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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 649-662
Src-Dependence and Pertussis-Toxin Sensitivity of Urokinase
Receptor-Dependent Chemotaxis and Cytoskeleton Reorganization in
Rat Smooth Muscle Cells
By
Bernard Degryse,
Massimo Resnati,
Shafaat A. Rabbani,
Antonello Villa,
Francesca Fazioli, and
Francesco Blasi
From DIBIT, Università Vita-Salute San Raffaele, Milano, Italy;
McGill University and Royal Victoria Hospital, Montreal, Quebec,
Canada; and the Dipartimento di Farmacologia, CNR and B. Ceccarelli
Centers, University of Milan.
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ABSTRACT |
The catalytically inactive precursor of urokinase-type plasminogen
activator (pro-u-PA) induced a chemotactic response in rat smooth
muscle cells (RSMC) through binding to the membrane receptor of
urokinase (u-PA receptor [u-PAR]). A soluble form of
u-PAR activated by chymotrypsin cleavage as well as a peptide located
between domain 1 and 2 of u-PAR reproduced the effect of pro-u-PA on
cell migration. The chemotactic pro-u-PA effect correlates with a
dramatic reorganization of actin cytoskeleton, of adhesion plaques, and
with major cell shape changes in RSMC. Pro-u-PA induced a decrease in
stress fiber content, membrane ruffling, actin ring formation, and
disruption leading to the characteristic elongated cell shape of motile
cells with an actin semi-ring located close to the leading edge of
cells. u-PAR effects on both chemotaxis and cytoskeleton were sensitive
to pertussis toxin and, hence, possibly require G proteins. u-PAR
effects are accompanied by a relocation of u-PAR, vitronectin receptor
(VNR)  v 3, 1 integrin subunit, and Src tyrosine kinase to the
leading membrane of migrating cells. In conclusion, our data show that pro-u-PA, via binding to u-PAR, controls a signaling pathway, regulated
by tyrosine kinases and possibly G proteins, leading to cell
cytoskeleton reorganization and cell migration.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
UROKINASE (u-PA) IS INVOLVED in a number
of physiological and pathological processes: fibrin degradation, tissue
involution, wound healing, inflammation, angiogenesis, rheumatoid
arthritis, and cancer invasion (for reviews, see Fazioli and
Blasi1 and Blasi2). These effects may be
reproduced in culture with catalytically active or inactive derivatives
of u-PA but require binding to the u-PA receptor (CD87/u-PAR). For
instance, cell adhesion and cell migration regulation mediated by the
membrane u-PA receptor may not require u-PA catalytic activity, because
either pro-u-PA, high molecular weight (HMW)-u-PA, or its
amino-terminal fragment (ATF) can exert the same influence on
cells.3-5 In myeloid cells, u-PAR-mediated chemotaxis
requires heterotrimeric G-proteins and tyrosine kinase
activity.5
The structure of u-PAR is organized in three different domains. The
first domain is involved in the binding of u-PA,6 whereas domains 2 and 3 are endowed with binding activity for
vitronectin.3 However, an intact receptor is important for
the high-affinity binding to both u-PA and vitronectin.7,8
u-PAR is anchored to the plasma membrane through a
glycosyl-phosphatidyl-inositol moiety and does not possess a
cytoplasmic domain. Thus, the problem arises of how u-PAR can mediate
intracellular signal transduction. A transmembrane adaptor, not yet
identified, has been hypothesized for the chemotactic
effect4,5; a direct binding to integrins and/or caveolin
may mediate its cell adhesion effects.9
Factors acting on cell motility regulate cytoskeleton organization by
polymerization and depolarization of actin filaments. Chemoattractants
induce reorganization of actin cytoskeleton concomitantly with
induction of cell motility.10,11 For instance, nonmotile fibroblasts exhibit higher stress fibers content than do motile fibroblats.12-14 CD 87/u-PAR has been proposed to be a
membrane-associated chemokine, because it can directly induce
chemotaxis2,5 as well as modify cell adhesion by undergoing
ligand-mediated conformational changes.3,9 Thus, like
chemokines, u-PAR might induce reorganization of the actin cytoskeleton.
Smooth muscle cells (SMC) represent the most abundant cell type in the
arterial blood vessels and appear to exist in two different states. In
intact arteries, SMC are in a contractile state and do not divide. They
are involved in the maintenance of the elasticity and rigidity of the
vessel wall and control blood pressure. When the endothelial wall is
damaged, these cells undergo a transition to a synthetic state
characterized by cell division and cell migration. The migration of SMC
from the tunica media to the neointima represents a key event in the
development and progression of vascular diseases participating to
intimal thickening in atherosclerotic lesions and to restenosis after
angioplasty (for reviews, see Casscells,15 Van
Leeuwen,16 and Schwartz17). In vivo, SMC are
embedded in the extracellular matrix. Migration from the tunica media
to the neointima needs disruption of contacts between SMC and
extracellular matrix proteins and also crossing of the basement
membrane that separates SMC from endothelium. In SMC, u-PA stimulates
migration and signal transduction through its specific
receptor.18,19
The importance of u-PA in these effects is shown by the phenotype of
u-PA / recombinant mice in which SMC do not
divide or migrate to cause restenosis. Activation of plasminogen into
plasmin, an enzyme able to degrade extracellular matrix and basement
membrane proteins, explains the role of u-PA in SMC migration in vivo
that does not appear to require binding to u-PAR.20
However, u-PAR and u-PA are required for basic fibroblast growth factor
(bFGF)-induced migration of murine SMC.21 Moreover, the
effect of u-PA could depend on its ability to activate growth factors
such as bFGF,22 pro-transforming growth factor-
(pro-TGF-),23 and pro-hepatocyte growth factor
(pro-HGF).24,25
The role of u-PAR in SMC migration is not known. Indeed,
u-PAR/ mice do not reproduce the phenotype of
u-PA/20,26,27 with respect to
restenosis, suggesting that its role may be not essential. On the other
hand, because the
u-PAR//t-PA/ double
knockout mice, unlike the t-PA/ mice, display
major fibrin deposits (K. Danø, personal communication, 1998), u-PAR must be involved in mediating u-PA functions in vivo. Thus, the data from knockout mice may not give the absolute answer and,
even in the absence of a u-PAR phenotype on SMC migration, the role of
u-PAR is still far from being elucidated. In any case, the elucidation
of the molecular mechanisms of u-PA functions is extremely important,
and SMC represent an exceptional tool to investigate the chemokine
action of u-PA and u-PAR, because they possess an impressive
cytoskeleton and good migration properties in vitro.
The aim of the present study is to determine the influence and
mechanisms of u-PA and u-PAR in rat SMC (RSMC) migration and RSMC
cytoskeleton organization. We show that pro-u-PA, through u-PAR,
induces cell migration, cell shape changes, and cytoskeleton reorganization. Pro-u-PA/u-PAR also induce a redistribution of integrins and u-PAR to the leading edge of migrating RSMC. The presence
of Src is essential in this process. Because Bordetella pertussis toxin
(BPT) blocks all pro-u-PA/u-PAR effects on cell migration and
cytoskeleton, we suggest that the pathway may include a G
protein-regulated step.
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MATERIALS AND METHODS |
Materials.
Rat smooth muscle cells were a kind gift of Dr M. Bertulli
(Bayer Research Laboratory, Milan, Italy). BPT and its mutant were a
kind gift of Dr M.G. Pizza (Sienna, Italy).
Receptor-associated protein (RAP) and human ATF were generously
provided respectively by Dr M. Nielsen (University of Aarhus, Denmark)
and Dr J. Henkin (Abbott Research Laboratories, Chicago,
IL). 3T3 Fibroblasts from wild-type and
Src/ mice, obtained through Dr K.B. Kaplan
(M.I.T., Cambridge, MA), were transfected with full-size
human u-PAR cDNA by standard procedures to generate 3T3/u-PAR clones.
The presence of cell surface u-PA-binding u-PAR was determined by
ligand-binding assays. Collagen I and fibronectin were purchased from
Boehringer Mannheim (Mannheim, Germany). Primary
antibodies used are the following: rabbit anti-c-Src (polyclonal;
Santa Cruz Biotechnology, Santa Cruz, CA), mouse antivitronectin receptor  v3 (monoclonal LM 609; Chemicon,
Temecula, CA), mouse anti-1 integrin subunit (kind
gift of Dr P.C. Marchisio, DIBIT, Milan, Italy), and
mouse antivinculin (monoclonal; Sigma, St Louis, MO). For the
production of rat u-PAR antibodies, cDNA encoding amino acids 25-114 of
rat u-PAR was subcloned into the expression vector pTrcHis A (InVitro,
San Diego, CA), and the recombinant protein was expressed and purified
to use for immunization of rabbits.28 Antirat u-PAR IgG
detects rat u-PAR by immunofluorescence and competes for the binding of
125I-labeled rat u-PA in receptor binding assay (S. Rabbani, data not shown). The secondary antibodies were
rhodamine-conjugated F(ab)'2 fragments of antirabbit
Ig (Protos Immunoresearch, San Francisco, CA) and
fluorescein-conjugated F(ab)'2 fragments of antimouse
Ig (Dako, Copenhagen, Denmark). Nonspecific rabbit
polyclonal Igs, non-specific monoclonal mouse IgG1 (MOPC-21),
tetramethylrhodamine isothiocyanate (TRITC), and
fluorescein isothiocyanate (FITC)-conjugated phalloidin
and fMLP were from Sigma. Peptide 1 (AVTYSRSRYLEC) and its scrambled
form (TLVEYYSRASCR) have been described previously.5 Peptide D, a shorter version of peptide H: of rat origin,
has the following sequence: PRGRY. Human su-PAR was purified by
affinity chromatography from conditioned media of Chinese hamster ovary cells transfected with a mutant of u-PAR.29
Chymotrypsin-cleaved su-PAR was prepared as previously
described.6
The cDNA encoding rat pro-u-PA was isolated from rat kidney cDNA
library and inserted into the baculovirus expression vector pFast Bac1
(GIBCO/BRL, Gaithersburg, MD).30 Rat pro-u-PA was expressed
according to manufacturer's instructions and purified by affinity
chromatography using antihuman u-PA antibody. Purity of rat pro-u-PA
was confirmed by sodium dodecyl sulfate (SDS)-gel electrophoresis and
sequence analysis.
Cell culture.
RSMC monolayers and mouse Src+ or
Src/ 3T3 fibroblasts were cultured in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum (FCS). In some chemotaxis or immunofluorescence experiments, cells were pretreated with blocking antibodies. Before cell treament with antibodies, RSMC were washed once with
phosphate-buffered saline (PBS) and detached at 37°C from support
using 0.05% (wt/vol) EDTA in Ca2+-Mg2+-free
PBS. The cells were then washed in serum-free medium and cell
suspensions were treated in serum-free DMEM either with antibodies against u-PAR (10 µg/mL), with antivitronectin receptor antibodies (0.5 µg/mL), or with unspecific control IgG (10 µg/mL) for 1 hour at 4°C, and chemotaxis assay was performed as described below.
Chemotaxis assay.
Chemotaxis assay was performed as previously described.4
Briefly, modified Boyden chambers were used with
polyvinylpyrrolidone-free polycarbonate filters (13-mm diameter; 5-µm
pore size; Nucleopore-Costar, Cambridge, MA). These filters were
treated with collagen I (100 µg/mL in 0.5 N acetic acid) and
fibronectin (10 µg/mL). Each step was followed by a wash with
serum-free DMEM containing 0.2% bovine serum albumin (BSA). RSMC,
grown to confluence, were detached from support (see above) and washed
twice in serum-free medium. Then, 20,000 to 40,000 cells in serum-free
DMEM were added to the upper well of the Boyden chamber. The molecules
to be tested as chemoattractant were diluted in serum-free medium and
added to the lower well of the Boyden chamber. When chemotaxis was
performed in the presence of antibodies or BPT, these molecules were
added in both wells of the Boyden chamber. Overnight migration was
allowed at 37°C in humidified conditions with 95% air/5%
CO2. The cells remaining on the upper surface of filters
were then scraped off, and filters were fixed in methanol and stained
in a solution of 10% (wt/vol) crystal violet in 20% (vol/vol)
methanol. Random cell migration, ie, migration in the absence of
chemoattractant, was given the arbitrary value of 100%. All
experiments were performed in triplicate. Results are the mean ± standard deviation of the number of cells counted in 10 high power
fields per filter and expressed as fold over control. The number of
cells that crossed the filter in random cell migration was
approximately 20 to 40 per field.
Wounding assay.
For wounding experiments, RSMC were grown to confluence in DMEM
supplemented with 10% FCS on a glass coverslip in a 2-cm2
well in 4-well plates. Monolayers were washed once with PBS and cultured for 24 hours in serum-free medium. Single-cross wounds were
then made by dragging a sterile pipette tip accross the monolayer to
create a cell-free space. Injured monolayers were washed with PBS and
allowed to recover for a further 24 hours in serum-free medium
supplemented or not with the molecule to be tested. RSMC were then
fixed and actin cytoskeleton was visualized with rhodamine-conjugated phalloidin as described below. Quantification was made by taking photographs at lower magnification and by counting the number of cells
that had migrated from the wound edge into the cell-free space.
Immunofluorescence microscopy.
Cells were seeded on glass coverslips at a density of 15,000 to 25,000 cells (20% to 40% confluence) per 2-cm2 well in 4-well
plates (Nunc, Roskilde, Denmark) cultured for 24 hours in
DMEM plus 10% FCS. Monolayers were then washed with PBS and cultured
for another 24 hours in DMEM without FCS. Cells were challenged for
different time periods with the appropriate stimulator. After
incubation, cells were fixed for 20 minutes at room temperature with a
solution of 3% paraformaldehyde and 2% sucrose in PBS, pH 7.5. After
3 washes with PBS-BSA 0.2%, cells were permeabilized with 20 mmol/L
HEPES, pH 7.4, 300 mmol/L saccharose, 50 mmol/L NaCl, 3 mmol/L
MgCl2, 0.5% (vol/vol) Triton X-100 for 3 minutes at
4°C. Monolayers were again washed three times with PBS-BSA 0.2%
and incubated with PBS-BSA 2% for 15 minutes at 37°C. Cells were
incubated for 30 minutes at 37°C with primary antibodies, washed
three times with PBS-BSA 0.2%, and further incubated with PBS-BSA 2%
for 15 minutes at 37°C. The cells were then stained with secondary
antibodies. For visualization of filamentous actin, coverslips were
incubated with fluorescein-conjugated phalloidin for 30 minutes at
37°C. After incubation of secondary antibodies, cells were washed
three times with PBS-BSA 0.2% and once with distilled water.
Coverslips were mounted with 20% (wt/vol) mowiol in PBS and analyzed
on a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen,
Germany). Fluorescence photographs were taken on T-Max 400 film (Eastman Kodak Co, Rochester, NY). Cytoskeleton
pictures were taken with a Zeiss 100 neofluar lens, whereas
quantification was performed using a Zeiss 40 neofluar lens.
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RESULTS |
Pro-u-PA induces RSMC migration through a mechanism requiring a
conformational change of u-PAR.
The migratory response of RSMC was determined with a chemotaxis assay
using modified Boyden chambers. In the first experiment (Fig 1A), rat
pro-u-PA was compared with the well-characterized attractant
fMLP.31 Rat pro-u-PA (1 nmol/L) induced RSMC to migrate threefold above control, an effect comparable to that of
107 mol/L fMLP (Fig 1A). Concentration-dependence
showed a bell-shaped dose-response curve with stimulation at
concentrations as low as 0.1 nmol/L, a maximum at 10 nmol/L reaching a
value sixfold above control, and inhibition at higher concentrations
(100 nmol/L; Fig 1B). The stimulation of migration by pro-u-PA (as well
as other attractants) varied with the age of the culture and in some experiments was rather low (50% to 60% over background), whereas with
cells kept in culture for a shorter time, it reached levels greater
than 300% to 600%. However, none of the properties described in the
following part was ever qualitatively affected.
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| Fig 1.
Chemotactic response of RSMC to pro-u-PA, C-su-PAR, and
peptide 1. Chemotaxis assay was performed as described in methods using
modified Boyden chambers. (A) Comparison of the chemotactic effects of
pro-u-PA (1 nmol/L) and fMLP (107 mol/L) on RSMC. (B)
Effects of increasing doses of pro-u-PA on RSMC migration. (C) Effects
of increasing concentrations of C-su-PAR on RSMC migration. (D)
Chemotactic activity of synthetic peptides on RSMC migration. Peptide 1 (AVTYSRSRYLEC), which corresponds to amino acids 84-95 of human u-PAR,
and a scrambled version of peptide 1 (TLVEYYSRASCR) were tested as
chemoattractant. Cells migrated towards increasing doses of peptide 1 or its scrambled version, as indicated. (E) Effect of the LRP antagonist RAP on RSMC
migration and its effect on pro-u-PA stimulation. (F) Influence of
either wild-type BPT or mutated BPT on pro-u-PA-induced or
bFGF-induced chemotactic response of RSMC. Toxins were present in both
chambers of the Boyden apparatus. Random cell migration of unstimulated
cells is considered to be 100% migration. Results are the mean ± SD
(n = 3).
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The effect was u-PAR-dependent as shown by the inhibition by
antibodies against u-PAR (see below), in agreement with previous results obtained with human and murine cells.4
Chymotrypsin-cleaved soluble u-PAR (C-su-PAR) was previously reported
to substitute for u-PA to stimulate cell migration, even in cells
devoid of u-PAR, indicating that u-PAR acts by interacting with other
cell surface molecules.4,5 We tested C-su-PAR and found
that it stimulates RSMC migration with a dose-dependent effect and
maximal response at 10 pmol/L. Higher concentrations decreased cell
migration (Fig 1C). These results are in perfect agreement with the
chemotactic effects of u-PA or ATF on myeloid and other cells that
causes a conformational change in u-PAR and exposes a chemotactic
epitope.4 Indeed, synthetic u-PAR peptides, corresponding
to a sequence located between domain 1 and domain 2 of u-PAR, can
reproduce the effects of both pro-u-PA and C-su-PAR on cell
migration.5 Peptide 1, a 12 amino acids peptide of human
u-PAR located between amino acids 84 and 95, greatly increases RSMC
migration with maximal effect at 1 pmol/L (Fig 1D). In the same
experiment, we determined the effect of a peptide that has the same
amino acid composition but a scrambled sequence. This peptide did not
induce a migratory response. Because the amount of u-PA and u-PAR is
controlled by PAI-1 and LDL-receptor related protein (LRP), which
internalize u-PA-PAI-1 complexes and u-PAR as well,32,33 we
tested whether the antagonist of LRP, RAP, had any effect on chemotaxis
of SMC. However (Fig 1E), RAP did not have an effect on its own and did not affect the migration induced by pro-u-PA.
BPT (50 ng/mL), which inhibits the activity of receptors dependent on
heterotrimeric G proteins, was found to inhibit pro-u-PA-dependent chemotaxis. As shown in Fig 1F, BPT inhibited the effect pro-u-PA on
RSMC migration. A similar effect was also noticed with fMLP (not
shown), as expected.31,34 However, BTP had no effect on random cell migration in the absence of stimulation. These data show
that also in RSMC the migratory response to pro-u-PA is pertussis toxin-sensitive and, hence, follows the same rules as in myeloid cells,
ie, being reproduced with soluble u-PAR fragments and being sensitive
to BPT. In fact, C-su-PAR-dependent chemotaxis was also BPT-sensitive.
We tested the effect of BPT on bFGF-induced migration; in this case,
the inhibitory effect was almost absent (Fig 1F). The differential
effect of BPT on pro-u-PA-dependent and bFGF-dependent migration
further supports the idea that pro-u-PA acts by mechanisms different
from those of bFGF.
Having defined the migratory response of rat SMC to pro-u-PA, we then
tested the effect on the cytoskeleton organization of these cells,
because they are particularly suitable for this study, due to their
well-developed cytoskeletal apparatus.
u-PA/u-PAR induce time-dependent cell shape changes and cytoskeleton
reorganization.
Cell shape change and cytoskeleton reorganization are observed as part
of a chemotactic motility response.10,11 To examine cell
shape changes and cytoskeleton reorganization, subconfluent cultures of
serum-starved RSMC were challenged with rat pro-u-PA (1 nmol/L) for
increasing incubation times from 5 to 120 minutes. Control conditions
were represented by unstimulated cells kept at 37°C for 120 minutes. Actin filaments were observed using fluorescein-conjugated phalloidin as an actin staining reagent. Cell-substratum contact sites
were visualized by immunofluorescence with antivinculin antibodies.
Upon stimulation with pro-u-PA, several time-dependent changes in
cytoskeleton occurred. The lower magnification pictures of
Fig 2 show the general changes that
appeared after the addition of pro-u-PA. Within 30 minutes, cell shape,
size, and actin cytoskeleton were affected, but the effect reversed
after 120 minutes (Fig 2). Higher magnification pictures give more
detailed information (Fig 3). In
control cultures, most cells show a high number of stress fibers,
showing a well-developed cytoskeleton (Fig 3A). Focal adhesions
containing vinculin were evenly distributed over the bottom surface of
RSMC (Fig 3B). Five minutes after the addition of pro-u-PA, membrane
ruffling responses were observed but cell-cell contacts were not
broken. A decrease in stress fibers content was also observed (Fig 3C),
and vinculin started to be redistributed at the periphery of cells (Fig
3D). Subsequently, actin was reorganized into a ring-like circular
structure with some of the actin filaments radially disposed (Fig 3E)
in contact with vinculin-containing focal contacts (Fig 3F). These
pictures also suggest that, through actin ring contractility, this
change of shape might serve to break cell-cell contacts (Fig 3G and H),
allowing the cell to move. Figure 3I and J show an actin ring in the
process of being cleaved and Fig 3K and L show a ring that has been
actually cleaved. Cleavage of the ring of actin correlated with a shape
resembling that of motile cells in which actin was organized in
semicircular structures. These stages are shown in Fig 3M through P. In
pro-u-PA-stimulated cells, this typical morphology of motile cells was
observed within 30 minutes. Close to the leading edge of the cell, a
semi-ring of actin filaments was formed concomitant with the generation of membrane ruffles (Fig 3Q). In many cells, distribution of vinculin also changed becoming organized in a double row, at the leading edge of
the cell, flanking the actin filament semi-rings (Fig 3R). These
effects gradually declined, and after 2 hours, the number of actin
stress fibers and the cell shape returned to levels similar to those
observed in control cells (compare Fig 3S and A). However, at this
time, the distribution of vinculin did not return to that of
unstimulated RSMC, appearing to be still organized in rows at the
periphery of cells (compare Fig 3A and T). Cells were aggregated anew
and new cell-cell contacts were formed (Fig 3S and T). These data show
that the effects of pro-u-PA on cell morphology and cytoskeleton
structure are transient.
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| Fig 2.
Effect of pro-u-PA on actin cytoskeleton organization and
on morphology of RSMC. Subconfluent (50% to 70%) cells were incubated
in the absence (A) or in the presence of 1 nmol/L pro-u-PA for 30 (B)
or 120 minutes (C). Actin filaments were visualized using FITC-coupled
phalloidin as described.
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| Fig 3.
Effect of pro-u-PA on actin cytoskeleton (A, C, E, G, I,
K, M, O, Q, and S) and vinculin distribution (B, D, F, H, J, L, N, P,
R, and T) on RSMC. Cells were incubated in the absence (A and B) or in
the presence of 1 nmol/L pro-u-PA for 5 minutes (C and D), for 10 to 15 minutes (E, F, G, H, I, and J), for 30 minutes (K, L, M, N, O, P, Q,
and R), or for 120 minutes (S and T). The cells were then fixed and
double-stained with fluorescein-conjugated phalloidin to visualize
actin filaments and with antibody against vinculin, which was
visualized with a rhodamine-conjugated secondary antibody.
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To test whether the shapes observed really represent intermediates
between a resting and a migrating morphology, we measured the frequency
of the different types of cellular shapes and cytoskeleton organization
at differents times. Photographs were taken at low magnification and
cells with different morphology were counted and classified
(Fig 4). Three stages were arbitrarily
defined. Stage 1 is a cell shape exhibiting numerous stress fibers with vinculin distributed under the surface of the cell in contact with
substratum, as shown in Fig 3A and B. These are features of
unstimulated cells. Stage 2 includes cells with membrane ruffling and
decreased stress fibers content, redistribution of vinculin, formation
or disruption of actin ring, or a motile cell shape with actin
semi-ring at the leading edge of the cell (Fig 3C through R). Cells
were classified in stage 3 when they showed actin stress fibers
reformation while vinculin remained still distributed at the periphery
of cells (see Fig 3S and T). In Fig 4A, the phalloidin-staining of the
three exemplified stages are shown. In unstimulated cultures, 50% of
RSMC were in stage 1 and 50% in stage 2 (Fig 4B). Within 5 minutes,
the proportion of cells in stage 1 decreased to approximately 25%,
whereas stage 2 cells increased by a similar value. No stage 3 cells
were observed (Fig 4B). At t = 30 minutes, the percentage of cells in
stage 1 remained constant, whereas cells in stage 2 slighty decreased.
At this time, approximately 20% of RSMC were classified in stage 3 (Fig 4B). After 120 minutes, the proportion of stage 2 cells decreased
to approximately 40% while the percentage of cells in stage 3 increased to approximately 40% (Fig 4B). These data therefore suggest
that the addition of pro-u-PA causes changes in cell morphology and
cytoskeleton structure that represent modification from a resting to a
migrating state.
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| Fig 4.
Pro-u-PA-induced actin cytoskeleton reorganization in
RSMC. Cells were stimulated with 1 nmol/L pro-u-PA for different time
periods at 37 °C, after which cells were fixed and stained with
fluorescein-conjugated phalloidin. Quantification of actin cytoskeleton
reorganization was performed by taking photographs at low magnification
and by counting cells in each stage of cytoskeleton organization. (A)
Examples of these stages are given (for more details see the text)
briefly. Stage 1 corresponds to resting cells that exhibit stress
fibers. Stage 2 coresponds to cells showing a reorganization of
cytoskeleton: a decrease of stress fibers content, membrane ruffling,
actin ring or actin semi-ring with elongated cell shape characteristic
of motile cells. Stage 3 corresponds to cells that reform stress fibers
but with a vinculin distribution different from that of cells in stage
1 (see Fig 3). (B) The percentage of cells exhibiting actin
cytoskeleton reorganization was recorded after incubation for 5, 30, and 120 minutes with pro-u-PA.
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Because cytoskeletal and shape changes occur during a chemotactic
response, we tested whether pro-u-PA effects on chemotaxis and cell
shape occurred through the same molecular mechanisms. To test this
point, we used u-PAR fragments or synthetic peptides. Both cleaved
su-PAR (C-su-PAR) and peptide 1 were able to reproduce the effects of
pro-u-PA on cell shape and cytoskeleton. Within the same time-range, 10 pmol/L C-su-PAR (Fig 5B and C) or 1 pmol/L peptide 1 (Fig 5D) induced membrane ruffling, reorganization of actin
filaments, and motile morphology, essentially as described above for
pro-u-PA. Taken together, these results confirm that pro-u-PA induces
cell shape changes and cytoskeleton reorganization through u-PAR using
the same signaling pathway that controls cell migration. Because the
experiments of Figs 3, 4, and 5 were performed under chemokinetic
conditions, the results indicate that the exposure of the same
chemotactic u-PAR epitope is involved in both chemotaxis and
chemokinesis.
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| Fig 5.
Effects of chymotrypsin-cleaved su-PAR and peptide 1 on
actin cytoskeleton on RSMC. Cells were incubated in the absence of
chemoattractant (A) or with 10 pmol/L C-su-PAR for 5 (B) or 30 minutes
(C) or with 1 pmol/L peptide 1 for 30 minutes. Effects of BPT on the
pro-u-PA-induced actin cytoskeleton reorganization on RSMC. Cells were
untreated or pretreated with 50 ng/mL pertussis toxin or with 50 ng/mL
of mutated pertussis toxin for 6 hours; the cells were then incubated
with or without 1 nmol/L pro-u-PA in the absence or in the presence of
toxin. (A) Untreated and unstimulated cells. (E) Unstimulated cells
treated with 50 ng/mL pertussis toxin. (F) Unstimulated cells treated
with 50 ng/mL mutated pertussis toxin. (G) Untreated cells stimulated
for 30 minutes with 1 nmol/L pro-u-PA. (H) Pertussis toxin (50 ng/mL)
-treated cells stimulated for 30 minutes with 1 nmol/L pro-u-PA. (I)
Mutated pertussis toxin (50 ng/mL)- treated cells stimulated for 30 minutes with 1 nmol/L pro-u-PA. Actin filaments were visualized using
fluorescein-conjugated phalloidin.
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Because cytoskeleton reorganization correlated with cell migration, we
examined the effect of BPT on the reorganization of actin filaments.
Treatment of control (untreated and unstimulated) cells either with BPT
or with a mutated BPT, which cannot ADP-ribosylate G proteins, had no
effect (Fig 5A, E, and F). However, pro-u-PA-induced actin
cytoskeleton reorganization of RSMC (Fig 5G) was inhibited by active
BPT (Fig 5H). In contrast, mutated pertussis toxin did not inhibit
pro-u-PA-induced cell shape change and cytoskeleton reorganization
(Fig 5I). The qualitative data of Fig 5 are representative of those
quantitatively obtained in cell migration assays. The pertussis-toxin
sensitivity of pro-u-PA-mediated cell shape change and cytoskeleton
reorganization, like chemotaxis, suggests that the u-PAR signaling
pathway may require G proteins.
Wounding experiments.
Using two different assays, chemotaxis and chemokinesis, we have shown
that pro-u-PA stimulates cell shape change, cytoskeleton reorganization, and cell migration through binding to u-PAR. An in
vitro wound-healing assay was used to confirm these results.
Single-cross wounds were made in confluent monolayers of serum-starved
RSMC grown on glass coverslips and cells were stimulated with 1 pmol/L
peptide D containing the rat u-PAR minimal chemotactic epitope. Peptide
D increased the number of migrating cells by approximately 50%
compared with control conditions (Fig 6A).
However, medium containing 1% FCS, which was used as positive control, gave a more than 100% increment. RSMC migrating from the edge of the
wound exhibited the same change in morphology and cytoskeleton organization described above in chemokinesis assays, like formation of
an actin ring with some actin filaments located radially to the ring
(Fig 6B), or motile morphology with cytoskeleton organized into an
actin semi-ring and membrane ruffles located at the leading edge of
cells (Fig 6C).
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| Fig 6.
Effect of peptide D on RSMC migration into a wound.
Single-cross wounds were made in confluent monolayers grown on glass
coverslip. After a wash with PBS, monolayers were then allowed to
recover for 24 hours in the absence or in the presence of 1 pmol/L
peptide D. Peptide D contains the minimum chemotactic epitope of rat
u-PAR, corresponding to the human peptide 1. One percent FCS was used
as a positive control. Quantification of migration was performed by
taking photographs at low magnification and counting cells that had
migrated into the cell-free space (A). Two examples of actin
cytoskeleton organization in migrating cells, visualized using
rhodamine-conjugated phalloidin, are shown in (B) and (C).
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|
We conclude, therefore, that pro-u-PA can activate chemotaxis,
chemokinesis, and wound-healing in vitro through the same mechanism(s) that includes a modification of u-PAR. However, the effect in the
wound-healing assay is only minor.
Pro-u-PA induces membrane relocation of integrins, u-PAR, and c-Src.
We next examined the effects of pro-u-PA on the subcellular
localization of u-PAR, VNR v3, 1 integrin subunit, and c-Src, using immunofluorescence microscopy.
In control cells, u-PAR, like VNR and integrin 1 subunit, was widely
distributed over the surface of the cell in contact with the substratum
(Fig 7A, B, and C). In pro-u-PA-stimulated RSMC exhibiting motile morphology, u-PAR redistributed to the leading
edge of the cell (Fig 7E). Similarly, VNR v3 and 1 subunit
were redistributed to the leading edge of migrating cells (Fig 7F and
G). Whereas in nonstimulated cells c-Src was distributed in the
cytoplasm (Fig 7D), pro-u-PA caused a c-Src redistribution to plasma
membrane. In motile RSMC, c-Src was mainly located at the leading edge
of cells (Fig 7H).
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| Fig 7.
Relocations of u-PAR, VNR, 1, and c-Src proteins to
membranes at the leading edge of migrating RSMC in response to
pro-u-PA. Subconfluent (50% to 70%) cultures were incubated in the
absence (A, B, C, and D) or in the presence of 1 nmol/L pro-u-PA for 30 minutes (E, F, G, and H). The cells were then fixed and stained with
antibodies against u-PAR (A and E), VNR (B and F), 1 (C and G), or
c-Src (D and H).
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In conclusion, these data show that pro-u-PA induces relocation of
u-PAR, integrins, and c-Src to the leading edge of motile RSMC. The
physiological relevance of this finding is supported by the observation
that, unlike Src+ 3T3/u-PAR fibroblasts,
Src/ 3T3/u-PAR fibroblasts do not
respond to ATF by cell shape changes and cytoskeletal reorganization
(Fig 8).
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| Fig 8.
Src is required for u-PA-dependent cell shape changes.
Mouse 3T3 cells from Src/ and wild-type mice were
transfected with a human u-PAR cDNA. u-PAR-positive cells (determined
by direct binding and immunofluorescence assays) were then supplemented
with 10 nmol/L ATF (the amino terminal fragment of human u-PA) and
incubated for 0 or 30 minutes at 37°C; at the end of the
incubation, the cells were washed, stained with phalloidin (A, C, E, G,
I, and K) or with antivinculin antibodies (B, D, F, H, J, and L), and
viewed and photographed under the immunofluorescence microscope. From
(A) to (F): Src+ cells incubated in the absence (A and B) or in the
presence of 10 nmol/L ATF for 30 minutes (C, D, E, and F). From (G) to
(L): Src/ cells incubated in the absence (G and H) or
in the presence of 10 nmol/L ATF for 30 minutes (I, J, K, and L).
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Cooperation of u-PAR and vitronectin receptor in pro-u-PA-induced
cell migration.
Polyclonal antibodies against domain 1 of rat u-PAR inhibit pro-u-PA
binding (S. Rabbani, data not shown). RSMC were first incubated with
either anti-u-PAR or control antibodies (10 µg/mL) and then subjected
to chemotaxis assay. Treatment with anti-u-PAR but not unspecific
control antibodies inhibited the response to pro-u-PA
(Fig 9). In addition, we found that
pro-u-PA-induced RSMC migration could also be blocked with LM 609, a
monoclonal antibody against VNR v3 (Fig 9). None of these
treatments altered the fMLP-dependent chemotaxis. Control monoclonal or
polyclonal antibodies had, respectively, no or slight inhibitory effect
on RSMC migration (Fig 9).
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| Fig 9.
Effect of antibodies anti-u-PAR and anti-VNR on RSMC
chemotaxis in response to pro-u-PA and fMLP. Cells were detached from
support and pretreated for 1 hour at 4°C in serum-free medium
without or with antibodies against u-PAR (10 µg/mL) or with
antibodies anti-VNR (LM 609) (0.5 µg/mL) or with unspecific
isotype-matched control antibodies (10 µg/mL). The cells were then
subjected to chemotaxis assay and migrated towards medium alone ( ),
10 nmol/L pro-u-PA ( ), or 107 mol/L fMLP ( ).
Antibodies at the same concentrations as in the pretreatment were added
in both chambers of the Boyden apparatus and were present during the
whole assay. Random cell migration of RSMC towards medium alone without
any chemoattractants or antibodies was considered to be 100%
migration. Results are the mean mean ± SD (n = 3).
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These results suggest that the relocation of u-PAR and integrins might
have also functional implication and that both u-PAR and vitronectin
receptor functions are involved in u-PAR-mediated RSMC migration.
| |
DISCUSSION |
Chemokines are proteins controlling leukocyte
migration.31,34 However, many cell types are required to
migrate and hence may use similar mechanisms as leukocytes. The
u-PA/u-PAR system, which has been shown to be essential in the
migration not only of T lymphocytes and macrophages but also of many
tumor cells, appears to control migration of many cell types. Indeed,
mice lacking u-PA are extremely sensitive to bacterial
infections,35,36 and metastasis in nude mice xenograft
models can be prevented by blocking the activity of u-PA or its
interaction with u-PAR.37-41
In this report, we show that in rat smooth muscle cells pro-u-PA can
induce chemotaxis, chemokinesis, and wound healing in vitro, along with
cytoskeletal and adhesion plaques rearrangements and rapid
relocalization of u-PAR, integrins, and c-Src to the leading edge of
the migrating cells. At least the migration and the cytoskeletal
rearrangement are blocked by pertussis toxin, implying that a
heterotrimeric G protein-coupled signal transduction mechanism is at
the basis of these effects. Addition of pro-u-PA can be substituted by
the exogenous supply of u-PAR fragments or of synthetic peptides that
contain the u-PAR chemotactic epitope and that have been shown to mimic
u-PA-induced chemotaxis in myelomonocytic cells.5 Thus,
the basic mechanisms involved are the same in RSMC, myelomonocytic
cells, and other cells. For all of these considerations, the real
inducer of migration is the membrane molecule, u-PAR. Because u-PAR has
neither transmembrane nor intracytoplasmic domain, it needs to contact
a transmembrane-signal transducer (the adaptor).5 Thus,
u-PAR is a cell-surface chemokine that becomes activated upon pro-u-PA
binding. The term chemokine, although improper for the lack of a
structural basis, is also justified by the inhibitory activity exerted
by BTP, ie, by the possible involvement of heterotrimeric G
proteins in u-PAR-dependent signaling.
The initial mechanism appears again to be the exposure of a specific
u-PAR epitope as previously demonstrated in nonadherent myeloid cells,
because pro-u-PA can be substituted by soluble u-PAR fragments or
synthetic peptides.5 That pro-u-PA induces a dose-dependent
chemotactic response in RSMC through a conformational modification of
u-PAR is shown by two sets of data. First, anti-u-PAR antibodies can
block the chemotactic effect of u-PA and, second, cleaved su-PAR as
well as peptides 1 and D can substitute for pro-u-PA and induce cell
migration, in agreement with previous data.4,5 This report
also shows that the same mechanism applies to different migration
models, chemotaxis, chemokinesis, and, to a lesser extent, wound
assays. Indeed, not only u-PAR dependent chemotaxis, but also the
cytoskeletal changes are induced by u-PAR fragments or peptides and are
blocked by BPT. This suggests that a u-PAR signaling pathway is
mediated by a Gi-coupled receptor. This is the case for the
fMLP-induced chemotaxis, which is mediated by a chemoattractant
receptor coupled to G proteins.31,32 However, we have found
no direct evidence of cross-talk between fMLP and u-PAR signaling. It
is also well known that G proteins play a key role in the regulation of
the actin cytoskeleton through intermediates signaling molecules of the
rho subfamily.42,43 In particular, membrane ruffles appear
to be regulated by the rac GTP-binding protein, whereas rho stimulates
actin stress fibers and focal adhesion formation.43-45
Clusters of integrins bound to proteins of the extracellular matrix
form adhesion plaques linked to intracellular proteins and constitute
the site of attachment of actin stress fibers.46,47 c-Src,
normally localized to endosomal membranes, is redistributed to focal
adhesions upon cell activation, where it regulates cell adhesion.48-52 Molecular interactions of u-PAR with
integrins and tyrosine kinases of the Src family has been reported and
functional cooperation has been suggested.4,9,53-55 We
demonstrate here that c-Src, v3, and 1 integrins, as well as
u-PAR, are redistributed to the leading edge upon pro-u-PA-induced
migration of RSMC.
Moreover, antibodies to v3, as well to u-PAR, inhibit
pro-u-PA-dependent chemotaxis, suggesting a functional cooperation between these molecules in promoting migration. Indeed, in SMC, adhesion depends on 1 integrins, whereas migration depends on v3.56,57 It is now well established that u-PAR is
able to provide cell adhesion by binding directly to
vitronectin.3,58 However, u-PAR also interacts with
integrins.9,53-55 Integrins may play different roles in
adhesion and migration,56,59-61 and u-PAR interaction with
integrins appears to modulate their functions.9,55 Important questions need to be answered to understand u-PAR-regulated cell migration. In particular, it is not known whether u-PAR affects chemotaxis and cell adhesion through consecutive, parallel, or connected pathways.
The presence of c-Src at the leading edge of RSMC demonstrates its
activation upon u-PAR-signaling. Involvement of c-Src in the signaling
pathway of u-PAR is supported by our finding that 3T3/u-PAR fibroblasts
from Src/ mice show an altered response to
ATF challenge. Compared with wild-type, 3T3/u-PAR Src
/ fibroblasts do not reorganize their actin
cytoskeleton or show shape changes.
Finally, the question still remains on whether cleavage of u-PAR in
vivo is required for signaling. Although many of the effects of u-PA
can be obtained with catalytically inactive derivatives (such as
pro-u-PA or ATF), nevertheless, the position of the chemotactic epitope
coincides with a u-PA-sensitive site of u-PAR.5 Indeed, at
physiological concentrations, u-PA cleaves u-PAR at residue 83 very
efficiently (50% in 30 minutes).8 Moreover, the linker region between domain D1 and D2 may not only be cleaved by u-PA, but
also by chymotrypsin, plasmin, and other proteolytic enzymes. It is
therefore possible that cleavage of u-PAR, even though required for
activity, may also occur after conformational change induced by
catalytically inactive u-PA derivatives, because it can be performed by
other proteases produced by the cell system used. Further studies will
be required to solve this problem.
| |
ACKNOWLEDGMENT |
The authors thank Drs M. Bertulli, M.G. Pizza, M. Nielsen, K.B. Kaplan,
and J. Henkin for providing cells and reagents. We are very grateful to
Dr P.C. Marchisio for helpful advice and comments. We also give our
friendly thanks to Drs M. Mazzotti, G. Cecchini, L. Spinardi, and T. Teesalu for technical support and stimulating discussion.
| |
FOOTNOTES |
Submitted October 1, 1998; accepted March 22, 1999.
Supported by grants from the Italian Association for Cancer research
(AIRC), The Italian Ministry of Education (MURST), the Istituto
Superiore di Sanità (ISS), and the EU (Biomed Grant No.
BMH4-CT96-0017).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Francesco Blasi, MD, Molecular Genetics,
DIBIT, H. S. Raffaele, via Olgettina, 58, 20132 Milano, Italy; e-mail:
blasi.francesco{at}hsr.it.
| |
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TOP
ABSTRACT
INTRODUCTION