, since these splicing events would change the spatial relationship between the binding sites for integrins and heparin.
We thank Drs. Richard Hynes for cDNA to rat FN and anti-FN antibodies, Leland Ellis for the pECE expression plasmid, and Randall Kramer for providing the HSC-3 and HOC313 cells and for critical review of the manuscript. We also thank Evangeline Leash for editing this manuscript.