From the Division Biology and Genetics, University of Brescia, Brescia 25123, Italy
Received for publication, November 25, 2002, and in revised form, January 22, 2003
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ABSTRACT |
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Fibronectin (FN) is an extracellular
matrix (ECM) protein involved in tumor growth and metastasis. Five
human FN cDNA segments encoding for FN fragments, all starting with
the II1 repeat and ending with different C-terminal extensions, have
been stably expressed in chick embryo fibroblasts (CEF). These FN
cDNAs induce the formation of an organized ECM in CEF as long as
they retain a sequence coding for a 13-amino acid stretch (FN13), with
collagen binding activity, localized between type II2 and I7 repeats.
An FN13 synthetic peptide induces in control CEF the assembly of an
FN-ECM comparable with that observed in CEF-expressing FN fragments. The activity of FN13 is specific for its amino acid sequence, although
the cysteine present in the 6th position can be substituted with a
polar serine without affecting the induction of a fibrillar FN-ECM. A
less fibrillar matrix is induced by FN13-modified peptides in which the
cysteine is methylated or substituted by a non-polar alanine. FN13
induces the assembly of an FN-ECM also in Rous sarcoma virus-transformed CEF lacking the ECM and in hepatoma (SK-Hep1) and
fibrosarcoma (HT-1080) human cell lines. FN13 also promotes the
adhesion of CEF and Rous sarcoma virus-CEF at levels comparable with
those obtained with purified intact FN. Finally, FN13 inhibits the
migratory and invasive properties of tumorigenic cells, whereas intact
FN favors their migration. All FN13-modified peptides show similar
effects, although with reduced efficiency. None of these activities is
supported by a scrambled peptide. These data suggest a possible role of
FN13 in tumor growth and metastasis inhibition and its possible use as
anti-tumorigenic agent.
Fibronectin (FN)1 is an
adhesive heterodimeric glycoprotein present in the extracellular matrix
(ECM) of connective tissues in disulfide cross-linked
insoluble fibrils and in the blood in dimeric soluble form. FN contains
three types of homologous repeats (I-III), organized in functional
domains, connected by flexible, protease-sensitive segments, which
allow the binding of the molecule to ECM components (fibrin, heparin,
collagen, FN, and integrin receptors) (1-3). Through these multiple
interactions, FN provides a scaffold for ECM assembly and takes part in
different physiological and pathological processes (4-7).
One of the earliest observations concerning FN was that in
vitro transformed and tumor-derived cells often fail to deposit a
matrix, whereas the normal counterparts do have a matrix (8, 9).
Because addition of FN to tumor-derived cultured cells improves cell
adhesion and induces ECM and cytoskeleton organization, supporting the
normal cell morphology, FN has been associated with the
normal cell phenotypes (6, 10, 11). The inability of the transformed
cells to deposit the ECM has been related to the proteolytic
fragmentation of FN generated by the enhanced levels of proteases
released by tumors and transformed cells (12-14), as well as to the
down-regulation of the expression of integrin receptors binding to FN
and supporting ECM assembly (7). Along these lines are also
observations that an excessive ECM, containing collagens and FN,
i.e. desmoplasia, is often deposited in stroma surrounding
invasive carcinomas (15). It has been proposed that this stromal
response temporarily antagonizes tumor growth and invasion (14, 16).
Thus, the absence of an ECM of FN is associated with the transformed
phenotype, whereas the presence of the ECM restricts cell invasion and
migration in many tumor cells. The transition from assembly to
non-assembly of the ECM may therefore be an important stage in cancer progression.
The assembly of FN into fibrils in vitro and in
vivo requires multiple binding sites within FN including an
N-terminal region consisting of the first five type I repeats (17-21),
the RGD cell-binding site (22-23), the synergistic cell adhesive
regions (24-25), the I12 repeat involved in the
stabilization of FN fibrils (26), and a site located at or near the
junction of type I9 and type III1 repeats
(27-28). In addition, an FN-FN-binding site has been identified in a
14-kDa FNfg containing the first two type III repeats (29). A 76-amino
acid FN peptide, including this site, corresponding to a C-portion of
the type-III1 repeat, has been shown to convert FN into a
polymeric fibrillar form called superfibronectin (sFN) (29). sFN
resembles the matrix fibrils produced by cultured fibroblasts, is
highly adhesive, can inhibit cell spreading and migration in
vitro, prevents tumor formation in nude mice injected with human
tumorigenic cells, and has a strong antimetastatic activity (30).
Recently, the 76-amino acid peptide assembling FN in vitro
has been reported to suppress tumor growth, angiogenesis, and
metastasis in mice even in the absence of FN (31).
The study of proteolytic FNfgs has disclosed cryptic biological
activities not shared by the native protein (32-36). In particular, the FN collagen binding domain (COL-domain), in addition to binding with collagens and gelatin (3, 37-40), has been shown to possess a
number of other biological activities including the expression of
collagenase activity (35, 41-42), the promotion of odontoblast differentiation (43), and the substratum-dependent stimulation of fibroblast migration (44).
In this work we expressed five human recombinant FNfgs, spanning from
the II1 to the III2 repeat of the molecule and
encompassing the COL-domain in CEF, and we studied their ability to
induce the FN-ECM organization. All FNfgs but one, which lacked the
sequence coding for a 13-amino acid stretch with collagen binding
activity, induced the organization of the FN-ECM either in control or
in RSV-CEF. Therefore, we restricted the ECM organization capability of
FNfgs to this 13-amino acid sequence. We demonstrate that the synthetic
peptide corresponding to this sequence (FN13) induces the assembly of
FN in cultured cells, enhances cell adhesion, and inhibits Matrigel
matrix invasion of RSV-CEF and human tumor cell lines.
Antibodies and Synthetic Peptides--
In this work we used an
anti-human FN rabbit polyclonal antibody (Ab) (Sigma) and three anti-FN
mAbs: the f33 anti-human FN (hFN), recognizing an epitope
located in the C terminus of the 120-140-kDa catheptic FNfg located
downstream of the COL-domain (45); the f25 mAb, recognizing an
epitope located at the N terminus of the same fragment, either in human
or, improved with efficiency, in chick
FN2; and the f29
anti-hFN mAb, recognizing an epitope in the 14-kDa hFN catheptic
fragment flanking the COL-domain (46). Anti-chick FN and anti-chick
COLI Abs were kindly provided by A. Colombatti (Aviano, Italy).
Five 13-amino acid synthetic peptides (Primm, Milan) were used:
FN13 (AHEEICTTNEGVM), containing 13 amino acids of hFN COL-site and
differing from this binding site as regards the absence of an
N-terminal A (47); FN13Ser and FN13Ala, differing from FN13 regarding
substitution of the cysteine in the 6th position from the N terminus
with a serine, or an alanine, respectively; FN13Mod, identical to the
FN13 peptide sequence with a methylated cysteine and FN13 scrambled
sequence peptide (ScrFN13) (ITCETNEGEVAMH). Mass spectrometry was
performed by the manufacturer for all peptides, with a 95% purity
assessed by high pressure liquid chromatography.
Cloning System--
The cloning system consisted of two vectors:
the miniplasmid Cla12NCO and the retroviral vector RCASBP (48).
Cla12NCO is an adaptor plasmid for RCASBP in which it is possible to
clone DNA in a polylinker flanked by ClaI restriction sites.
After ClaI restriction, the cloned DNA can be inserted in
the unique RCASBP ClaI site. RCASBP contains the 5'- and
3'-long terminal repeats and the complete coding sequence for Gag, Pol,
and Env proteins. The expression of the DNA inserted in this site is
under the control of the long terminal repeat promoters and is
translated from the start site present in the ClaI
polylinker; after transfection and integration in CEF, RCASBP genes are
expressed at high levels and direct the production of non-transforming
retroviruses that infect all cells in culture.
FN cDNA Constructs--
The cDNA coding for the human FN
type II1-III2 repeats
(II1-III2 fragment, 1.47 kb) (Fig. 1) was
obtained by pFH134 plasmid HindIII and PvuII
restriction (49) and dimerized with T4 ligase. A palindromic
self-annealing SacI linker (5'GTGTGGAGCTCCACAC3') was
ligated to the PvuII blunt ends; the modified
II1-III2 cDNA was digested with
HindIII and SacI and inserted in the
SacI- and HindIII-linearized/Cla12NCO plasmid. A
95-bp cDNA fragment coding for the hFN secretion signal peptide was
obtained by PCR on pgHF3.7 plasmid (50) with the primers
5'dACATGCTTAGGGGTCCGG3' and 5'rCCTCTTGCTCTTCGAGGC3' and Vent
Taq DNA polymerase (New England Biolabs, Beverly, MA), and
inserted in-frame upstream of the II1-III2
cDNA in the filled-in Cla12NCO polylinker EcoRI site.
From this recombinant plasmid, 4 FN cDNA fragments, maintaining a
common 5' end and lacking progressive portions at the 3' end, were
generated by restriction digestion (Fig. 1) as follows: the
II1-I9 (0.9 kb), lacking type III1
and III2 cDNA; the II1-I8 (0.7 kb), lacking the I9 cDNA; the
II1-II2 (0.5 kb), lacking I8, and a
portion of I7 (114 bp from the 3' end) cDNA. The
II1-II2 cDNA contains 30 bp of
I7 5' end and maintains the COL-site sequence
(AAHEEICTTNEGVM) spanning from the type II2 to the adjacent
type I repeat (47). The last cDNA fragment, the
II1-II2del (461 bp), derives from the
II1-II2 construct without the 39 bp at the 3'
end encoding for the COL-site without the first alanine
(47).
Recombinant Cla12NCO plasmids were transformed in KH802
Escherichia coli strain (51); after amplification, they were
purified by Endo Free Plasmid Maxi and QIAprep Spin Miniprep kits from Qiagen (Diagen, GmbH, Germany); the FN cDNA inserts were excised by
ClaI digestion and ligated in the ClaI-linearized
RCASBP. After transformation in E. coli, the recombinant
retroviral vectors, carrying the FN cDNA inserts in the correct
orientation, were selected by restriction mapping and PCR analysis.
Five recombinant RCASBPs containing the different FN cDNAs were
generated (RCASBPFN constructs) with FN cDNAs inserted in-frame, as
ascertained by sequence analysis.
Cell Cultures--
Primary cultures of CEF were prepared as
described previously (52). Secondary CEF were grown in Hanks' MEM with
5% (v/v) newborn calf serum (Invitrogen), 10% (v/v) tryptose
phosphate broth, 100 µg/ml streptomycin, and 100 IU/ml ampicillin.
Normal human adult skin fibroblasts were prepared in our laboratory
from control donor skin biopsy. Human fibrosarcoma (HT-1080), hepatoma (SK-Hep1), cervix epithelioid carcinoma (HeLa), and rhabdomyosarcoma tumor-derived cell lines were from the ATCC. Human cells were grown in
Earle's MEM, 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a 5% CO2 incubator. For
SK-Hep1 cells the medium was supplemented with 1% (v/v) sodium
pyruvate and non-essential amino acids.
CEF Transfection and Infection--
Secondary cultures of CEF
were transfected using the calcium phosphate method (53) with empty
RCASBP expression vector or with RCASBPFN constructs using the
CellPhect transfection kit (Amersham Biosciences). In particular, 15 µg of retroviral vectors were co-precipitated with calcium phosphate
for 30 min and added to monolayers of CEF. After 4 h of incubation
at 37 °C, the media were removed. The cells were washed twice with
PBS, and complete medium was added. The cells were maintained at
37 °C in a 5% CO2 incubator.
Secondary CEF were seeded on round coverslips (1.5 cm inner diameter in
a 24-well plate (Corning Costar Corp., Cambridge, MA)) (40,000 cells/well) in Hanks' MEM supplemented as above and infected with 30 focus-forming units of PA1 SR-RSV, subgroup D mutant (52).
After 24 h of incubation at 35 °C, the medium was changed, and
after 24 h, the cultures were treated with purified FN and
FN13.
RT-PCR--
The expression of human FN cDNAs, transfected in
CEF in the RCASBP vector, was analyzed by RT-PCR on total RNAs and
purified from control and transfected CEF at the 4th in
vitro passage after transfection, with Trizol reagent
(Invitrogen). One µg of total RNA was reverse-transcribed by standard
procedures; PCR was carried out using the primers
5'dGCGGTAGCTGGACGTGC3', corresponding to 17 nucleotides of Cla12NCO
linker, and 5'rCCTCTTGCTCTTCGAGGC3', corresponding to 18 nucleotides of
FN secretion signal sequence, present in all constructs, using a
standard protocol. The product of the reaction was a 133-bp DNA
fragment in all transfectants integrating and expressing the RCASBP
constructs. The endogenous FN mRNA expressed by control and by
transfected CEF was evaluated by RT-PCR performed with a set of primers
amplifying a 419-bp segment of chick FN cDNA encoding for a
fragment that spans from the III8 to the III10
repeat absent in hFNfgs (54).
Quantitative in Situ Hybridization (QISH)--
The expression of
human FN cDNAs in CEF transfected with the different RCASBP
constructs was analyzed at different in vitro passages by
in situ hybridization with a 461-bp FN cDNA probe (i.e. the II1-II2del fragment)
labeled by nick translation (Invitrogen) in the presence of unlabeled
dCTP, dGTP, and dTTP (6 µM each) and of an excess of
[ FN-ECM Analysis by Immunofluorescence Microscopy
(IF)--
8 × 104 CEF not transfected and
transfected with the different RCASBPFN constructs were seeded on
22 × 22-mm glass coverslips and grown in Hanks' MEM, 10%
tryptose phosphate broth, and 5% newborn calf serum at 37 °C in a
5% CO2 incubator. After 48 h, the cells were washed
in PBS, methanol-fixed twice for 20 min at 4 °C, air-dried, and
immunoreacted with anti-chick FN and anti-chick-COLI Ab and with the
f25 mAb. Anti-FN and anti-COLI Ab, 1:100 in 0.3% bovine serum
albumin, 0.01% NaN3 (diluting buffer), and f25 mAb, 1:10 in diluting buffer, were reacted for 30 min at room temperature. After washing 3 times for 3 min in PBS, the cells were reacted with
rhodamine- or fluorescein-conjugated anti-rabbit or anti-mouse IgG
(1:100 in diluting buffer) for 30 min at room temperature, washed 3 times for 5 min in PBS, mounted on glass slides in 1:1 PBS/glycerol
solution, and photographed with a Leitz fluorescence microscope.
The assembly of the FN-ECM was also analyzed by IF in non-transfected
CEF cultured in the presence of purified hFN (New York Blood Center
Inc.) or of FN13, FN13Mod, FN13Ser, FN13Ala, and ScrFN13 synthetic
peptides. Increasing concentrations of peptides were added to the
medium (from 5 to 80 µg/ml) for 48 h, and the cells were
immunoreacted either with anti-hFN f25 mAb or with the
anti-chick COLI Ab.
The FN-ECM IF analysis was also performed on secondary CEF, before and
after RSV infection, and on human skin fibroblasts, hepatoma (SK-Hep1),
and fibrosarcoma (HT-1080) tumor-derived cell lines, grown in the
absence and in the presence of conditioned media recovered from
transfected CEF expressing the different FNfgs, or of 5-10 µg/ml
hFN, or of 40 µg/ml FN13. After 48 h treatment, the cells were
immunoreacted with f25 (CEF) and f33 (human cells) FN mAbs as
reported above.
Quantitative evaluation of the fluorescence (associated with the ECM
organized by control, transfected, and peptide-treated CEF and by
SK-Hep1 and HT-1080 tumor cells) was performed as follows. The
fluorescent images, with the same spatial resolution and comparable light intensity, were captured by a CCD black and white TV camera mounted on a Zeiss Axiovert 10S/H fluorescence microscope. By using the
Image-Pro Plus program (Media Cybernetics, Silver Spring, MD), a task
list file was developed that could be executed in semi-automatic mode
on different images. Each image was processed by applying Sharpen and
Rank nonlinear filters to optimize the image contrast and to remove the
background noise. A binary image, visualized as a red overlay,
corresponding to the fluorescence signal, was obtained by setting two
thresholds in the fluorescence peak corresponding to the gray tones of
the image visualized, with the lighter tones at the highest value of
the peak and the darker ones below the slice background. The sequence
of options in the task list was repeated on four images captured on
each slide; the area without cells was subtracted from the field area containing the fluorescence signal, and the integrated optical density
(IOD), related to the field area containing the signal, was measured
and normalized to the cell number present in each image.
Cell Adhesion Assay--
The effect of FN13 on CEF and CEF-RSV
adhesion was tested by seeding 4 × 104 cells in
24-well plates (Corning Costar, Italy) coated with 0.5-10 µg/ml FN13
or with 10 µg/ml hFN. The adhesion assay was also performed on human
control skin fibroblasts, HT-1080 and SK-Hep1 tumor cell lines in the
presence of 10 µg/ml hFN or FN13. Coating of hFN and FN13 diluted in
PBS was performed for 2 h at 37 °C followed by blocking with
0.5% bovine serum albumin for 1 h at room temperature. After
medium removal, the cells were allowed to adhere for 1 h at
37 °C in a humidified 5% CO2 incubator. Cell adhesion
was quantified by staining the cells with crystal violet in 20%
methanol, rinsing with water, and counting the cells with an optic
microscope. The reported values are means ± S.D. of three
independent measurements.
Cell Migration Assay--
The effect of FN13, FN13Mod, FN13Ser,
FN13Ala, and ScrFN13 peptides on cell migration was studied using the
Transwell 8-µm filter (Corning Costar, Cambridge, MA) migration
assay. 5 × 104 control and RSV-CEF, human control
skin fibroblasts, HT-1080 and SK-Hep1 cells, resuspended in serum-free
culture medium, were plated onto the upper chamber and allowed to
migrate for 6 h through the polycarbonate filter into the lower
chamber. The bottom wells were filled with culture medium supplemented
with or without 10 µg/ml hFN and the different FN13 peptides.
Migrating cells collected in the bottom chamber were counted, and their
number is reported as the average of three independent experiments.
Cell Invasion Assay--
Transwell 8-µm filters were coated
with 150 µg/filter Matrigel basement membrane matrix (BD Biosciences)
diluted in cold PBS. 2 × 105 human control
fibroblasts, HT-1080, and SK-Hep1 tumor cells, suspended in
Dulbecco's modified Eagle's medium containing 10% heat-decomplemented FBS supplemented or not with 40 µg/ml FN13, FN13Mod, FN13Ser, FN13Ala, ScrFN13 peptides or purified hFN, were added
to the upper Transwell chamber. Conditioned medium from human control
fibroblasts, grown in the absence of FBS, was placed in the lower
chamber. The assays were carried out at 37 °C in 5% CO2
atmosphere for 48 h; the non-invasive cells were removed with a
cotton swab, and the filters were fixed in methanol and stained
with 5% (v/v) Giemsa. All cells adhering to the filters were counted.
Each assay was performed in triplicate.
Construction and Expression of Recombinant FN
cDNAs--
Five cDNAs encoding for human FNfgs with a
common N terminus and different C termini (Fig.
1) were inserted in the retroviral vector
RCASBP and transfected in secondary CEF. The transfected cells
integrated and expressed the recombinant FN cDNAs, as shown by
RT-PCR performed on RNAs purified at the 4th in vitro cell passage (Fig. 2A). The level
of endogenous FN mRNA was similar in control and transfected CEF,
as shown by RT-PCR performed with a set of primers amplifying an FN
mRNA region between type III8 and III10
repeats absent in the hFN cDNAs (not shown) (54).
By using the RCASBP vector and the CEF as a cloning system, selection
of the transfectants was not necessary because the cells expressing the
retroviral RNA also produced recombinant viruses that infected the
non-transfected cells present in the cultures. In order to obtain the
different CEF populations homogeneously expressing the recombinant FN
cDNAs, the transfectants were analyzed at increasing in
vitro passages by QISH, using the
II1-II2del radiolabeled FN cDNA as a probe.
Starting from the 4th in vitro passage, QISH showed in all
transfectant populations the presence of a given percentage of CEF
expressing high levels of FN mRNA hybridizing with the
II1-II2del probe (about 400 pixels per cell) and a low hybridization signal (less than 50 pixels per cell) in
non-transfected CEF, corresponding to the basal endogenous FN mRNA
level detected in these cells with the human probe (Fig. 2B). The number of CEF expressing high levels of hFN
mRNAs increased following the in vitro passages and
reached 100% between the 8th and the 10th culture passage.
Extracellular Assembly of FN and COLI in Transfected
CEF--
Cultures of CEF, CEF transfected with the empty RCASBP and
with the different RCASBPFN constructs at the 10th in vitro
passage, were analyzed by IF using the f25 mAb (Fig.
3A) and the anti-COLI Ab (Fig.
3B). In control CEF, as well as in CEF transfected with RCASBP empty vector, f25 mAb detected extracellular FN not
organized in a fibrillar ECM (Fig. 3A, a); on the
contrary, in CEF transfected with the different cDNA fragments,
a well organized and fibrillar FN-ECM was detected (Fig. 3A,
b-e), the only exception being the FNII1-II2del (Fig. 3A,
f). FNII1-II2del-transfected cells
expressed and organized an FN-ECM only when they were maintained for 1 week in culture; at this time the matrix was still absent in control CEF (not shown). Quantitative evaluation by image analysis of the
fluorescent FN signals shown in Fig. 4
(a and b) shows that the highest level of the IF
signal was present in CEF transfected with the
FNII1-III3 cDNA, overlaid by an FN-ECM with
a 2.7-fold average increase in fluorescence compared with CEF
transfected with the empty RCASBP vector. The fluorescent signal was
slightly decreased (from 2.6- to 1.9-fold) in CEF transfected with the shorter FN cDNAs. On the contrary, a slight reduction (0.8-fold increase), compared with CEF transfected with the RCASBP vector, was
measured in cells expressing the FNII1-II2del.
An FN organization very similar to that reported above was obtained by
detecting FN with a polyclonal anti-chick FN Ab. Similar FN-ECM
structures were also obtained by adding to culture medium of control
CEF 10 µg/ml purified hFN or by growing control CEF for 2 days in the
presence of the conditioned media from the five transfectants (not
shown). These results indicate that the transfected CEF express FN
polypeptides capable of inducing the assembly of endogenous FN in an
organized ECM, as well as intact FN. The expression of the human FNfgs
could only be verified in cells transfected with the largest FN
cDNA containing an epitope recognized by the f29 anti-hFN
mAb (45), but not in the other transfectants expressing FNfgs, for
which antibodies have never been isolated, due to the evolutionary
conservation of this sequence (not shown).
Comparable results were also obtained following IF analysis of COLI
(Fig. 3B, g-n). Whereas control CEF mainly
showed COLI at a cytoplasmic level, transfected CEF showed the
organization of this protein into the ECM. In the
FNII1-II2del transfectant COLI was mainly
detectable as an intracellular signal. Quantitative evaluation of the
COLI organized by the transfectants, performed by image analysis on the
immunoreacted cells and shown in Fig. 4 (c and
d), shows that irrespective of the degree of COLI
organization, all transfectants expressed about a double amount of the
protein compared with control CEF.
Identification of an FN Peptide Inducing FN and COLI
Organization--
The FNfgs expressed by CEF, the only exception being
the FNII1-II2del fragment, contain the main
COL-site of the molecule (47), the AHEEICTTNEGVM peptide absent in the
FNII1-II2del fragment. This 13-amino acid
peptide (FN13) was synthesized, and its effect on the assembly of FN
was evaluated. Increasing concentrations of FN13 were added to the
culture medium of control CEF, and the organization of FN into the ECM
was analyzed by IF with the f25 anti-FN mAb. As shown in Fig.
5, FN13 starting from 5-10 µg/ml was
capable of inducing the assembly of a fibrillar FN-ECM. At increasing
peptide concentrations a thicker and closer FN-ECM network was
organized; in particular, at 40 µg/ml, FN13 induced in control CEF a
matrix comparable with that detected in transfected CEF (Fig. 3,
b-e). This peptide concentration was therefore used in the
following experiments.
Because FN13 contained a cysteine in the 6th position, to ascertain
whether its possible effect on FN assembly could be due to the
SH-reactive group, FN13 activity was analyzed in comparison with that
exhibited by the following: a modified peptide carrying the SH residue
methylated (FN13Mod); an FN13 peptide carrying a serine (FN13Ser) in
place of the cysteine; an FN13 with a hydrophobic alanine (FN13Ala)
instead of cysteine. Finally, the activity of these 4 peptides was
compared with that of an FN13 scrambled-sequence peptide (ScrFN13)
containing a cysteine in the 3rd position.
At a concentration of 40 µg/ml, FN13, FN13Ser, and
FN13Mod induced the organization in cultured CEF, analyzed by IF with
the f25 mAb, of a fibrillar network of FN overlaying the cells
(Fig. 6A, b-d),
which was absent in untreated cells. FN13Ala induced the formation of
an abundant ECM that was less fibrillar (Fig. 6A,
e) than that detected after treatment with the other FN13 peptides. The ScrFN13 peptide did not induce either enhancement or
organization of FN into the ECM (Fig. 6A,
f). Fig. 6B (a and b)
shows the quantitative evaluation, performed by image analysis of the
fluorescent FN signals, detected by IF with f25 mAb, in CEF in
basal conditions and after treatment with the different peptides. The
highest level of labeled FN was detected in CEF treated with
FN13, showing about a 6-fold increase in FN compared with the control
cells. The other peptides all induced a 4-fold increase in FN in CEF,
whereas ScrFN13 had no effect on the organization of FN. Similar
results were obtained when 10 µg/ml peptides were added to cultured
CEF, although the signals were lower than those shown in Fig.
6A (not shown). These results indicate that induction of the
FN-ECM in CEF is due to the FN13 sequence, although the presence of a
reactive group (SH- or OH-) in the 6th position is associated with
the formation of a more fibrillar FN-ECM.
IF analysis of COLI-ECM in CEF treated with the different peptides
showed that they were not capable of inducing a fibrillar matrix,
although they did induce the organization of COLI into a structure
comparable with that observed in the transfected CEF (Fig.
3B, i-m). The ScrFN13 peptide had no effect
on COLI organization (not shown).
FN13 Induces FN-ECM Assembly in RSV-CEF and in Human Tumor
Cells--
The effect of FN13 on FN-ECM organization was studied in
RSV-CEF and in human tumor-derived cell lines. Cultured CEF were massively infected with the mutant PA1 of the SR-RSV strain, and, after
the appearance of transformation foci they were grown in the
absence and in the presence of FN13 and analyzed by IF with the
f25 mAb. Fig.
7A, c, shows that
RSV-CEF are round-shaped and not overlaid by the ECM of FN before
treatment with FN13, whereas in the presence of the peptide a well
organized fibrillar ECM of FN overlaying the cells can be observed
(Fig. 7A, d).
A human hepatoma- (SK-Hep1) and a human fibrosarcoma-derived cell line
(HT-1080), unable to organize the FN-ECM (Fig. 7A, g and i) after treatment with FN13, deposited FN in the
extracellular environment. Whereas in SK-Hep1 cells FN was organized in
a compact extracellular structure (Fig. 6A, h),
differing from that observed in human control fibroblasts (Fig.
7A, e and f), in HT-1080 it was
deposited in thick extracellular aggregates and lacked a fibrillar appearance (Fig. 7A, l). Quantitative evaluation
of FN overlaying the cells, which was performed by image analysis,
showed an increase in fluorescence after FN13 cell treatment (Fig.
7B). The enhancement of FN assembled in the ECM was highest
in SK-Hep1 and HT-1080 tumor cells. Similar results were obtained when
treating normal and malignant cells with 10 µg/ml purified hFN. This
indicates that FN13 is able, to a varying extent, to promote the
formation of an FN network surrounding transformed and tumor-derived cells.
Effect of FN13 on Cell Adhesion--
In order to
ascertain whether the ECM induced by FN13 in control and RSV-CEF
influenced their adhesive properties, we performed an adhesion assay on
these cells with and without the peptide. Increasing amounts of FN13
were coated on plastic; hFN was coated as a positive control. Fig.
8A shows that the number of
adherent control and RSV-CEF increases with hFN and FN13 concentration, indicating that FN13 can support the adhesion of these cells as well as
purified FN.
Human control fibroblasts and tumor-derived cell lines were also tested
for their adhesive properties in the absence and in the presence of 10 µg/ml hFN and FN13. As shown in Fig. 8B, control fibroblasts adhere to the substrate more efficiently than HT-1080 and
SK-Hep1 cells. In the presence of hFN and FN13, the adhesiveness of
control fibroblasts was slightly enhanced (1.3- and 1.5-fold, respectively). The adhesiveness of SK-Hep1 and HT-1080 tumor cells increased several times (10.4- and 8.4-fold, respectively) in the
presence of hFN, and 13- and 16-fold, respectively, in the presence of
FN13. Therefore, FN13 effectively favors the adhesion of human tumor
cells not organizing an FN-ECM in basal conditions. The effect of FN13
on the adhesion of these cells is comparable with that produced by
purified intact hFN.
Effect of FN13 on Cell Migration and Invasion--
We studied the
effect of FN13 on the migration of control CEF, RSV-CEF, human control
fibroblasts, HT-1080, and SK-Hep1 tumor-derived cells in Transwell
chambers. Fig. 9 shows that, in the
absence of any treatment, control CEF and human control fibroblasts
migrated less efficiently than RSV-CEF and tumor cells.
FN13 and hFN stimulated the migration of CEF and human control
fibroblasts about 6- and 2-3-fold, respectively. On the contrary, the
ScrFN13 peptide did not have any effect on the migration of either type
of control cell. FN13 had the opposite effect on the migration of
RSV-CEF and human tumor cells compared with control cell strains.
Indeed, the peptide inhibited the migration of RSV-CEF, SK-Hep1, and
HT-1080 cells 2-3-fold (Fig. 9). The ScrFN13 peptide did not show any effect on the migration of these cells, whereas hFN slightly enhanced their migration. In order to elucidate the different effects on cell
migration produced by FN13 and hFN, FN13Ser, supporting fibrillar FN-ECM assembly, and FN13Mod and FN13Ala, inducing the aggregation of
FN in a non-fibrillar meshwork, were also tested. These peptides induced the migration of control cells, although to a lower extent than FN13 and hFN, and inhibited the migration of transformed and tumor
cells.
Human control fibroblasts and tumor cells were also tested for their
ability to invade a Matrigel basement membrane matrix. In this system,
FN13, all FN13-derived peptides, and hFN, but not ScrFN13, slightly
enhanced the invasive properties of control cells by 1.5-2.6-fold
(Fig. 10). FN13 reduced by 4- and
5-fold the invasive capability of SK-Hep1 and HT-1080 tumor cell lines, respectively; the inhibitory activity of FN13Mod, FN13Ser, and FN13Ala
was lower than that of FN13 both on SK-Hep1 (by 1.4-, 3.9-, and
1.8-fold, respectively) and on HT-1080 (by 1.3-, 4.7-, and 1.7-fold,
respectively) (Fig. 10). ScrFN13 had no effect on the tumor cell
invasion, whereas hFN slightly enhanced the invasiveness of these cells
by 1.3- and 1.4-fold, respectively. Therefore, FN13 but not hFN, shows
an anti-invasive effect on in vitro transformed avian cells
and on human tumor-derived cells, whereas it favors the invasion of
control avian and human cells. FN13 also showed an anti-invasive effect
in HeLa (9.0-fold reduction) and in rhabdomyosarcoma (2.0-fold
reduction) tumor cell lines (not shown). All FN13-modified peptides,
but not ScrFN13, inhibited the invasive properties of transformed avian
cells and human tumor-derived cells. In particular, FN13Ser, carrying a
reactive -OH on the 6th amino acid, inhibited cell invasion to the
highest extent, suggesting that the anti-migratory activity of FN13
peptide is associated not only with its sequence but also with a
reactive group on the amino acid in the 6th position. Taken altogether,
these data indicate that FN13 and FN13-related peptides have an
anti-invasive potential on tumor and transformed cells which is not
associated with the intact FN molecule.
The adhesive ECM protein FN, in synergy with its integrin
receptors, plays an important role in several stages of embryo and tumor development. Embryonic and tumor cells are less adhesive than the
normal adult counterpart and deposit less ECM. A diminished adhesion to
the ECM may contribute to the migratory properties of embryo and tumor
cells in the surrounding tissues (7).
FN is a multidomain protein containing binding sites for the cell
(i.e. integrin receptors), for other ECM components, and for
FN itself; through these interactions FN participates in many physiological and pathological processes (3, 7). The proteolysis of
purified FN with several enzymes, as well as the construction and
expression of recombinant FNfgs, has provided evidence that FNfgs can
perform activities that are not present in the intact molecule
(32-36).
In this work we investigated the involvement of FN COL-domain (37-39)
and its adjacent regions in ECM formation by using a set of FN cDNA
deletion segments that were stably expressed in CEF after cloning in a
retroviral vector. All FN cDNAs started with the sequence encoding
the first type II repeat of the molecule and encompassed downstream
regions of different lengths. The data reported here show that
recombinant hFN cDNA, retaining the sequence coding for the
13-amino acid stretch, falling in the boundary between the end of type
II2 and the beginning of type I7 repeat, and
corresponding almost completely to the major COL-site of FN (39, 47),
was capable of inducing in CEF the assembly of an organized ECM of FN
and of COLI. Only the largest FNfg, spanning to the end of type
III2 repeat, retains the 76-amino acid C-terminal sequence
acting as an FN-FN binding region (29). However, the other three FN
cDNAs also lacking this sequence induced the assembly of the
FN-ECM. The absence in hFN cDNA of the sequence, coding for the
last 13 amino acids, was associated with a strong reduction of the
FN-ECM, indicating that this sequence probably plays an important role
in the organization of the FN-ECM. The hFN cDNAs expressed in CEF
also to different extents supported the organization of COLI into
the ECM. This indicates that COLI is assembled in the ECM of CEF even
in the absence of the major COL-site, in agreement with previous
reports showing that the two type II FN repeats, upstream of the
COL-site, share a 50% homology with the three type III repeats of type
IV collagenase, which notoriously binds to COLs (56). Furthermore, it
is noticeable that a gelatin binding activity has also been disclosed
in a 21-kDa FNfg containing the I8-I9 repeats
(39). Therefore, FN also contains multiple COL-sites, three of which
are clustered between the type II1 and the type I9 repeats. This might explain the differential deposition
of COLs into the ECM observed in the transfected CEF expressing FNfgs containing one or more COL-sites.
The 13-amino acid peptide FN13, which corresponds almost exactly to the
main FN COL-site, was synthesized and analyzed to determine its effect
on the assembly of FN and of COLI in cultured cells. FN13 is able to
direct the organization in cultured CEF of an FN-ECM comparable with
that observed in transfected CEF. FN13 also induces in CEF the
formation of a COLI-ECM in structures lacking a clear-cut fibrillar
organization. The linking activity of FN13 is partly due to the
presence of a central cysteine possibly involved in disulfide bonds
with other cysteines distributed in FN type I and II repeats (3) and on
pro- The 13-amino acid FN13 sequence is fully conserved in six different
species from Homo sapiens to zebrafish (ExPASy BLAST2 Interface), indicating that this amino acid stretch must be maintained intact in order to allow the accomplishment of very important and
common functions in all Vertebrata. This sequence is very ancient since
it has also been found, entirely or for internal portions, with a
47-96% homology in all living organisms. The conservation of this
sequence, mainly found in FN molecules, also explains the lack of
immunogenicity of the COL-domain of FN (3).
The induction of the FN-ECM by recombinant FNfgs and FN13 has also been
studied in RSV-CEF and in human tumor-derived cell lines. Transformed
and tumor cells do not assemble the FN-ECM (4, 6, 8, 9, 23) and, due
either to FN degradation by tumor proteases or to integrin receptors
modulation (12-14, 16, 58), this phenotypic trait has been associated
with tumor growth, invasion, and metastasis (30, 31). We observed that all conditioned media from transfected CEF, with the only exception of
that expressing the II1-II2del FNfg, were able,
with comparable efficiency, to induce the organization of FN-ECM
even over the foci of RSV-transformed cells, which are
normally deprived of FN-ECM. A similar FN-ECM network overlaying the
transformation foci was observed in RSV-CEF grown in the
presence of FN13 even with a low serum concentration. Following
these treatments, the morphology of transformed CEF shifted from
round-shaped to fibroblast-like, indicating that the FN-ECM induced in
these cells temporarily restored a "normal" cell phenotype. SK-Hep1
and HT-1080 human tumor cell lines, which do not organize FN, assembled
an FN-ECM in the presence of FN13. These results show that FN13 is
sufficient to induce FN-ECM formation in ECM-transformed and tumor
cells. Through its ability to induce an organized FN-ECM, the FN13
peptide also influenced the adhesive properties of control and
transformed CEF. The stimulation of adhesiveness was much higher for
transformed RSV-CEF, compared with control CEF. Indeed, FN13 treatment
enhanced the adhesion of RSV-CEF to a level comparable with that
obtained after hFN treatment. These data show that FN13 induces an
FN-ECM supporting cell adhesion in a manner comparable with that
retained by a hFN-induced matrix and suggest a similar organization for these structures in the different cell types.
FN13 also acted on the migration of control, transformed, and tumor
cells in a Transwell chamber. In this assay, FN13 enhanced the
migration of control avian and human fibroblasts as well as hFN. On the
contrary, FN13 inhibited the migration of RSV-CEF and of tumor cells,
whereas hFN slightly stimulated the migration of these tumorigenic cells.
The anti-migratory property of FN13 was also confirmed in a Matrigel
matrix invasion assay. In this case too, hFN and FN13 both favored the
migration of control fibroblasts, whereas FN13 strongly reduced the
migration of SK-Hep1 and HT-1080 cells, unlike hFN, which enhanced the
Matrigel invasion of these tumor cells as well. Comparable effects on
the migratory and invasive properties of control, transformed, and
tumor cells were obtained with FN13-related peptides, but not with
ScrFN13, indicating that the FN13 sequence plays a crucial role in
these biological functions, although, to different extents, the residue
present on the 6th amino acid affects the efficiency of cell invasion
and migration. Because HeLa (cervix epithelioid carcinoma) and
rhabdomyosarcoma tumor cell lines, following FN13 treatment, also
showed a drastic reduction in their migratory activity in the Matrigel
matrix, FN13 seems capable of inhibiting the invasion both of ectoderm-
and of mesenchyme-derived tumor cells.
Similar results were obtained when treating tumor cells of different
embryonic origin with sFN (29) and with the III1 FNfg, which is also defined anastellin and organizes FN in sFN
(31). Either sFN or anastellin blocked tumor cell spreading
and migration in vitro through the organization of FN.
Although FN13 organizes FN, as well as COLI in cell cultures, its
effect on the adhesion and migration of control, transformed, and tumor
cells is comparable with that of anastellin. The different
effect of purified hFN and of FN13 or anastellin on tumor
cell migration can be explained by assuming that the matrix organized
by these short FN peptides has a different structure, if compared
with the physiological FN-ECM. Because anastellin and sFN
have been reported to exert in mice strong tumor formation-prevention,
together with antimetastatic and anti-angiogenetic activity (30, 31),
we can hypothesize that FN13, which induces FN-ECM organization and
shows an anti-migratory effect on tumor but not on normal cells and low
immunogenic activity, will have similar biological activities in
vivo. Treatment of mice with FN13 is in progress in order to
evaluate its toxicity, its turn-over after intravenous and
intraperitoneal injection, and its role in tumor growth and metastasis
in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-33P]dATP (2000 Ci/mmol) (PerkinElmer Life Sciences),
according to the supplier's instructions. In situ
hybridization was performed as reported previously (55) on CEF cultured
on microscope slides, and the hybridization grains were quantitatively
evaluated with the Magiscan Image Analysis System (Joyce Loebl,
Gateshead, UK).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
FNfgs coded by the cloned hFN cDNAs
segments aligned with a portion of the FN monomer showing its
structural organization. All FNfgs start at amino acid 316 and end
at the amino acid indicated below each fragment.
View larger version (22K):
[in a new window]
Fig. 2.
FN recombinant cDNAs are expressed by
transfected CEF. The expression of FN mRNA fragments was
detected by RT-PCR using primers specifically recognizing these inserts
(A) and by QISH (B). A, DNA markers
(lane 1); control CEF (lane
2); CEF transfected with RCASBP empty vector
(lane 3), with
RCASBP-FNII1-III2 (lane
4), with RCASBP-FNII1-I9
(lane 5), with
RCASBP-FNII1-I8 (lane 6),
with RCASBP-FNII1-II2 (lane
7), and with RCASBP-FNII1-II2del
(lane 8) recombinant vectors. B,
percentage of transfected cells expressing the different hFN mRNA
fragments at increasing in vitro passages as
detected by QISH performed with the II1-II2del
radiolabeled fragment.
View larger version (59K):
[in a new window]
Fig. 3.
Transfected CEF expressing hFNfgs
organize the FN-ECM. IF on RCASBP (a and g)
and on RCASBPFNs transfected CEF (all other panels) using
the f25 anti-FN mAb (A) and the anti-COLI Ab
(B). The transfectants, at the 10th in vitro
passage, contain and express the II1-III2
(b and h), the II1-I9
(c and i), the II1-I8
(d and l), the II1-II2
(e and m), and the
II1-II2del (f and n)
cDNAs. Scale bar, 5-10 µm.
View larger version (21K):
[in a new window]
Fig. 4.
Quantitative image analysis of the FN-ECM
(a and b) and COLI-ECM (c
and d) organized by CEF transfected with hFN
cDNAs. The quantitative evaluations were performed in
triplicate and are reported as IOD; each evaluated field contained on
average 23 ± 1 cells. FI, fold increase.
View larger version (120K):
[in a new window]
Fig. 5.
Effect of increasing concentrations of FN13
on the FN-ECM assembled by control CEF, as detected by IF
with the f25 anti-hFN mAb. a, untreated CEF;
b-f, CEF treated with 5, 10, 20, 40, and 80 µg/ml FN13.
Scale bar, 8 µm.
View larger version (49K):
[in a new window]
Fig. 6.
Effect of FN13 and of FN13-derived peptides
on the organization of FN of cultured CEF. A, IF was
performed with the f25 mAb on control CEF (a) and on
CEF treated with 40 µg/ml FN13 (b), FN13Ser
(c), FN13Mod (d), FN13Ala (e), and
ScrFN13 (f). Scale bar, 8 µm.
B, quantitative image analysis of the FN ECMs (a)
are reported as IOD. The quantitative evaluation of the FN-ECM was
performed in triplicate; each evaluated field contained on average
23 ± 1 cells (b). FI, fold increase.
View larger version (26K):
[in a new window]
Fig. 7.
FN13 organizes the FN-ECM in avian and human
normal and malignant cells. A, IF of FN organized by
control (a and b) and RSV-CEF (c and
d), by human control fibroblasts (e and
f), by SK-Hep1 (g and h), and
by HT-1080 (i and l) malignant cells, before
(a, c, e, g, and i) and
after treatment with 40 µg/ml FN13 (b, d,
f, h, and l). The avian cells were
immunoreacted with the f25 mAb, whereas the human cells were
reacted with f33 mAb. Scale bar, 8 µm.
B, quantitative evaluation of the FN-ECM performed by image
analysis is indicated as IOD. The number of cells for each evaluated
field is as follows: 23 ± 1 CEF, 33 ± 6 ± RSV-CEF,
14 ± 1 SK-Hep1, and 27 ± 2 HT-1080. FI, fold
increase.
View larger version (15K):
[in a new window]
Fig. 8.
FN13 and hFN favor the cell adhesion of CEF,
RSV-CEF (A), human control fibroblasts, HT-1080, and
SK-Hep1 tumor cells (B). A, increasing
concentrations of FN13 and hFN (from 0.5 to 10 µg/ml) were coated on
the plates before cell adhesion. B, 10 µg/ml FN13 or hFN
was used for coating.
View larger version (29K):
[in a new window]
Fig. 9.
FN13 inhibits the migration of malignant
cells. Effect of FN13 and of hFN on the migration in a Transwell
chamber of control and RSV-CEF, of human control fibroblasts, and of
SK-Hep1 and HT-1080 tumor-derived cell lines. The values reported in
a are the average ± S.D. of the measurements performed
in three independent experiments. b, the enhancement of cell
migration is indicated as stimulation factor (SF) and its
inhibition as inhibition factor (IF), compared with the
untreated cells corresponding to 1.
View larger version (23K):
[in a new window]
Fig. 10.
FN13 inhibits the invasion of malignant
cells. Invasion assay on Matrigel matrix performed with human
control fibroblasts, HT-1080 and SK-Hep1 tumor-derived cells, in the
absence and in the presence of FN13 peptides and of hFN. The values
reported in a are the average ± S.D. of the
measurements performed in three independent experiments. b,
the enhancement of cell invasion is indicated as stimulation factor
(SF) and its inhibition as inhibition factor
(IF), compared with the untreated cells corresponding to
1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(I) and pro-
2(I) collagen chains (57). When the cysteine is
methylated (FN13Mod) or substituted with a non-polar amino acid such as
alanine (FN13Ala), FN and COLI are still organized by CEF in compact
and less fibrillar aggregates than those induced by FN13. Fibrillar FN
organization into the ECM is also induced by another peptide carrying a
reactive -OH group on the 6th amino acid (FN13Ser). These findings
indicate that a reactive amino acid in the 6th position of FN13 is
important for the formation of a fibrillar FN- and COLI-ECM and that
the presence of a cysteine, which might be involved in the formation of
disulfide bonds, is not required for the assembly of a fibrillar FN-
and COLI-ECM. The sequence of FN13, therefore, is sufficient to induce
the aggregation of an organized ECM, whereas a reactive amino acid in
the 6th position favors the organization of fibrillar structures. The
role of FN13 sequence in FN assembly is emphasized by the observation
that the ScrFN13 peptide, which contains a cysteine in the 3rd
position, does not induce FN and COLI organization into the
ECM.
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ACKNOWLEDGEMENTS |
---|
We thank S. H. Hughes, who provided the cloning system vectors, and B. Arici and A. Ghinelli for their expert technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by Ministero dell'Istruzione, dell'Università e della Ricerca, Centro di Eccellenza Innovazione Diagnostica e Terapeutica, and by CNR as part of the Progetto Strategico Oncologia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Division of Biology
and Genetics, Dept. of Biomedical Sciences and Biotechnology, Medical
Faculty, University of Brescia, Viale Europa 11, 25123 Brescia, Italy.
Tel.: 39-030-3717240; Fax: 39-030-3701157; E-mail: barlati@med.unibs.it.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M211997200
2 S. Barlati and A. Vaheri, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: FN, fibronectin; Ab, polyclonal antibody; CEF, chick embryo fibroblasts; COL-domain, collagen-binding domain; COLI, type I collagen; COL-site, collagen-binding site; ECM, extracellular matrix; FNfgs, fibronectin fragments; FN13, 13-amino acid collagen-binding fibronectin peptide; hFN, human fibronectin; IF, immunofluorescence microscopy; QISH, quantitative in situ hybridization; RCASBPFN, retroviral fibronectin constructs; RSV, Rous sarcoma virus; sFN, superfibronectin; mAb, monoclonal antibody; RT, reverse transcriptase; FBS, fetal bovine serum; MEM, minimum Eagle's medium; PBS, phosphate-buffered saline; IOD, integrated optical density; sFN, superfibronectin.
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