Articles |
2ß1 Integrin and Disassembly of Actin Filaments
From the John P. Robarts Research Institute and London Health Sciences Centre, Departments of Medicine (Cardiology) (J.G.P., C.M.F., M.A.L., L.H.C.), Medical Biophysics (J.G.P., C.G.E., J.F.), Biochemistry (J.G.P.), and Microbiology and Immunology (S.U., T.C., B.M.C.C.), University of Western Ontario, London, Canada.
Correspondence to J. Geoffrey Pickering, London Health Sciences Centre, 339 Windermere Rd, London, Ontario N6A 5A5, Canada. E-mail gpickrng{at}rri.uwo.ca
| Abstract |
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2ß1,
3ß1, and
5ß1
integrins on human SMCs, as assessed by flow cytometry. The greatest
increase was for the collagen-binding
2ß1
integrin. Despite this, FGF-2 did not increase SMC adhesion to type I
collagen but instead promoted SMC elongation and SMC motility. The
latter was evaluated by using a microchemotaxis chamber and by digital
time-lapse video microscopy. Although FGF-2 was not chemotactic for
human SMCs, cells preincubated with FGF-2 displayed a 3.1-fold increase
in migration to the undersurface of porous type I collagencoated
membranes and a 2.1-fold increase in migration speed on collagen.
Furthermore, chemotaxis to platelet-derived growth factor-BB on
collagen was significantly greater in SMCs exposed to FGF-2.
FGF-2induced elongation and migration on collagen were inhibited by a
blocking anti-
2ß1 antibody; however, SMC
adhesion to collagen was unaffected. SMC migration on fibronectin was
also enhanced by FGF-2, although less prominently: migration through
porous membranes increased 1.8-fold, and migration speed increased
1.3-fold. Also, FGF-2 completely disassembled the smooth muscle
-actincontaining stress fiber network contemporaneously with the
change in integrin expression and cell shape. We conclude that (1)
exogenous FGF-2 promotes SMC migration and potentiates chemotaxis to
PDGF-BB; (2) the promigratory effect of FGF-2 is especially prominent
on type I collagen and is mediated by upregulation of
2ß1 integrin; and (3) FGF-2 disassembles
actin stress fibers, which may promote differential utilization of
2ß1 integrin for motility but not
adhesion. This dynamic SMC-ECM interplay may be an important mechanism
by which FGF-2 facilitates SMC motility in vivo.
Key Words: smooth muscle cell integrin collagen actin fibroblast growth factor
| Introduction |
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FGF-2 (basic FGF) is a potent mitogen for SMCs13 14 and a proven mediator of SMC proliferation after injury to the rat carotid artery.15 A role for FGF-2 in mediating SMC migration has also been suggested by in vivo studies. Injection of FGF-2 following gentle deendothelialization of the rat carotid artery significantly stimulated SMC migration from the media to the intima. Furthermore, SMC migration induced by balloon disruption of the arterial media was abrogated by a blocking antibody to FGF-2.16 However, the mechanism by which FGF-2 mediates SMC migration is unclear. Unlike PDGF, which is a potent chemoattractant for SMCs,3 17 FGF-2 has no,3 or at best weak,17 18 chemotactic activity for SMCs. A potential role for FGF-2 in modulating the interactions between SMCs and the ECM has been suggested16 but not explored.
Integrins are cell surface receptors that serve to bridge the ECM with
the cell cytoskeleton.19 In SMCs, integrins of the
ß1 family appear to be the dominant integrins responsible
for cell adhesion to the ECM, and several ß1 integrins
have been identified on SMCs.10 20 21 22 23 24 25 Migration of SMCs
has been noted to be dependent on ß3
integrins,26 27 but a number of recent studies have also
implicated ß1 integrins in SMC migration. Antisera
against ß1 integrin blocked fibronectin-promoted
migration of rat SMCs,27 and
2ß1 integrin has been implicated in
PDGF-induced chemotaxis on collagen.25 On the other hand,
excessive ß1 integrinmediated adhesion may impair SMC
migration, as demonstrated by recent studies with a function-activating
antiß1 integrin antibody.10 28
Expression of ß1 integrins is regulatable by growth
factors. Transforming growth factor-ß1 upregulated
1 and
5 integrin subunits in rabbit
SMCs,29 and PDGF-BB has been reported to increase the
5 integrin subunit in bovine and rabbit
SMCs.29 30 Klein et al31 have demonstrated
that FGF-2 increases the expression of
2ß1,
3ß1,
5ß1, and
6ß1
integrins in microvascular endothelial cells. However,
the effect of FGF-2 on ß1 integrin expression in vascular
SMCs is unknown.
In the present study, we have examined the mechanism by which FGF-2
controls migration of human SMCs, focusing particularly on the
interplay between SMCs and the ECM. Using flow cytometry, we found that
FGF-2 upregulates the expression of a number of ß1
integrins, most prominently the collagen-binding
2ß1 integrin. Associated with this altered
integrin repertoire was morphological elongation of the cell and
complete disassembly of the actin stress fibercontaining
cytoskeleton. Consistent with other reports,3
FGF-2 was not chemotactic for human SMCs. However, SMCs treated with
FGF-2 displayed enhanced migration on type I collagen, including
augmented chemotaxis to PDGF-BB, that was inhibited by a blocking
antibody to
2ß1 integrin. This
2ß1 integrinmediated increase in
migration was not accompanied by an increase in adhesion to collagen,
suggesting a complex response to FGF-2 designed to selectively enhance
motility. The findings indicate that coordinated alterations in the
ß1 integrincytoskeletal axis may be a critical means by
which FGF-2 promotes SMC migration and potentiates chemotaxis to
PDGF.
| Materials and Methods |
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1ß1
integrinspecific mAb, Ts2/732 ; the
2ß1 integrinspecific mAb,
BHA2.133 (Chemicon Inc); the
3ß1 integrinspecific mAb, P1B5
(GIBCO/BRL); an
5ß1 integrinspecific
mAb, HA5; a ß1 integrin subunitspecific mAb,
HB1.1(Chemicon Inc); and an
v integrin subunitspecific
mAb, 23C6 (Serotec). Isotype-matched control antibodies were used for
flow cytometry and blocking experiments. The F(ab')2
fragment of FITC-conjugated anti-mouse antibody was purchased from
Becton-Dickinson. Human recombinant FGF-2 and PDGF-BB were purchased from GIBCO/BRL. Rat-tail collagen solubilized in acetic acid was used as a source of type I collagen and was prepared as previously described.34 Human fibronectin was isolated from human plasma by gelatin-Sepharose chromatography,35 and EHS tumorderived laminin was obtained from GIBCO/BRL.
SMC Culture
Primary cultures of human arterial SMCs were
initiated by explant outgrowth from unused segments of internal mammary
artery retrieved at the time of coronary artery bypass surgery,
as previously described.36 37 The identity of vascular
SMCs was confirmed morphologically and by positive
immunostaining for smooth muscle
-actin (clone 1A4,
Dako). Cells were grown in medium (M199, GIBCO/BRL) supplemented with
the designated concentration of FBS. All experiments were performed
using human SMCs in the third or fourth subculture.
Immunofluorescence
SMCs seeded on to multiwell slides were fixed in ice-cold
acetone and stained for smooth muscle
-actin (clone 1A4, Dako) as
previously described.36 F-actin stress fibers were also
visualized using FITC-conjugated phalloidin (Sigma Chemical Co). Cells
were coverslipped using glycerol/PBS (9:1) containing Hoechst 33258
(2.5 µg/mL, Sigma) to identify cell nuclei and evaluated by
fluorescence microscopy.
Flow Cytometry
Flow cytometry for detection of integrin expression was carried
out by indirect immunofluorescence staining as
described previously.33 38 Early confluent SMCs were
trypsinized and washed in cold PBS with 1% BSA. Cells were incubated
for 30 minutes on ice with control or integrin-specific mAbs at
predetermined saturating concentrations. Washed cells were incubated
with FITC-labeled anti-mouse F(ab')2 fragment, and
fluorescence staining was analyzed using a
Becton-Dickinson FacScan.
SMC Adhesion Assay
Cell adhesion was assessed using SMCs labeled with BCECF
(Sigma), as described previously.33 39 Briefly,
5x104 BCECF-labeled cells were allowed to adhere to
matrix-coated wells for 20 minutes at 37°C. Unbound cells were
removed by gentle washing. Bound fluorescence was measured by a
Fluorescence Concentrator Analyzer (IDEXX). Bound
fluorescence from adhesion to BSA-coated wells served as
control for background adhesion. Net fluorescence was
determined by subtraction of background and expressed as a percentage
of the total fluorescence yielded from 5x104
labeled SMCs minus background fluorescence. For inhibition
experiments, mAb BHA2.1 was used at 10 to 20 µg/mL, a concentration
based on prior titering to inhibit adhesion of HT1080 cells to
collagen.33
SMC Elongation Assay
SMC shape change after seeding was evaluated using a Boyden-type
microchemotaxis chamber (Neuroprobe, Cabot John), as previously
described.28 Polycarbonate filters (Nucleopore) were
precoated overnight at 4°C with 10 µg/mL of either fibronectin or
collagen type I in PBS. Control or FGF-2treated SMCs were washed in
PBS and trypsinized, and 2000 SMCs (40 000 cells/mL in DME with 1%
BSA) were placed in the upper well. The lower well was filled with the
same medium with or without FGF-2. The chamber was incubated at 37°C
for 2 hours, and SMCs adherent to the upper surface of the filter were
fixed in methanol and stained with hematoxylin. Prior experiments
demonstrated that cell migration to the undersurface of the membrane
was negligible (<1 cell/HPF) under these conditions for both control
and FGF-2treated SMCs. The lengths of the major and minor axes
transecting the nucleus were measured from digitized calibrated images,
using morphometry software (Jandel Scientific).
SMC Migration Assay
Bulk migration was measured using a microchemotaxis chamber, as
previously described.25 40 Control or FGF-2treated SMCs
were washed in PBS and trypsinized, and 25 000 cells (500 000
cells/mL in DME with 1% BSA) were added to the upper well of the
microchemotaxis chamber. Polycarbonate filters with 10-µm pores were
precoated with fibronectin or collagen type I as described above. The
lower well of the chamber was filled with DME with 1% BSA with or
without growth factor. Migration was allowed to proceed for 6 hours at
37°C under 5% CO2. Cells remaining on the upper surface
of the filters were mechanically removed, and then filters were fixed
in methanol and stained with Harris's hematoxylin. The number of cells
that had migrated to the lower surface was determined by counting under
high-power microscopy (x40 objective). All conditions for a given
experiment were performed in triplicate.
Time-Lapse Video Microscopy
SMC migration speed was evaluated using a digital time-lapse
video recording system. Cells incubated with or without FGF-2
were seeded onto culture dishes coated with matrix proteins at 10
µg/mL. Migration was monitored with an inverted microscope (Diaphot
300, Nikon) using a x10 objective and a halogen light source. A CCD
video camera (C72, Dage-MTI) attached to the microscope was used to
generate video images, which were digitally acquired over a 5- to
8-hour recording period using a Silicon Graphics Indy
workstation and custom-written time-lapse software. Ambient temperature
was maintained at 37°C by mounting the culture dish in a Styrofoam
sleeve with transparent heating elements (MINCO Products, Inc)
placed above and below the dish. Cells were incubated in
bicarbonate-reduced medium (M199 with Hanks' salts and 25 mmol/L
HEPES, GIBCO/BRL) to maintain physiological pH in
room air. Migration was measured from digital images by tracking the
location of cell centroids at hourly intervals. Migration speed was
determined as the sum of hourly distances divided by the total time, as
described previously.41
Statistics
Data are expressed as mean±SD. Comparisons were made by
t test or ANOVA with Scheffé's post hoc test.
Statistical significance was set at P<.05.
| Results |
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ß
heterodimers differed. FGF-2 produced either no effect or a slight
reduction in
1ß1 integrin level, whereas
it increased the expression of
2ß1,
3ß1, and
5ß1
integrins. Expression of these integrins in cells exposed to 10 ng/mL
FGF-2 was intermediate, indicating that the effect was dose dependent
(data not shown).
4ß1 integrin was not
detected in the cultured human SMCs, similar to the results of Skinner
et al.25 Expression of the
v integrin
subunit was detected and was increased by FGF-2 treatment (data not
shown).
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The quantitatively greatest change in expression induced by FGF-2 was
observed for
2ß1 integrin; thus, this
response was characterized further. The kinetics of the increase in
2ß1 integrin was evaluated by harvesting
SMCs at various times after the addition of 25 ng/mL FGF-2. As shown in
Fig 1B
, increased expression was detected at 12 hours and was maximal
by 35 hours.
2ß1 integrin is known to bind collagens
and laminin.19 To determine if the ability of FGF-2 to
increase
2ß1 integrin surface expression
was dependent on specific interaction with the ECM, SMCs were seeded
onto dishes coated with either fibronectin, laminin, or type I collagen
and stimulated for 48 hours with FGF-2. The relative increase in
2ß1 integrin expression induced by FGF-2
was not different for the three substrates: 3.7±0.7-fold for cells on
fibronectin, 2.2±0.5-fold for cells on laminin, and 2.4±1.3-fold for
cells on type I collagen (P=NS). However, in absolute terms,
SMCs seeded on collagen expressed more
2ß1
integrin under both basal and FGF-2stimulated conditions than did
cells seeded on fibronectin or laminin (P<.05) (Fig 1C
).
FGF-2 Increases SMC Elongation but Not Adhesion to Type I Collagen
via
2ß1 Integrin
As shown in Fig 2
, neither acute exposure to FGF-2
(25 ng/mL) nor 48 hours of treatment with FGF-2 at this dose had a
significant effect on SMC adhesion to type I collagen. This was
interesting in light of the observation that FGF-2 increased surface
expression of the collagen-binding
2ß1
integrin. Of note, however, addition of mAb BHA2.1, which blocks
2ß1 integrin function,33 had
no detectable effect on adhesion of SMCs to collagen in either
untreated or FGF-2treated SMCs (Fig 2
).
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The effect of FGF-2 on the morphology of SMCs was assessed first
by evaluating changes in cell shape over 48 hours. As shown in Fig 3
, SMCs incubated with 25 ng/mL FGF-2 acquired a
distinct spindle-like shape that was evident after 24 hours of
treatment and most prominent after 48 hours. This shift to a
spindle-like morphology was observed in SMCs growing on collagen type
I, fibronectin, or laminin. To quantitatively examine the effect of
FGF-2 on SMC elongation, SMCs were treated with FGF-2 (25 ng/mL) for 48
hours and then trypsinized and allowed to spread on matrix-coated
filters for 2 hours. The length and width of 80 SMCs were determined
for each condition, and data were obtained in triplicate. As shown in
Fig 3C
and 3D
, the ratio of length to width was significantly higher in
SMCs treated with FGF-2, indicating that FGF-2 promoted elongation of
SMCs. This effect of FGF-2 was observed for cells seeded on collagen
(P<.05) and fibronectin (P<.01). mAb BHA2.1
inhibited the FGF-2mediated increase in elongation of SMCs on
collagen (P<.05) but had no effect on elongation on
fibronectin (Fig 3
). Further evidence for the role of
2ß1 integrin in mediating shape changes
came from visual assessment of the SMCs seeded onto matrix.
FGF-2treated SMCs seeded on collagen with mAb BHA2.1
displayed atypical thin cytoplasmic processes in an arborized
pattern, suggesting a failed attempt at reshaping the cell (data not
shown).
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FGF-2 Disassembles the Actin Stress Fiber Cytoskeleton
Actin microfilaments are a major component of the
cytoskeleton and are known to interact with integrins on the inner face
of the cell membrane. To determine if FGF-2 influenced cytoskeletal
organization, and hence its relationship with integrins in focal
adhesions, SMCs were fixed and immunostained for smooth
muscle
-actin. As shown in Fig 4
, there was a gradual
loss of smooth muscle
-actin microfilament bundles traversing the
cell body. This was evident by 24 hours and complete in almost all SMCs
after 48 to 72 hours. The disassembly of smooth muscle
-actincontaining stress fibers was seen in cells growing on either
collagen or fibronectin. Furthermore, the response did not require
2ß1 integrin function, as mAb BHA2.1
failed to prevent actin disassembly. Similar results were seen when
F-actin was visualized using FITC-phalloidin (data not shown).
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FGF-2 Is Not Chemotactic for Human SMCs but Enhances Migration
Through Type I CollagenCoated Membranes and Increases Migration Speed
on Type I Collagen
Having identified that FGF-2 alters ß1 integrin
expression, SMC shape, and the actin cytoskeleton, we determined
whether SMCs incubated with FGF-2 would also manifest altered migratory
properties. Control SMCs and SMCs treated for 48 hours with 25 ng/mL
FGF-2 were seeded onto ECM-coated porous polycarbonate filters, and
migration to the lower surface was evaluated after 6 hours. For these
experiments, FGF-2 (0 to 25 ng/mL) was placed in the lower chamber. As
illustrated in Fig 5
, several observations were made.
First, there was no evidence for chemotaxis of human SMCs toward FGF-2;
ie, for a given set of experimental conditions, the number of cells
that migrated to the undersurface of the membrane when FGF-2 was in the
lower well was not different than when vehicle alone was in the lower
chamber. This lack of chemotaxis to FGF-2 was observed with SMCs seeded
on membranes precoated with fibronectin or type I collagen. PDGF-BB (10
µg/mL) served as a positive control for chemotaxis and produced SMC
migration when placed in the bottom but not upper well of the chamber
(eg, see Fig 8
). FGF-2 in the upper well also had no effect on
migration. A second observation was that SMCs pretreated with FGF-2
migrated through collagen-coated filters in significantly greater
numbers than did untreated control SMCs. The number of FGF-treated SMCs
that migrated to the lower surface, in the absence of growth factor in
the lower chamber, was 3.1±0.8-fold greater than control SMCs
(P<.001, n=5 experiments). Similar results were obtained
when FGF-2 (1 to 25 ng/mL) was present in the bottom well of the
chamber. The third observation was that the FGF-2induced augmentation
of migration was more prominent for SMCs on type I collagen than on
fibronectin. Migration through fibronectin-coated membranes was
increased 1.8±0.2-fold by FGF-2 pretreatment (P<.05, n=3
experiments), but this increase was significantly less than that for
SMCs migrating on collagen-coated membranes (P<.05).
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To directly measure the speed of SMC migration, SMC movement was
monitored by digital time-lapse video microscopy. The results of image
quantification including representative migration paths
are shown in Fig 6
. SMCs preincubated for 48 hours with
FGF-2 migrated on collagen at speeds that, on average, were 2.1-fold
higher than those of untreated SMCs (19.4±5.2 versus 9.3±3.8
µm/h, P<.05). FGF-2treated SMCs also migrated on
fibronectin faster than did untreated SMCs, although the relative
increase was only 1.3-fold (11.2±0.4 versus 8.4±0.8 µm/h,
P<.05). These findings thus corroborate those of the
chemotaxis chamber assay and implicate augmented crawling speed, likely
together with cell shape changes, as components of the enhanced
migratory response of SMCs to FGF-2.
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FGF-2Mediated Enhancement of SMC Migration on Collagen Is
Inhibited by Anti
2ß1 Integrin
Antibody
Although inhibition of
2ß1 integrin
had no significant effect on SMC adhesion to collagen,
2ß1 integrin function was important for
FGF-2stimulated morphological changes. Therefore, the role of
2ß1 integrin in mediating the
FGF-2induced increase in SMC migration was evaluated using the
blocking mAb BHA2.1. As shown in Fig 7
, the addition of
10 µg/mL mAb BHA2.1 to the upper well prevented the FGF-2mediated
increase in migration through collagen-coated membrane
(P<.01), whereas the isotype-matched control antibody had
no effect. The same dose of mAb BHA2.1 had no effect on basal or
FGF-2enhanced migration through fibronectin-coated membranes.
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FGF-2 Enhances Chemotaxis to PDGF via
2ß1 Integrin
Both FGF-216 and PDGF17 have been found
to regulate SMC migration in vivo. Therefore, we next determined if
exposure of SMCs to exogenous FGF-2 altered the chemotactic response to
PDGF. As illustrated in Fig 8
, PDGF induced
dose-dependent chemotaxis through collagen-coated filters, as has been
previously described.3 25 When cells were pretreated for
48 hours with 25 ng/mL FGF-2, the migratory response to 1 and 10 ng/mL
PDGF was significantly increased (P<.01). At these doses of
PDGF, migration of SMCs not preincubated with FGF-2 was unaffected by
the anti
2ß1 integrin mAb BHA2.1.
However, mAb BHA2.1 blocked the incremental increase provided by FGF-2
treatment (P<.01). Thus, FGF-2 treatment potentiated the
chemotactic response to PDGF on collagen, by effectively supplementing
an
2ß1 integrinindependent process with
an
2ß1 integrindependent process. The
response to high-dose PDGF (30 ng/mL) differed in that FGF-2 did not
augment the chemotactic response in this setting. Furthermore,
migration of SMCs not preincubated with FGF-2 to high-dose PDGF was
partially inhibited by mAb BHA2.1 (P<.01), indicating that
an
2ß1-mediated mechanism already
existed.
| Discussion |
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2ß1
integrin, the expression of which was increased by over 2-fold by
FGF-2. Finally, FGF-2induced motility was also associated with
postintegrin alterations in the cytoskeleton, including disassembly of
actin stress fibers. The regulation of cell migration by growth factors is often considered and studied in a time frame of a few hours. SMC chemotaxis, for example, typically is manifested in vitro within 4 to 6 hours.12 18 25 However, SMC migration within the arterial wall follows a more prolonged time course. Indeed, the rationale for examining the consequences of a more prolonged exposure to FGF-2 comes from in vivo studies. After balloon injury to the rat carotid artery, SMC proliferation begins after 24 hours,43 and SMC migration is detectable by 3 days.16 Both responses have been shown to be regulated by FGF-2,15 16 which implies that in this setting SMCs may be exposed to FGF-2 over a matter of days. The fact that FGF-2 binds with low affinity to the ECM is also compatible with sustained delivery of FGF-2 to the SMC, during conditions of active remodeling.44
FGF-2 increased the surface expression of several ß1
integrins, including
2ß1,
3ß1, and
5ß1
integrins. However, the effect was not generalized, since
1ß1 integrin expression was not increased.
The quantitatively greatest increase in expression was seen for
2ß1 integrin.
2ß1 integrin is capable of binding
collagen types I and IV as well as laminin,19 and there is
evidence that expression of
2ß1 integrin
is dependent on SMC phenotype. Specifically, expression of
2ß1 integrin was not observed in SMCs
within the aortic media but was induced after growth in culture,
suggesting that
2ß1 integrin expression is
a feature of an "activated" SMC
phenotype.25 Although one study did not detect
2ß1 integrin after injury to the rat
carotid artery,45 neoexpression of
2ß1 integrin has been identified in human
SMC tumors.46 The effect of growth factors on
2ß1 integrin expression by SMCs has not
previously been reported, although it is of interest that FGF-2 has
been shown to increase
2ß1 integrin
expression in microvascular endothelial
cells.31
It was noteworthy that the FGF-2mediated increase in the expression
of
2ß1 integrin was not associated with
increased adhesion to collagen but was associated with cell elongation
and increased motility on this substrate. Under basal or
FGF-2stimulated conditions, adhesion to collagen was not inhibited by
mAb BHA2.1, an anti-
2ß1 antibody
previously shown to block adhesion to type I collagen.33
Similar results were obtained using another
2ß1 integrin mAb (data not shown). Skinner
et al25 also did not observe a role for
2ß1 integrin on SMC attachment to type I
collagen. On the other hand, Lee et al24 observed a 31%
decrease in adhesion of human venous SMCs to type I collagen by an
anti
2ß1 integrin antibody, implying that
the
2ß1 receptor is capable of at least
partly mediating adhesion of SMCs to type I collagen. Other receptors
besides
2ß1 integrin may be involved in
binding to type I collagen, including
1ß1
and
3ß1 integrins,19 38 and
cooperativity has been demonstrated between
1ß1 and
2ß1
integrins.24 Therefore, it is conceivable that inhibition
of
2ß1 integrin alone may be insufficient
to impair adhesion and that inhibition of multiple receptors is
necessary before a functional consequence on adhesion is manifested.
Even so, it is of interest that in the face of a 2.4-fold increase in
2ß1 integrin expression, FGF-2 did not
increase SMC adhesion to collagen whatsoever. FGF-2 did, however,
promote SMC elongation and migration on collagen in a manner that was
dependent on the increase in
2ß1 integrin.
The ability of the cell to modulate its shape and extend over its
substrate is fundamental to locomotion and morphogenesis, and a role
for
2ß1 integrin in these processes has
been suggested in a few cell lines.47 48 For example, in a
mammary epithelial cell line, inhibition of
2ß1 integrin significantly reduced cell
spreading and migration speed as well as the ability of these cells to
wrap around collagen fibers.47 The present studies
support a similar role for
2ß1 integrin in
human SMCs, specifically in the setting of sustained stimulation by
FGF-2.
The ability of FGF-2 to induce integrin-mediated changes in cell shape
without augmenting adhesivity may be precisely the scenario required to
optimize cell locomotion. Theoretical analysis has suggested
that maximum migration speed will occur at an intermediate
adhesiveness,49 and subsequent experimental evaluation of
SMC migration on fibronectin and type IV collagen has supported this
concept.5 Furthermore, using an antiß1
integrin antibody that augments the binding affinity of
ß1 integrins, Seki et al10 and Koyama et
al28 observed enhanced adhesion of SMCs to ECM substrates
but decreased SMC migration to PDGF on Matrigel. Thus, if
integrin-mediated adhesion is excessive, the cell may be effectively
"frozen" in location. Therefore, it appears that utilization of
2ß1 integrin for shape change (elongation)
but not for adhesion is a coordinated response to FGF-2 designed to
facilitate motility.
What is the potential mechanism by which a FGF-2mediated increase in
2ß1 integrin increases SMC elongation and
migration but does not increase adhesion to collagen? Presumably, there
are other effects of FGF-2 that effectively dissociate the expression
of
2ß1 integrin from its adhesive
function. In this regard, it was noteworthy that incubation of SMCs
with FGF-2 led to disassembly of the actin stress fiber network,
evidenced by staining for smooth muscle
-actin and F-actin. It is
well established that integrins associate with the actin-based
cytoskeleton,19 including the actin stress fibers that
traverse the cell. These actin microfilament bundles are structurally
linked to integrins in focal adhesion contacts, and their presence is
felt to be an important feature of the stationary cell
phenotype50 ; eg, actin stress fibers are prominent
in corneal fibroblasts attached and flattened over a planar substrate
but are absent in fibroblasts migrating in three-dimensional collagen
gels51 or in vivo.52 In vivo, actin stress
fibers are present in endothelial cells subjected
to high shear stress,53 where strong attachment to the
underlying ECM is required. Given the association between actin stress
fiber network and strong surface adhesion, disassembly of actin stress
fibers may be one means by which the cell liberates itself for
movement. We speculate that disassembly of actin stress fibers in SMCs
by FGF-2 minimizes or even prevents increased adhesivity in the setting
of increased integrin expression, by disrupting the intracellular
structural network required for stable attachment. Other possible
mechanisms underlying the differential response include selective
activation of intracellular signaling cascades that mediate migration
but not adhesion or selective modulation of integrin function or
activation state through conformational changes in specific receptor
domains.10 54
Disassembly of actin stress fibers by FGF-2 was observed in cells
cultured on collagen as well as on fibronectin, and the process did not
require
2ß1 integrin function. Therefore,
this particular effect of FGF-2 does not appear to be specific for a
given ECM environment. However, actin disassembly could reflect an
overall reduced level of interaction between the cell and the ECM. It
has recently been shown that cellular interaction with ECM is necessary
for stress fiber attachment to focal adhesion contacts.55
Furthermore, we have recently determined that FGF-2 induces expression
of matrix metalloproteinase-1 (collagenase) in human
SMCs.56 This raises the possibility that local degradation
of collagen, induced by FGF-2, may reduce the available ECM ligands for
integrins, with disorganization of actin stress fibers as a downstream
consequence. This remains speculative, and whether a similar
degradation of fibronectin occurs in response to FGF-2 is not known.
However, the interplay between ECM degradation and integrin function
has recently been highlighted by the observation that signaling via
2ß1 integrin induces matrix
metalloproteinase-1 expression.57 Other potentially
relevant mediators of stress fiber integrity include members of the Rho
family of GTP-binding proteins.58 59 Some growth factors,
such as PDGF and epidermal growth factor, induce stress fiber assembly
via functional Rho.58 The effect of FGF-2 on Rho function
is unexplored.
Both FGF-216 and PDGF17 have been shown to
mediate SMC migration following balloon injury to the rat carotid
artery. Previous studies have indicated that PDGF-mediated release of
FGF-2 plays a role in chemotaxis to PDGF.18 42 However,
during vigorous vascular remodeling, eg, after mechanical injury, FGF-2
may also be liberated from dead or injured cells as well as from ECM
depot sites. The present findings of augmented chemotaxis to PDGF
following FGF-2 exposure may be particularly relevant to such
circumstances. By endowing the SMC with the capacity to migrate faster
on collagen, FGF-2 induced a functional augmentation of PDGF-induced
migration. Interestingly, this cooperative effect was dose dependent
and apparent when SMCs were exposed to 1 to 10 ng/mL PDGF but not to
high-dose PDGF (30 ng/mL). In the absence of FGF-2, SMC migration to
the lower concentrations of PDGF was largely independent of
2ß1 integrin function. However,
preincubation of SMCs with FGF-2 effectively supplemented an
2ß1 integrinindependent process with an
2ß1 integrindependent process. In
contrast, migration to high-dose PDGF in the absence of FGF-2 was
partly dependent on
2ß1 integrin,
consistent with that reported by Skinner et al.25
Under these circumstances, FGF-2 did not provide an additional effect,
likely because the
2ß1-mediated component
of migration had already been recruited. Other effects of PDGF relevant
to cell motility have also been shown to depend on PDGF concentration.
In particular, the effects of low-dose PDGF (5 ng/mL) on membrane
ruffling, actin organization, and tyrosine
phosphorylation of focal adhesion kinase and
phosphatidylinositol 3-kinase were found to differ from those of
high-dose (30 ng/mL) PDGF.60 Furthermore, we have observed
that high-dose PDGF induced a small increase in
2ß1 integrin expression, but this was not
detectable at lower concentrations (data not shown).
Although the promigratory effect of FGF-2 was greatest for SMCs on
collagen, an enhancement in migration was also seen with SMCs on
fibronectin. The mechanism for this response is not presently
defined; however, both disassembly of actin stress fibers and cell
elongation occurred on fibronectin and may be contributing factors.
Furthermore, integrins other than
2ß1 may
be relevant. FGF-2 effected a modest increase in expression of
5ß1 integrin and the
v
integrin subunit, both of which are capable of interacting with
fibronectin.19 A role for
vß3
integrin in SMC migration and vascular lesion formation has been
previously suggested.61 62
In summary, we have determined that FGF-2 increases the speed
with which SMCs migrate and have identified two associated subcellular
processes: upregulation of
2ß1 integrin
and disassembly of the actin stress fiber network. The former process
is necessary for the increase in migration on collagen; the latter may
underlie the differential utilization of
2ß1 integrin for migration but not for
adhesion. These coordinated changes in the ß1
integrincytoskeletal axis may be a critical means by which FGF-2, and
possibly other growth factors, regulate SMC migration in vivo.
Furthermore, they provide a unique basis of cooperativity between PDGF
and FGF-2 in the control of SMC migration.
| Selected Abbreviations and Acronyms |
|---|
|
| Acknowledgments |
|---|
Received September 18, 1996; accepted February 20, 1997.
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