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INTRODUCTION |
Fibronectins (FNs)1 are
extracellular matrix glycoproteins that play a role in cell adhesion
and migration during embryonic development, wound healing, and tumor
progression (1). Proteolytic digestion of FN has revealed that FN has
several cell binding domains: the central cell binding domain (CCBD),
the COOH-terminal heparin binding domain (Hep2), and the CS1 region
within the type III connecting segment (IIICS) (2). CCBD is the major
cell-adhesive domain of FN and contains the Arg-Gly-Asp (RGD) motif
that is recognized by members of the integrin family of cell adhesion receptors including
5
1,
v
1,
v
3,
v
5,
v
6,
IIb
3, and
8
1 (3-6). There is evidence that FN acts not
only as a cell-adhesive substrate but also transduces biochemical
signals across the plasma membrane via integrin receptors, thereby
regulating cell proliferation, differentiation, and apoptosis (7-9).
Interaction of FN with integrins stimulates tyrosine phosphorylation of
several cellular proteins including FAK (10-12) and
p130Cas (13-15) and also stimulates activation of Src
family protein tyrosine kinases (7) and the extracellular
signal-related kinases ERK1 and ERK2 (16-19). Furthermore, FN induces
expression of cyclin D1 and subsequent inactivation of pRb by
hyperphosphorylation and thus mediates cell cycle progression through
the G1 phase (20).
FN contains three alternatively spliced segments: EDA, EDB, and IIICS
(21-24). The EDA and EDB segments are each encoded by a single exon,
and both comprise an intact type III repeat (22). FNs expressed in
fetal and tumor tissues contain a greater percentage of EDA and EDB
segments than those expressed in normal adult tissues (25-29).
Increased expression of FNs containing the EDA and/or EDB segments has
also been observed during wound healing (30). Because FNs containing
the EDA and/or EDB segments are highly expressed in tissues that are
populated with cells showing high proliferative and migratory
potential, it seems likely that FNs containing these extra segments
play an important role in promoting cell proliferation and migration.
Despite accumulated evidence for regulated expression of EDA- and/or
EDB-containing FNs in vivo, the biological functions of
these isoforms are poorly understood (31).
Recently, we found that FN isoforms containing the EDA segment
(EDA+ FN) were more potent than those lacking the EDA
segment (EDA
FN) in promoting cell adhesion and
migration, irrespective of the presence or absence of the EDB segment.
The increased cell adhesion and migration were caused by increased
binding of EDA+ FN to integrin
5
1 (32). In the
present study, we investigated whether alternatively spliced EDA and
EDB segments regulate the potential of FNs to promote cell
proliferation. We found that insertion of the EDA, but not EDB, segment
potentiated the ability of FN to promote cell cycle progression. The
enhanced cell cycle progression was associated with increased tyrosine
phosphorylation of p130Cas, activation of ERK2, increased
expression of cyclin D1, and hyperphosphorylation of pRb. These results
indicated that alternative splicing in the EDA region regulates
FN-mediated extracellular signals and subsequent cell cycle progression
via modulation of binding affinity to integrin
5
1.
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EXPERIMENTAL PROCEDURES |
Purification of Recombinant FN Isoforms--
Construction of the
cDNA expression vectors for three different full-length FN
isoforms, EDA
/EDB
FN,
EDA+/EDB
FN, and
EDA+/EDB+ FN, was described previously (32). To
construct the expression vector for EDA
/EDB+
FN, the whole inserts of pHCF93B+ encoding
Val527-Arg1449 including the EDB segment (32)
were excised by double digestion with SalI/BamHI
and ligated to SalI/BamHI-cleaved pAIFNC, which encodes a full-length FN isoform lacking both EDA and EDB segments. The
resulting cDNA expression vector for
EDA
/EDB+ FN was designated pAIFNBC.
Recombinant FNs were expressed in CHO DG44 cells and purified from the
culture supernatants of the stable transfectants by gelatin-affinity
chromatography as described previously (32).
Cell Culture--
CHO-K1 and CHO DG44 cells were obtained from
the Japanese Cancer Research Resources Bank (Tokyo, Japan) and Dr.
Lawrence Chasin (Columbia University), respectively. CHO-K1 cells were
grown in Ham's F-12 medium supplemented with 10% FBS. CHO DG44 cells
deficient in dihydrofolate reductase activity were maintained in
-minimal essential medium containing
ribonucleosides/deoxyribonucleosides and 10% FBS. Stable transfectants
of CHO DG44 cells overexpressing recombinant FNs were maintained in
-minimal essential medium without
ribonucleosides/deoxyribonucleosides in the presence of 10% FBS (for
routine passage) or 1% FN-depleted FBS (for large scale culture to
purify recombinant proteins). All cells were maintained under a
humidified 5% CO2 atmosphere at 37 °C.
Antibodies--
Polyclonal antibodies against cyclin D1 (R-124),
p130Cas (N-17), and ERK2 (C-14), and horseradish
peroxidase-conjugated anti-phosphotyrosine mAb (PY20) were obtained
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The mAb against
pRb (G3-245) was obtained from Pharmingen (San Diego). Anti-FAK mAb 2A7
was obtained from Upstate Biotechnology (Lake Placid, NY). Horseradish
peroxidase-conjugated secondary antibodies against mouse or rabbit IgG
were from Cappel Worthington Biochemicals (Malvern, PA).
Cell Spreading Assay--
Cell spreading assays were performed
using 96-well microtiter plates coated with 5 µg/ml recombinant FNs
and blocked with 1% bovine serum albumin. Amounts of recombinant FNs
immobilized on plates were determined by enzyme-linked immunosorbent
assay using anti-human FN antiserum or anti-FN mAbs as described
previously (32). CHO-K1 cells grown in Ham's F-12 medium containing
10% FBS were trypsin treated and washed with serum-free medium. The cells were plated at a density of 3 × 104 cells/well
in serum-free medium and incubated at 37 °C for 40 min. Nonadherent
cells were removed by washing with serum-free medium, and attached
cells were fixed with 3.7% formaldehyde and then stained with Giemsa.
Cells adopting a well spread morphology (i.e. cells that had
become flattened with the long axis more than twice the diameter of the
nucleus) were counted/mm2.
Integrin-liposome Binding Assay--
Integrins
5
1 and
v
3 were purified and reconstituted into liposomes as described
previously (32). Integrin-liposomes in TBS(+) (25 mM
Tris-HCl, pH 7.5, 0.13 M NaCl, 1 mM
CaCl2, and 1 mM MgCl2) containing
0.2% bovine serum albumin were added to the wells of microtiter plates
precoated with recombinant FNs (20 µg/ml) and incubated for 6 h
at room temperature. The wells were washed with TBS(+), and bound
liposomes were recovered in 1 N NaOH. The radioactivity of
bound liposomes was quantified using an Aloka LSC-3500 scintillation
counter (Aloka Co., Ltd., Tokyo).
Induction of G1-S Phase Transition in Synchronized
Cells--
CHO-K1 cells were grown to confluence and synchronized at
G0 by serum starvation for 48 h in Ham's F-12
supplemented with 0.2% FN-depleted FBS, 50 µg/ml streptomycin, and
50 units/ml penicillin (starvation medium) according to Orren et
al. (33). The cells were dissociated with phosphate-buffered
saline containing 0.2% trypsin and 2 mM EDTA, and trypsin
treatment was stopped by adding soybean trypsin inhibitor (1 mg/ml).
The cells were washed with serum-free medium, incubated in suspension
in starvation medium for 90 min at 37 °C, and seeded on plates that
had been precoated with 5 µg/ml recombinant FNs or 20 µg/ml
poly-L-lysine and then blocked with 1% bovine serum
albumin. The amounts of immobilized recombinant FNs on the plates were
determined by enzyme-linked immunosorbent assay using anti-human FN
antiserum to confirm the equality of adsorbed FNs.
Determination of S Phase Entry--
Quiescent cells were kept in
suspension for 90 min and then plated at a density of 2 × 104 cells/well in starvation medium.
[3H]Thymidine (5 µCi/ml) was added 12-24 h after cell
plating. The cells were washed three times with serum-free medium at
the indicated time points, fixed with ice-cold 10% trichloroacetic
acid for 1 h, and washed once with 10% trichloroacetic acid. The
acid-insoluble precipitates were dissolved with 0.1% SDS and 0.2 N NaOH, neutralized with 0.2 N HCl, and
transferred to vials containing scintillation fluid. Radioactivities in
the precipitates were determined with an Aloka LSC-3500 scintillation
counter. To determine the percentage of cells entering S phase,
bromodeoxyuridine was added to the cultures 12-24 h after plating, and
the cells were incubated with anti-bromodeoxyuridine mAb followed by
visualization of bound mAb with 3,3'-diaminobenzidine
tetrahydrochloride according to the manufacturer's instructions
(Amersham Pharmacia Biotech).
Immunoprecipitation and Immunoblotting--
Quiescent cells
cultured on the substratum were extracted in lysis buffer (1% Nonidet
P-40, 20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 40 mM
-glycerophosphate, 2 mM orthovanadate,
0.5 mM sodium fluoride, 20 µg/ml leupeptin, 5 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride). The cell
lysates were clarified by centrifugation at 14,000 × g
for 10 min at 4 °C, and protein concentration was determined using a
DC protein assay kit (Bio-Rad). The whole lysates were boiled in
SDS-sample buffer (8% SDS, 100 mM dithiothreitol, 266 mM Tris, pH 6.8, 40 mM EDTA), and equal amounts
of protein were subjected to SDS-polyacrylamide gel electrophoresis
with 8, 12, or 12.5% gels for detection of tyrosine-phosphorylated
proteins and pRb (8%), cyclin D1 (12%), and ERK2 (12.5%). Proteins
were detected immunologically after electrotransfer onto nitrocellulose membranes as described (32).
For immunoprecipitation of FAK, cell lysates were incubated with
protein A-conjugated anti-FAK mAb 2A7 for 5 h. For
immunoprecipitation of p130Cas, cell lysates were incubated
with anti-p130Cas antibody for 1 h followed by
incubation with protein G-Sepharose (Amersham Pharmacia Biotech) for
4 h. The immune complexes were precipitated by brief
centrifugation and washed four times with lysis buffer. The
immunoprecipitates were separated on 8% polyacrylamide gels to
determine the levels of tyrosine phosphorylation and the amounts of
precipitated proteins as described above.
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RESULTS |
EDA but Not EDB Regulates Cell Adhesive Functions of FN--
We
showed previously that the presence or absence of the EDB segment did
not affect the ability of EDA+ FN isoforms to promote cell
adhesion and spreading (32), although we could not exclude the
possibility that inclusion of the EDB segment alone might potentiate
the cell-adhesive activity of FN. To explore this possibility, we
produced a recombinant FN isoform containing the EDB but not the EDA
segment (designated rFN(BC); see Fig. 1)
and compared its cell attachment and spreading activities with those of
rFN(C), rFN(AC) and rFN(BAC). CHO-K1 cells were used in these assays
because they have long been used as a standard model in cell cycle
analysis and are more easily synchronized at the G0 state
than HT1080 cells, which were used in the previous study (32).
Significant differences were observed in both cell attachment and
spreading among the four different FN isoforms, EDA+ FNs
(i.e. rFN(AC) and rFN(BAC)) being more potent in promoting cell attachment than EDA
FNs (i.e. rFN(C) and
rFN(BC)) (Fig. 2A).
EDA+ FNs were also more than twice as effective as
EDA
FNs in inducing cell spreading (Fig. 2B).
No significant differences were observed in cell spreading activity
between rFN(AC) and rFN(BAC) or between rFN(C) and rFN(BC) (Fig.
2B), indicating that the EDB segment is not involved in
regulation of the cell-adhesive activity of FN. Because CHO-K1 cells
adhere to FN-coated substrates predominantly via integrin
5
1 (34)
and the increased cell-adhesive activity of EDA+ FNs is the
result of enhanced binding of CCBD to integrin
5
1 (32), the
integrin binding activity of EDB+ FN was compared with that
of EDB
FN. There were no significant differences in
integrin
5
1 binding between rFN(C) and rFN(BC), although rFN(AC)
was more than twice as active as rFN(C) and rFN(BC) in integrin
5
1 binding (Fig. 3). These results
support the previous conclusion that the cell-adhesive functions of FN
are specifically regulated by insertion of the EDA but not the EDB
segment.

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Fig. 1.
Structure of recombinant FNs. Modular
structures of recombinant FNs are shown schematically on the basis of
internally homologous modules termed types I, II, and III. The EDA and
EDB segments are shown by filled rectangles, and the IIICS
segment is shown by a hatched oval. All recombinant FNs
contain the complete IIICS sequence of 120 amino acids. Functional
domains that interact with heparin (Hep1, Hep2), fibrin (Fib1, Fib2),
bacteria, collagen, and integrin 5 1 are indicated above the
figures.
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Fig. 2.
Attachment and spreading of CHO cells on
recombinant FN-coated substrata. Panel A, CHO cells
(3 × 104) were seeded on 96-well microtiter plates
coated with 5 µg/ml of different FN isoforms and incubated for 40 min
at 37 °C. The cells were rinsed, fixed, and stained with Giemsa.
Bar, 100 µm. Panel B, spreading of
CHO cells was quantified as described under "Experimental
Procedures." The S.D. of multiple determinations (n = 3) are indicated at the top of the bars.
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Fig. 3.
Binding of integrin
5 1 to recombinant
FNs. Integrin 5 1 was purified from human placenta and
reconstituted in phosphatidylcholine liposomes as described under
"Experimental Procedures." The integrin 5 1-liposomes were
added to microtiter plates precoated with 20 µg/ml of different FN
isoforms and incubated for 6 h at room temperature. Quantities of
bound integrin 5 1-liposomes are expressed as percentages of the
total input radioactivity after subtraction of the radioactivity bound
to plates coated only with bovine serum albumin. Each bar
represents the mean S.D. (n = 6).
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EDA+ FN Is More Potent than EDA
FN in
Promoting Cell Cycle Progression--
The in vivo
expression patterns of EDA+ FN suggested a potential role
of EDA+ FN in cell proliferation (31). To investigate
whether insertion of the EDA and/or EDB segment affects the ability of
FN to promote cell proliferation, CHO-K1 cells were synchronized at the
G0 phase by serum starvation, incubated in suspension for
90 min, and replated on substrates precoated with different FN isoforms
to stimulate the cells to reenter the cell cycle. To confirm that the
effects of FN isoforms on cell proliferation were primarily mediated
via integrins, the cells were also plated on the substrate coated with
poly-L-lysine, which mediates integrin-independent cell
adhesion. All recombinant FNs significantly promoted entry into S phase when compared with poly-L-lysine (Fig.
4). The levels of DNA synthesis were
different among the FN isoforms; rFN(AC) and rFN(BAC) were more potent
than rFN(C) and rFN(BC) in inducing DNA synthesis. No significant
differences were observed between rFN(AC) and rFN(BAC) or between
rFN(C) and rFN(BC). Labeling of cells with bromodeoxyuridine showed
that more than 70% of the plated cells entered the S phase by 24 h after replating onto the FN-coated substrata (data not shown). These
results indicated that EDA+ FN has a greater potential than
EDA
FN to induce cell proliferation irrespective of the
presence or absence of the EDB segment.

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Fig. 4.
Induction of DNA synthesis upon adhesion of
cells onto recombinant FN-coated substrata. Quiescent CHO cells
were replated onto wells precoated with 5 µg/ml rFN(C), rFN(BC),
rFN(AC), rFN(BAC), or 20 µg/ml poly-L-lysine
(PLL) in starvation medium. [3H]Thymidine was
added to the wells 12 h after replating, and the cells were
cultured for the indicated periods. S phase entry was quantified by
measuring [3H]thymidine incorporation into DNA as
described under "Experimental Procedures." Each bar
represents the mean S.D. (n = 6).
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EDA Segment Up-regulates Cell Cycle-associated Signal
Transduction--
It has been shown that pRb plays a key role in the
G1-S phase transition. Inactivation of pRb by
hyperphosphorylation occurs at the restriction point in G1
as the cell enters the S phase (35). We investigated whether the
presence or absence of the EDA segment could modulate FN-induced
phosphorylation of pRb. Quiescent cells were trypsin treated, held in
suspension to reset anchorage-dependent signals, and
replated onto dishes coated with rFN(C), rFN(AC) or
poly-L-lysine. Hypo- and hyperphosphorylated forms of pRb
were distinguished by the difference in their mobility on
SDS-polyacrylamide gel electrophoresis. Hyperphosphorylation of pRb was
induced on the substrates coated with rFN(C) and rFN(AC) compared with
those with poly-L-lysine (Fig.
5, upper panel). The
level of pRb hyperphosphorylation was higher with cells plated on
rFN(AC) than on rFN(C) through 16-24 h after plating, indicating that
the increased cell cycle progression driven by EDA+ FN was
associated with the increased hyperphosphorylation of pRb. Although pRb
hyperphosphorylation was detectable even on poly-L-lysine upon prolonged incubation, this could be the
result of deposition of endogenous FN by CHO cells.

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Fig. 5.
pRb phosphorylation and cyclin D1
accumulation in response to cell adhesion onto recombinant FN-coated
substrata. Quiescent CHO cells were seeded onto substrates coated
with 5 µg/ml rFN(C) or rFN(AC) or 20 µg/ml
poly-L-lysine (PLL), or they were kept in
suspension in starvation medium for varying periods as indicated. Cells
were then solubilized in Nonidet P-40 lysis buffer, and equal amounts
of whole cell lysates were subjected to SDS-polyacrylamide gel
electrophoresis. Protein levels of pRb (upper panel) and
cyclin D1 (lower panel) were detected by Western blotting
with antibodies specific for pRb or cyclin D1 after electrotransfer
onto nitrocellulose membranes. The positions of hyper- and
hypophosphorylated pRbs are indicated as ppRb and
pRb, respectively. The reproducibility of the results was
confirmed in three separate experiments.
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pRb has been shown to be phosphorylated by cyclin D1·cdk4/6
complexes, with concomitant accumulation of cyclin D1 during the G1 phase (35). Therefore, we examined whether the level of
expression of cyclin D1 was also modulated by the EDA segment upon cell
adhesion onto FN-coated substrates. When cells were plated onto
substrates coated with recombinant FNs with or without the EDA segment,
the expression of cyclin D1 was significantly induced by 24 h
after replating, although no obvious induction of cyclin D1 expression was observed on the poly-L-lysine-coated substrate (Fig. 5,
lower panel). The expression of cyclin D1 was more
pronounced with cells plated onto rFN(AC) than onto rFN(C) with a time
course profile similar to that of pRb hyperphosphorylation. These
results indicated that expression of cyclin D1 and phosphorylation of
pRb were coordinately regulated by the EDA segment upon cell adhesion
to FN-coated substrates.
EDA Segment Enhances FN-dependent MAP Kinase
Activation--
Because activation of MAP kinase has been shown to
induce expression of cyclin D1 and subsequent hyperphosphorylation of
pRb (36), we investigated whether ERK2 MAP kinase was stimulated differently by EDA+ and EDA
FNs. When
quiescent cells were detached and either held in suspension or replated
on dishes coated with rFN(C), rFN(AC), or poly-L-lysine, the activated form of ERK2 was detectable as early as 10 min after replating and persisted for over 60 min (Fig.
6). In contrast, no significant
activation was observed in cells kept in suspension or replated on the
substratum coated with poly-L-lysine. Activation of ERK2
was more prominent on surfaces coated with rFN(AC) than on those coated
with rFN(C), indicating that the EDA segment potentiates the ability of
FN to activate MAP kinases via integrin-dependent signaling
and thereby promotes cell cycle progression.

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Fig. 6.
MAP kinase activation induced by recombinant
FNs. Quiescent cells were either held in suspension or plated on
substrates coated with 5 µg/ml rFN(C) or rFN(AC) or 20 µg/ml
poly-L-lysine (PLL) in starvation medium for
various times. Cells were lysed, and MAP kinase activity was analyzed
by a band shift assay. The phosphorylated, activated forms of ERK2
(pp42) display more reduced electrophoretic mobility than
the unphosphorylated, nonactivated forms of ERK2 (p42). The
reproducibility of the results was confirmed in three separate
experiments.
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EDA Increases FN-induced Tyrosine Phosphorylation of
p130Cas--
Protein tyrosine phosphorylation triggered by
integrin-mediated cell-matrix interaction has been shown to be caused
by activation of protein tyrosine kinases including the Src family
kinases, FAK and FAK-related kinases (7). These kinases phosphorylate tyrosine residues of their specific substrate proteins, providing binding sites for SH2-containing molecules that are involved in cell
proliferation (7). We examined whether the EDA segment modulates the
ability of FNs to stimulate tyrosine phosphorylation of cellular
proteins. In parallel with the analysis of FN-dependent activation of ERK2, quiescent cells were kept in suspension or replated
on dishes precoated with rFN(C), rFN(AC), or poly-L-lysine and incubated for different periods. As shown in Fig.
7A, proteins migrating at
positions corresponding to 150, 130, and 80-60 kDa were tyrosine
phosphorylated irrespective of the type of substrate. Among these,
tyrosine phosphorylation of the 130-kDa protein was significantly
induced in cells plated on substrates coated with recombinant FNs but
not in cells either plated on poly-L-lysine-coated substrates or kept in suspension. The kinetics of tyrosine
phosphorylation of the 130-kDa protein were similar to those of ERK2
activation (Fig. 6). The 130-kDa protein was tyrosine-phosphorylated at
10 min after replating on FN-coated surfaces, and the levels of
tyrosine phosphorylation persisted for more than 60 min. The 130-kDa
protein was more prominently tyrosine-phosphorylated in cells on
rFN(AC) than those plated on rFN(C).

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Fig. 7.
Tyrosine phosphorylation of
p130Cas and FAK upon cell adhesion onto recombinant
FNs. Panel A, quiescent cells were either held in
suspension or plated on substrates coated with 5 µg/ml rFN(C) or
rFN(AC) or 20 µg/ml poly-L-lysine (PLL) in
starvation medium for various times. Whole cell lysates were prepared,
and tyrosine phosphorylation of cellular proteins was analyzed by
immunoblotting with anti-phosphotyrosine antibody. The positions of
molecular weight marker proteins are indicated in the left
margin. Panel B, whole cell lysates prepared 30 min after
replating were immunoprecipitated with anti-p130Cas or
anti-FAK antibody. The immunoprecipitates were subjected to
immunoblotting with antibodies against phosphotyrosine
(upper), FAK (middle), and p130Cas
(bottom). The reproducibility of the results was confirmed
in two separate experiments.
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Cell adhesion to FN-coated substrates has been shown to induce tyrosine
phosphorylation of p125FAK (10-12) and p130Cas
(13-15). Immunoprecipitation with anti-FAK and
anti-p130Cas antibodies showed that p130Cas was
phosphorylated in CHO-K1 cells plated onto FN-coated surfaces, although
no significant tyrosine phosphorylation was observed with FAK in the
same cells (Fig. 7B, upper panel). These results indicated
that the tyrosine-phosphorylated 130-kDa protein was p130Cas but not FAK. Tyrosine phosphorylation of
p130Cas was more pronounced on the substrates coated with
rFN(AC) than on those coated with rFN(C). The enhanced phosphorylation
of the 130-kDa protein was not the result of an increase in
p130Cas expression because the amount of immunoprecipitated
p130Cas was decreased rather slightly on rFN(AC) than that
on rFN(C) (Fig. 7B, bottom panel). Although
we could not exclude the possibility that FAK was
tyrosine-phosphorylated at levels below the limits of detection, it is
likely that p130Cas is the main target of protein
tyrosine kinases activated by integrin-mediated adhesion onto FN-coated
substrates and therefore is involved in activation of ERK2 and
subsequent cell cycle progression.
 |
DISCUSSION |
Regulation of FN-mediated Signal Transduction by the EDA
Segment--
It has been shown that the EDA segment is included in FN
species expressed in embryonic tissues, but is spliced out of the molecule as embryonic development proceeds (28, 31). In adults, EDA+ FN reappears at sites of tissue injury and
inflammation (30, 38). Despite the close association of the expression
of EDA+ FN with tissues populated with cells showing high
proliferative potential, the physiological significance of this
phenomenon is not known.
In this study, we found that EDA+ FN was more potent than
EDA
FN in inducing G1-S phase transition. The
increased ability of EDA+ FN to induce G1-S
phase transition was closely associated with enhanced ERK2 activation,
cyclin D1 expression, and pRb phosphorylation. Given that
EDA+ FN is more potent than EDA
FN in
promoting cell adhesion through enhanced binding to integrin
5
1
(32) and that ligand ligation of integrin
5
1 promotes DNA
synthesis through activation of intercellular signal transduction pathways involving ERK2, cyclin D1, and pRb (1, 20), it is conceivable
that inclusion of the EDA segment up-regulates FN-dependent mitogenic signal transduction through increased binding of FN to
integrin
5
1. Because EDA+ FN is expressed
preferentially in embryonic and tumor tissues, alternative splicing at
the EDA region of FN may be instrumental in supporting the high
proliferative potential of cells in these tissues.
FN contains two major integrin binding regions: CCBD and CS-1. CCBD is
recognized by integrin
5
1 and
v-containing integrins, whereas
CS-1 is recognized by integrin
4
1. Besides the distinct binding
specificities, the integrin binding activities of these two regions are
regulated differently at the level of RNA splicing. Thus, the CS-1
region itself is excluded from the FN molecule by alternative splicing
(39-41), regulating its cell adhesive activity in an all-or-none
manner. In contrast, CCBD is encoded by constitutive exons, and its
cell adhesive activity is regulated by the alternatively spliced EDA
segment in a range of 2~3-fold increments. The difference in the mode
of regulation of their activities by alternative splicing appears to
reflect the physiological roles of these cell adhesive regions.
Integrin
5
1, the major receptor for CCBD, is expressed on a wide
variety of cell types and plays a central role in
adhesion-dependent signaling events involved in
proliferation, migration, and survival of cells (1, 9). The interaction
of integrin
5
1 with CCBD may provide housekeeping signals that
need to be maintained strictly at the minimal level required for cell
survival. In contrast, integrin
4
1, the specific receptor for
CS-1, is expressed on restricted cell types such as lymphocytes (39,
42) and elicits a cell type-specific proliferation signal (43).
All-or-none regulation of CS-1 activity by alternative splicing may be
suitable for such an auxiliary signal, leaving the basal survival
signal from CCBD uncompromised.
The interaction of cells with FN is regulated not only by the
alternative splicing at the EDA region but also by the affinity states
of integrin
5
1. Integrins exist at different affinity states
ranging from inactive to fully activated forms (44). The affinity
states of integrins are modulated by cellular stimulation by
chemoattractants (44) and interaction with membrane-associated proteins
such as IAP (integrin-associated protein) (45). Modulation of the
ligand binding affinity of integrin
5
1 has been shown to affect
FN-dependent cell proliferation (46) and differentiation (47) by modulation of integrin
5
1-mediated signal transduction. It is conceivable, therefore, that alternative splicing of FN and
affinity modulation of integrin
5
1 coordinately regulate the
integrin-mediated, extracellular signals required for proliferation, differentiation, and survival of cells.
Role of p130Cas in FN-mediated Signal
Transduction--
We found that cell adhesion to FN-coated substrata
activates ERK2 without significant tyrosine phosphorylation of FAK.
Although there is some evidence suggesting that FAK can activate the
ERK signaling pathway in response to cell adhesion (16, 17, 19), it is
unclear whether FAK plays a physiological role in the ERK signaling
pathway. In fact, recent studies have shown that the activation of ERK
in response to integrin ligation is mediated by the adaptor protein Shc
independently of FAK (48). In support of this observation, introduction
of a dominant-negative version of FAK did not impair the ERK activation
elicited by cell adhesion to FN (49). Our results also showed that
p130Cas, but not FAK, was prominently
tyrosine-phosphorylated in response to cell adhesion onto FN-coated
substrata with kinetics similar to those of ERK2 activation. Although
the precise molecular mechanisms of
p130Cas-dependent activation of ERK remain to
be defined, tyrosine phosphorylation of p130Cas was
suggested to be involved in ERK activation by the following observations. First, overexpression of p130Cas or
FN-stimulated tyrosine phosphorylation of p130Cas promotes
the SH2-mediated binding of the adaptor proteins Crk and/or Nck to
p130Cas and subsequent association of Crk and/or Nck with
the GDP-GTP exchange protein Sos, which could result in Ras activation
(50, 51). Second, overexpression of protein tyrosine phosphatase 1B,
which can also associate with p130Cas, resulted in
dephosphorylation p130Cas and inhibition of ERK activation,
whereas a protein tyrosine phosphatase 1B mutant unable to bind
p130Cas failed to dephosphorylate p130Cas and
therefore failed to inhibit ERK activation (52). Although these
observations seem to support the involvement of p130Cas in
ERK activation, further studies are required to define the role
of tyrosine phosphorylation of p130Cas in
EDA-dependent enhancement of cell cycle progression.
Adhesive Function of FN Is Independent of the EDB Segment--
Our
results showed that the abilities of rFN(BC) to promote cell adhesion,
integrin
5
1 binding, and cell cycle progression were the same as
those of rFN(C) and were significantly lower than those of rFN(AC),
excluding the possibility that inclusion of the EDB segment alone might
potentiate the adhesive activity of FN. Adhesive functions of FN are
therefore regulated exclusively by alternative splicing at EDA but not
EDB. Based on the three-dimensional structure of a recombinant FN
fragment consisting of III7-III10 modules
(53), we proposed that insertion of the EDA segment alters the global
conformation of the FN molecule through rotating the
NH2-terminal portion of the FN polypeptide relative to the COOH terminus, thereby resulting in the enhanced binding of
EDA+ FN to integrin
5
1 because of an increased
exposure of the RGD motif in III10 module and/or local
unfolding of the module (32). Failure of the EDB segment to substitute
for EDA in altering the global conformation of the FN so as to increase
the binding affinity to integrin
5
1 indicates that the
conformational activation of FN by insertion of an extra type III
module is dependent on the position and/or sequence of the inserted
module, making the EDA segment most suited for modulation of the
binding affinity to integrin
5
1.
Although the presence or absence of the EDB segment did not affect the
cell adhesive activity of FNs, the EDB segment may modulate other
biological functions of FN. Expression of EDB+ FN in
vivo is more restricted than that of EDA+ FN (28),
suggesting that the EDB segment may have an important function only in
highly specific situations such as angiogenesis (54) and lung
development (55). Regulated in vivo expression of
recombinant FN isoforms differing with respect to the presence or
absence of the EDA and EDB segments should provide insight into the
physiological and pathological roles of the alternatively spliced
segments during development, tumorigenesis, and inflammatory processes.