From the Departments of Oncology and
§ Pharmacology at the Hadassah-Hebrew University Hospital,
Jerusalem 91120, Israel, the ¶ Department of Hematology, Medical
Center, Tel Aviv 64239, Israel, and the
Tumor Immunology
Program, German Cancer Research Center,
D-69120 Heidelberg, Germany
Received for publication, August 3, 2000, and in revised form, January 25, 2001
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ABSTRACT |
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The first prototype of the protease
activated receptor (PAR) family, the thrombin receptor PAR1, plays a
central role both in the malignant invasion process of breast carcinoma
metastasis and in the physiological process of placental implantation.
The molecular mechanism underlying PAR1 involvement in tumor invasion and metastasis, however, is poorly defined. Here we show that PAR1
increases the invasive properties of tumor cells primarily by increased
adhesion to extracellular matrix components. This preferential adhesion
is accompanied by the cytoskeletal reorganization of F-actin toward
migration-favoring morphology as detected by phalloidin staining.
Activation of PAR1 increased the phosphorylation of focal adhesion
kinase and paxillin, and the induced formation of focal contact
complexes. PAR1 activation affected integrin cell-surface distribution
without altering their level of expression. The specific recruitment of
The ability of tumor cells to invade beyond controlled hemostatic
boundaries and re-emerge from blood vessels to establish new metastatic
colonies continuous to present a major obstacle in cancer cure. It is
well known that in tumor invasion and metastasis, the pericellular
proteolytic systems, consisting of proteases and their specific cell
surface receptors, are tightly regulated to modulate cellular functions
and degrade selective matrix barriers (1, 2). We have previously
demonstrated that the proteolytically activated receptor 1 (PAR1,1 thrombin receptor)
plays a central role in the malignant and physiological invasion
processes of both breast carcinoma metastasis and placental
implantation (3). At the molecular level, tumor invasion is mediated
via the combined interactions of the host cell signaling machinery and
the regulation of the stromal extracellular matrix (ECM). Extensive
proteolysis in the tumor microenvironment is also responsible for the
activation of several enzymatic precursors, like plasminogen,
pro-matrix metalloproteinase, and prothrombin (4-6). In addition, the
extravascular deposition of fibrin within the tumor microenvironment is
well established (7), pointing to the significant role of the
coagulation proteins in tumor progression. Indeed, the tissue factor
(TF), a protease receptor that plays a central role in hemostasis, has
also been implicated in angiogenesis and tumor cell metastasis by means
of intracellular events mediated by its cytoplasmic tail and by the
perivascular extracellular proteolysis (8-10). These features are
shared by other cellular receptors involved in the proteolytic
modification of the tumor environment. Among these is the receptor for
the serine protease urokinase, which, when bound to its cell surface
receptor (uPAR), converts plasminogen to plasmin; plasmin, in turn, is
known to effectively degrade various matrix glycoproteins (1, 11). It
has been shown also that uPAR serves as an adhesion receptor for
vitronectin (12) and that the vitronectin receptor
Since solid tumors are in close contact with ECM components, malignant
cell invasion into the surrounding tissues is facilitated by mutual
interactions that convey essential signaling cues to the cells (19,
20). These cell-ECM interactions are mediated by integrins, a family of
adhesion receptors that mediate the attachment of the cell to both
structural and matrix-immobilized proteins to promote cell survival,
proliferation, and migration (21, 22). It is widely recognized that
integrins perform a significant function in cellular invasion and
metastasis (23-26). Non-ligated integrins are generally distributed
diffusely over the cell surface with no apparent linkage to the actin
cytoskeleton. However, ECM-bound integrins frequently cluster into
specialized structures termed focal adhesion complexes (FACs), thus
providing a convergence site for multiple signaling components (26, 27) and also physically linking the receptors to the actin filaments (28-30). The known signaling events that follow receptor clustering include tyrosine phosphorylation of proteins like focal adhesion kinase
(FAK) and paxillin (31), as well as the recruitment of other FAC
components like vinculin, talin, tensin, and p130 Cas (32-36). A
growing number of studies indicate that signals driven by integrins act
in concert with signals initiated by the G-protein-coupled receptors
and with receptors for tyrosine kinase to promote the pathological
tumor cell invasion process, on the one hand, and physiological
activities like angiogenesis and wound healing (37, 38) on the other.
The combined signals involved with the activation of focal adhesion
proteins indicate that the cooperation between the signaling pathway
takes place most likely within these FAC structures.
In the present work, we have studied the molecular mechanism of PAR1
involvement in tumor cell invasion. We show here that PAR1 modulates
the invasive phenotype of melanoma cell lines, inducing the otherwise
non-invasive cells to migrate effectively through Matrigel barriers.
This process is accompanied by the increased adherence of the cells to
various matrix components, actin stress fiber formation, and
adhesion-triggered signaling, with no alteration of the cell surface
integrin levels. We demonstrate now, for the first time, that PAR1
mediates these functions via selective cross-talk with the
Cells--
SB-2 non-invasive human melanoma and A375-SM
"super-metastatic" human melanoma cells (kindly provided by J. Fidler and M. Bar-Eli, Department of Cell Biology, University of Texas,
M. D. Anderson Cancer Center, Houston, TX) were grown in 10%
FCS-DMEM supplemented with 50 units/ml penicillin and streptomycin
(Life Technologies, Inc.) and maintained in a humidified incubator with 8% CO2 at 37 °C. The PAR1 stable transfectants, clone
13 and clone Mix L, were grown under the same conditions; for long term
maintenance, these were supplemented also with 200 µg/ml G418
antibiotics. MCF-7 cells were maintained as previously described
(3).
Cell Transfection--
Cells were grown to 30-40% confluence
and then transfected with 0.5-2 µ g/ml plasmid DNA in Fugene 6 transfection reagent (Roche Molecular Biochemicals) according to the
manufacturer's instructions. After 10 days of selection, stable,
transfected clones were established in medium containing 400 µg/ml
G418. Antibiotic-resistant cell colonies were transferred to separate
culture dishes and were grown in 200 µg/ml G418 medium. Forty-eight
hours after transfection, transiently transfected cells were collected
and tested by immunoprecipitation analyses (see below).
Preparation of Truncated PAR1--
Using polymerase chain
reaction, we constructed a PAR-1 mutant protein truncated in its
cytoplasmic tail after the amino acid Leu-369. As a template, we used
PAR-1 cDNA in the pCDNA 3 vector. For amplification, we used
a T7 sense primer and the reverse primer GGTCTAGAAAACTATAGGGGGTCGATGCACGAGCT containing a STOP codon and an
XbaI site. The amplified DNA fragment was subcloned using
the polymerase chain reaction-blunt technique (Invitrogen) and
confirmed by DNA sequencing. The insert was released from the vector by XbaI digestion and cloned into plasmid pCDNA3. To
confirm the functional integrity of the DNA constructs, wild type and
mutant cDNAs were transiently expressed in 293 cells that were
subsequently stained with a PAR-1-specific antibody (WEDE15,
Immunotech, Cedex, France).
Western Blot Analysis--
Cells were solubilized in lysis
buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and
protease inhibitors (5 µg/ml aprotinin, 1 µM
phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin) at 4 °C for
30 min. The cell lysates were subjected to centrifugation at
10,000 × g at 4 °C for 20 min. The supernatants
were saved and their protein contents were measured; 50 µg of the
lysates were loaded onto 10% SDS-polyacrylamide gels. After the
proteins were separated, they were transferred to an Immobilon-P
membrane (Millipore). Membranes were blocked and probed with 1 µg/ml
amounts of the appropriate antibodies as follows: anti-PAR1 thrombin
receptor mAb, clone II aR-A (Biodesign Int.); anti-paxillin monoclonal
antibody (mAb), clone 349 (Transduction Laboratories, Lexington KY);
anti-human focal adhesion kinase, rabbit polyclonal IgG (Upstate
Biotechnology Inc., Lake Placid, NY); anti-phosphotyrosine mAb, clone
4G10 (Upstate Biotechnology Inc.); anti-vinculin mAb (Transduction
Laboratories). The antibodies were suspended in 1% BSA in 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.5%
Tween 20. After washes with 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.5% Tween 20, the blots were incubated with
secondary antibodies conjugated to horseradish-peroxidase.
Immunoreactive bands were detected by the enhanced chemiluminescence
(ECL) reagent using Luminol and p-cumaric acid (Sigma).
Immunoprecipitation--
Cells were treated for 30-60 min with
thrombin at a concentration of 1 NIH unit/ml of serum-free DMEM medium
(0.5% BSA), and then lysed as described above. We used 400 µg of
total protein for immunoprecipitation of
Matrigel Invasion Assay--
We used blind-well chemotaxis
chambers with 13-mm diameter filters. Polyvinylpyrrolidone-free
polycarbonate filters with 8-µm pores (Costar Scientific Co.,
Cambridge, MA) were coated with basement membrane Matrigel (25 µg/filter) as described previously (39). Briefly, the Matrigel was
diluted to the desired final concentration with cold distilled water,
applied to the filters, dried under a hood, and reconstituted with
serum-free medium. In the upper compartment of the Boyden chamber, we
placed 2-3 × 105 cells suspended in DMEM containing
0.1% bovine serum albumin. As a chemo-attractant, into the lower
compartment of the Boyden chamber, we put 3T3 fibroblast conditioned
medium. Assays were carried out in 5% CO2 at 37 °C.
After 2 h of incubation, we observed that more than 90% of the
cells were attached to the filter. At this time, the cells on the upper
surface of the filter were removed by wiping with a cotton swab. The
filters were fixed in DifQuick system (American Scientific Products)
and stained with hematoxylin and eosin. Cells from various areas of the
lower surface were counted. Each assay was performed in triplicate. For
chemotaxis studies (a control of Matrigel invasion), the filters were
coated with collagen type IV alone (5 mg/filter) to promote cell
adhesion. Cells were added to the upper chamber and conditioned medium
to the lower compartment.
Adhesion Assay--
The medium of cells grown in 10% FCS was
replaced by DMEM with 0.5% BSA, and the cells were detached from
the plate by treating with 0.05% trypsin in a solution of 0.02% EDTA
in 0.01 M sodium phosphate, pH 7.4 (Biological Industries,
Beit Ha'emek, Israel). After washing, 0.5 × 106
cells/ml cells were re-suspended in a serum-free DMEM medium (as above)
and laid on 13-mm culture dishes pre-coated with either 100 µg/ml
fibronectin or Th-1, a thrombin-derived RGD (arginine-glycine-aspartic acid) peptide. After a 45-min incubation period to allow cell adhesion,
the excess cells were washed away. The adhered cells were fixed to the
plates with 4% formaldehyde in PBS, pH 7.4, for at least 2 h.
After fixation, the plates were washed in 1% boric acid solution and
the cells were stained with 1% methylene blue reagent (Sigma) in 1%
boric acid for 30 min. After extensive washing with tap water, the
methylene stain was eluted by the addition of 500 µl of 1 M HCl. The intensity of the color staining was measured by
color spectrometry at a wavelength of 620 nm.
Immunofluorescence--
Cells were plated on glass coverslips in
16-mm culture dishes; after the cells had grown to subconfluence, they
were washed with PBS, permeabilized in 0.5% Triton X-100-containing
3.5% paraformaldehyde/PBS solution on ice for 2 min, and finally fixed
with 3.5% paraformaldehyde/PBS for 20 min. Reactions with the
appropriate antibodies were performed in room temperature for 60 min,
after which the cells were washed extensively in PBS. The antibodies
included the following: anti- Flow Cytometry Analysis--
The medium of cells grown in 10%
FCS-DMEM was replaced by serum-free DMEM containing 0.5% BSA. Thrombin
at a concentration of 1 IU/ml was added to the plates that were
activated by incubation for 60 min. The plates were washed with PBS,
and the cells were detached from the plates by treatment with 0.05%
trypsin in a solution of 0.02% EDTA in 0.1 M sodium
phosphate at pH 7.4 (Biological Industries). After being washed twice
in PBS, the cells were re-suspended in 200 µl of PBS and the
appropriate antibodies were added to a concentration of 10 µg/ml.
These reactions, performed at room temperature for 60 min, were
followed by extensive washing in PBS. A 1-h incubation with a secondary
antibody goat-anti-mouse IgG (Jackson Immunoresearch Laboratories)
conjugated with FITC and diluted 1:500 was carried out in the dark. The
treated cells were washed extensively, re-suspended in 100 µl of PBS,
and analyzed by FACS.
Altering the Expression of PAR1 Affected Tumor Cell
Invasiveness--
In previous work (3), we showed that there is a
direct correlation between PAR1 expression and the metastatic potential of primary tumor biopsies and tumor cell lines, as reflected by their
in vitro potential to invade through a Matrigel
barrier.2 In a physiological
invading model system of placenta trophoblast implantation, we have
also shown that PAR1 is part of the invasive program of trophoblast, as
evaluated by their villi extension and matrix metalloproteinase
synthesis.3 Here, to clarify
how high levels of PAR1 may confer invasiveness, we transfected a
non-invasive melanoma cell line (SB-2 cells) with PAR1 cDNA and
compared the properties of the transfected cells to those of the highly
invasive melanoma cell line A375SM. We used PAR1 cDNA under the
control of the cytomegalovirus viral promoter in the pCDNA3
expression vector. We selected several stable clones that expressed
high levels of PAR1, as evaluated by Western blot analysis (Fig.
1a) and Northern blot analysis (data not shown). The selected clones were then tested for their ability to invade through Matrigel-coated filters. Indeed, clones expressing high levels of PAR1 had an increased ability to invade the
Matrigel layer, as compared with control clones transfected with empty
vectors or SB-2 cells that had not been transfected at all (Fig.
1b). In addition, we observed that, whereas highly invasive
A375SM cells invaded Matrigel coated membranes more efficiently than
did non-metastatic cells (Fig. 1b, SB-2),
activating the A375SM cells with PAR1 increased their ability to invade
Matrigel to an even higher level (Fig. 1b, activ.
A375SM). In addition, the invasiveness of PAR1-transfected cells
was further increased when they were either activated by thrombin, as
shown in two separate PAR1-transfected clones (Fig. 1b,
clones 13 and Mix L), or
when they were treated with the thrombin-receptor-activating peptide (TRAP) that corresponds to PAR1 internal ligand SFFLRN (data not shown).
Circulating tumor cells can invade into a new metatastic site only if
they can adhere to the basement membrane. We analyzed the adhesion
properties of cells suspended in a serum-free medium and then incubated
for 60 min on plates coated with either fibronectin, a major component
of the ECM, or with Th-1, an 11-amino acid peptide, corresponding to
the thrombin RGD motif (40). Highly invasive A375 SM melanoma cells
adhered strongly to both Th-1 and fibronectin; however, under the same
conditions, the non-invasive SB-2 cells failed to adhere. We observed a
marked increase in the adherence to both of these matrices of
PAR1-transfected SB-2 cells (Fig. 2,
a and b). The level of adherence of these PAR1
transfectants was directly correlated both with their level of PAR1
expression and with their ability to invade the Matrigel barrier. To
assure that this increase in their adherence was actually caused by the presence of PAR1, we asked if reducing the expression of PAR1 in
malignant cells would reduce the adhesion properties of these cells. To
do this, we evaluated the effect of transfection by PAR1 antisense DNA
on the adhesion properties of the invasive A375SM cells. We used a
462-base pair oligonucleotide fragment corresponding to the 5' region
of PAR1 that included part of the near promoter sequence and the coding
region for the internal ligand. We cloned this DNA segment into
pCDNA3 mammalian expression vector in an antisense orientation,
selecting for stable clones expressing the plasmid bearing the PAR1
antisense DNA as compared with cells transfected by empty vectors or
non-transfected control cells. Northern blot analysis (Fig.
2d) indicated that, whereas empty vector transfection (Fig.
2d, B) had no significant effect on PAR1
expression (Fig. 2d, A), clones AS -3 (Fig.
2d, C) and AS-4 (Fig. 2d,
D), which were transfected by the PAR1 antisense DNA, did
exhibit reduced PAR1 expression. When we analyzed clones AS-3 and AS-4
for their adhesion properties, we found that the cell adherence
properties to fibronectin (Fig. 2c) and to Th-1 (data not
shown) of both of these clones were significantly lower than those of
the A375 SM parental cells.
The organization of the cytoskeleton is critically influenced by
adhesion interactions. To explore the effect of PAR1 activation on
cytoskeletal reorganization, we plated PAR1-transfected cells (clone
13) and control non-transfected cells (SB-2 cells) on glass coverslips
and then treated them with TRAP for various periods of time (Fig.
2e). After activation by TRAP, the cells were permeabilized, fixed, and stained with FITC-labeled phalloidin to detect filamentous actin (F-actin). Cytoskeletal reorganization was observed as early as
15 min after activation by TRAP (Fig. 2e). Thirty to 60 min after PAR1 activation, we observed a transition in the PAR1
transfectants from elongated spindle-like shapes to spreading,
jellyfish-like structures. Ninety minutes to 2 h after activation,
the cells became rounder and we observed the appearance of a ringlike
bundle of actin filaments at the base of the cells (typical of
migrating cells). These changes occurred more rapidly and were more
dramatic in PAR1-overexpressing cells than they did in the
non-transfected control cells. Altogether, these data show that the
adhesive properties of tumor cells were affected by changes in PAR1 expression.
PAR1 Activation Induced Signaling and Led to Establishment of Focal
Contacts--
Integrin activation typically leads to the assembly of
focal adhesion contacts; this takes place by phosphorylation on
tyrosine leading to the recruitment of various signaling and structural molecules. FAK and paxillin are the most common signaling components of
FAC that are phosphorylated upon integrin activation. To analyze the
phosphorylation levels of FAK and paxillin in PAR1-transfected cells,
we immunoprecipitated these proteins from cell lysates of either
thrombin-activated or non-activated control cells. The immunoprecipitated proteins were blotted onto a nylon membrane and
probed with anti-phosphotyrosine mAb to detect their phosphorylation levels. FAK and paxillin proteins from parental, non-invasive SB-2
cells exhibited low levels of phosphorylation (Fig.
3a). On the other hand, FAK
and paxillin from PAR1 transfectant cells exhibited increased
phosphorylation to high levels similar to those observed in the
metastatic line A375 SM (Fig. 3a). By immunofluorescent analysis using anti-phosphotyrosine mAb followed by a Cy-3
fluorescence-labeled secondary antibody, we detected FAC formation as
soon as 15 min following PAR1 activation, reaching a maximum after 60 min (Fig. 3b). Together, these data demonstrate that,
following activation, overexpressed PAR1 is capable of initiating high
levels of integrin signaling.
We characterized FAC assembly by immunofluorescent staining, using mAb
anti-vinculin, anti-paxillin, and FAK polyclonal antibodies. Following
activation, we observed some FAC formation in all of the cell types
that we examined; however, the complexes in the activated PAR1
transfectants were far more distinct and larger than those that we
observed in non-activated cells (Fig. 3c), in cells that had
been transfected by empty vectors, or in cells that had not been
transfected (data not shown). In the activated PAR1 clones, vinculin
staining of FACs was intense; however, we also observed clear, although
less intense, vinculin staining of FACs in activated non-transfected
and mock-transfected cells. This may be explained by the fact that,
rather than having a signaling function, vinculin functions mainly as a
structural protein, and it has been reported to play a role in the
maintenance of the FAC and adherence junctions (41). It has also been
reported that both vinculin and talin are phosphorylated even under
basal conditions (42).
The
We then asked which of these integrins would respond to PAR1 by
participating in the induction of the cytoskeleton signaling events. We
examined the cell surface integrins by immunofluorescent visualization
before and after PAR1 activation. Although
To substantiate the cooperative cross-talk and the recruitment of
In this study, we have shown that changes in the expression of
PAR1 in a cell affect its invasive capabilities. These changes come
about through the specific recruitment of the
It is well established that there is cooperation between integrins and
other cell surface receptors, and further, that this cooperation may
operate at several levels (47-50). Physical interactions between the
extracellular domains of integrins and non-integrin receptors may
result in mutual or sequential activation. For example, the results of
several parallel studies demonstrate a physical link of uPAR with the
integrin Thrombin contains a cryptic RGD epitope that can potentially be
recognized by integrins (40). The transient binding of thrombin to its
receptor, prior to receptor cleavage, may serve as an RGD-exposing event that enables integrin binding during PAR1 activation. Thus, it
seems that, at least theoretically, cooperation between PAR1 and the
vitronectin receptor The adhesion of tumor cells to the basement membrane is an essential
step in the process of invasion. In contrast to the passive, non-active
nature of non-malignant cells, the dynamic nature of tumor cell
adherence to the underlying ECM precedes matrix degradation and
migration. The interactions of the cell matrix involve the activation
of integrins as well as the initiation, through focal adhesion
structures, of signaling cascades that lead to cytoskeletal reorganization. This has been shown to be the case during tumor progression where TF supports cell adhesion, migration, and spreading through the action of the cytoplasmic portion of the TF molecule (10).
The interaction of uPA with its cell surface receptor uPAR is necessary
for vitronectin-dependent human pancreatic carcinoma (FG) cell adhesion and migration mediated via the
v
5 to focal contact sites, but not of
v
3 or
5
1, was observed by immunofluorescent
microscopy. PAR1 overexpressing cells showed selective reciprocal
co-precipitation with
v
5 and paxillin but
not with
v
3 that remained evenly distributed under these conditions. This co-immunoprecipitation failed
to occur in cells containing the truncated form of PAR1 that lacked the
entire cytoplasmic portion of the receptor. Thus, the PAR1 cytoplasmic
tail is essential for conveying the cross-talk and recruiting the
v
5 integrin. While PAR1 overexpressing
cells were invasive in vitro, as reflected by their
migration through a Matrigel barrier, invasion was further enhanced by
ligand activation of PAR1. Moreover, the application of
anti-
v
5 antibodies specifically attenuated this PAR1 induced invasion. We propose that the activation of PAR1 may lead to a novel cooperation with the
v
5 integrin that supports tumor
cell invasion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
3 not only supports the migration of
tumor cells on various matrix-proteins but also binds matrix
metalloproteinase-2, thus presenting an immobilized enzyme with
improved matrix-collagen degradation properties at the invasive front
(13). Additional cell surface protease receptors include the PAR
family, which are proteolytically cleaved G-coupled receptors of seven
transmembrane-spanning domains. Unlike most cellular growth factor
receptors, the PAR family members do not require the traditional
ligand-receptor complex formation for activation. Instead, they are
activated by a specific cleavage within their extracellular N-terminal
portion to unmask a new amino acid terminus, which serves then as an
internal ligand for activation (14-18). Until now, four members of the
PAR family have been identified and of these, three (PAR1, PAR3, and
PAR4) have been established collectively as "thrombin receptors,"
possibly serving as a redundant receptor system for the coagulation
protease cellular response (14).
v
5 integrin to confer FAC formation, distinct signaling events, and cytoskeletal reorganization. Combined, these processes promote tumor cell invasion.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
3,
v
5,
paxillin, FAK, or both paxillin and FAK. All the antibodies were used
at a concentration of 10 µg/ml. After overnight incubation, Protein
A-Sepharose beads (Amersham Pharmacia Biotech) were added to the
suspension (50 µl) that was subsequently rotated at 4 °C for
1 h. Elution of the reactive proteins was made by re-suspending
the beads in protein 2× sample buffer (63 mM Tris-HCl, pH
6.8, 20% glycerol, 20% SDS, 0.01% bromphenol blue, 5%
-mercaptoethanol, 0.02 M dithiothreitol) and boiling for
5 min. The supernatant was loaded on a 10% SDS-polyacrylamide gel
followed by the same procedure as in Western blotting.
v
3 mAb clone
LM609, anti-
v
5 clone P1F6, and
anti-
5
1 clone JBS5, (all from Chemicon
Int.). After the 60-min incubation with the primary antibodies,
followed by extensive washes in PBS, an additional 60-min incubation
was carried out in the dark with secondary antibodies, goat-anti-rabbit
or goat-anti-mouse IgG each conjugated with Cy-3 (Jackson
Immunoresearch Laboratories) diluted 1:700. Labeling of filamentous
actin by 1 µ g/ml FITC-conjugated phalloidin (Sigma) was performed
similarly. The labeled cells were visualized and photographed by
fluorescent confocal microscopy (MRC-1024 confocal imaging system,
Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Transfection by PAR1 DNA confers invasive
properties on non-metatastic melanoma cells. The expression of
PAR1 and cellular invasive properties were measured in SB-2
non-metatastic human melanoma cells, in A375 SM highly metatastic human
melanoma cells, in stable PAR1 transfectants clone 13 and clone Mix L,
and in mock-transfectant SB-2 cells transfected by empty vectors.
Stable PAR1 clones were screened for PAR1 expression (a)
using anti-PAR1 thrombin receptor mAbs on a Western blot of 50 µg of
lysates total protein. b, the invasive capabilities of the
selected clones were determined by the Matrigel invasion assay. Cell
lines marked "activ." were activated by 1 unit/ml
thrombin for 1 h before being used in the invasion assay. The data
presented here are the averages of data from at least three replicate
experiments.
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Fig. 2.
Altering PAR1 expression affected cell
adhesion and actin fiber re-organization. Stable
PAR1-transfected clones and PAR1 antisense selected clones were
analyzed for their adhesive properties to substrates coated with
fibronectin (a and c) or Th-1 RGD peptide
(b). Cell adhesion was measured by Methylene blue staining
of formaldehyde-fixated cells. The eluted stain was detected by
spectrophotometry using a = 620 nm filter. The cells tested
(a and b) were the same as those described in
Fig. 1. In addition, we show that in highly metastatic human melanoma
A375SM cells stably transfected by PAR1 antisense cDNA (AS clone 4 and AS clone 3), reduced adhesion was observed (c) as
compared with A375SM cells that were not transfected or that were
transfected by an empty vector. These clones exhibited low PAR1 levels
as shown by Northern blot analysis (d) of RNA samples from
A375SM (A), A375SM cells transfected with vector only
(B), AS clone 3 (C), and AS clone 4 (D). The data
presented here are the averages of data from at least three replicate
experiments. L32 is a ribosomal RNA that we have used as a housekeeping
control gene for these experiments. e, SB-2 cells and PAR1
transfectant clone13 were subjected to actin staining by
FITC-phalloidin after PAR1 activation by TRAP. Note that the PAR1
transfectants exhibited a more rapid change in actin fiber
re-organization and cellular morphology than did the naive SB-2
cells.
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Fig. 3.
Activation of PAR1-induced phosphorylation of
FAK and paxillin and their recruitment to FACs. a, the
tyrosine-phosphorylated levels of immunoprecipitated FAK
(upper section) and paxillin (lower
section) were measured by anti-phosphotyrosine mAb (4G10,
UBI) in SB-2 naïve cells, in A375SM metastatic cells, and in
the stable PAR1 transfectant clone 13. Note that FAK was observed to
co-precipitate with paxillin (lower section).
b, immunofluorescent staining of phosphotyrosine in
PAR1-transfected clone 13 was performed by specific incubations with
mAbs (4G10) at 0, 15, and 60 min. Detection was made by Cy3-labeled
goat anti-mouse IgG, using confocal microscopy. Following activation by
100 µM TRAP, the FACs were observed to be enriched with
phosphorylated proteins with a peak at 60 min. c,
immunofluorescent staining with anti-vinculin, anti-FAK, and
anti-paxillin in the stable PAR1 transfectant clone 13 activated with
100 µM TRAP for 1 h or not (NA). When the
cells were stained with anti-FAK and anti-paxillin, distinct FAC
staining was observed only in the activated cells. When the cells were
stained with anti-vinculin, FACs were detectable at a low level in the
non-activated (NA); this level was increased following
receptor activation by TRAP.
v
5 Integrin Is Specifically
Recruited to FACs in Response to PAR1 Activation without Alteration of
the Cell-surface Integrin Level--
Having established that signaling
was induced by PAR1 ligand activation, that also led to establishment
of focal contacts, we asked whether altering the adhesive phenotype
would be accompanied by de novo integrin expression. Here we
used flow cytometry analysis carried out with a battery of
anti-integrin antibodies directed against the
v
3,
5
1, and
v
5 integrins. Following activation, we
observed no significant differences between the cell-surface integrin
profiles of the PAR1 transfectants and of the parental cells (Fig.
4a). Nevertheless, the fact
that PAR1 activation did not alter integrin expression does not exclude
the possibility of affinity modulation of the integrins in an
inside-out manner.
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Fig. 4.
PAR1 activation did not alter the levels of
integrin expression but did induce
v
5
recruitment to FACs. a, integrin expression levels were
measured by flow cytometry in SB-2 naive cells and in PAR1 clone13,
each activated with thrombin at a concentration of 1 unit/ml. The
levels of
5
1 (A and
B),
v
5 (C and
D),
v
3 (E and
F), and the integrin
v chain (G
and H) were detected by incubating the cells with the
appropriate specific mAb, followed by incubation with FITC-labeled
anti-mouse IgG. The white peaks correspond to the
expression levels of control secondary isotype-specific mouse IgG
antibodies. Note that no significant changes were observed in the
levels of any of the integrins examined. b, the distribution
of integrins was detected by immunofluorescent staining
(upper panel). Cy-3 red fluorescence was
visualized by confocal microscopy. The lower
panel shows the same cells as in the upper
panel, but visualized by phase-contrast microscopy.
Non-activated (NA) PAR1 clone 13, stained by
anti-
v
5 mAbs revealed a diffused pattern
(A). After activation by TRAP, the integrin
5
1 was detected in a random perinuclear
position (B); the integrin
v
3
was randomly scattered over the cell membrane (C); the
integrin
v
5 was localized to distinct
"spikes" of focal adhesion contacts (D).
5
1 and
v
3
(Fig. 4b, B and C) are distributed
diffusely over the cell surface both before and after PAR1 activation,
after PAR1 activation we found that
v
5
was localized to distinct sites of FACs (Fig. 4b,
D). However, we only detected
v
5 within the focal contacts in the
activated PAR1-overexpressing cells (Fig. 4b, E)
but not in the PAR1-transfected cells prior to PAR1 activation cells
(Fig. 4b, A), nor in the mock transfectants or in the
parental non-transfected cells (data not shown). Based on our results, we hypothesized that the
v
5 integrin
would respond to signals conveyed by the activated PAR1. It seemed that
v
5 was specifically recruited to the
focal adhesion contacts, where it played a major role in the
reorganization of the cytoskeleton. Our hypothesis was confirmed by the
results of the following reciprocal co-precipitation experiments. We
analyzed the co-precipitation of paxillin with either
v
5 or with
v
3 in cell lysates of naive SB-2 cells
and of the stable PAR1 transfectant clone 13 that was either
thrombin-activated or not. The blotted membranes were incubated with
the mAb of the anti-
5 subunit. As we expected, in the
parental cells, we found only basal levels of paxillin precipitation
with either of the two integrins (Fig. 5,
a and b). In the PAR1 clone 13, we found that
paxillin co-precipitated with
v
3 at a low
level, and that level was not increased significantly by thrombin
activation of PAR1 (Fig. 5b). However, in PAR1 clone 13, we
did find a high level of co-precipitation of
v
5 with paxillin, and that level was
significantly increased by thrombin activation of PAR1. We also
analyzed co-immunoprecipitation of paxillin and FAK from cell lysates
of SB-2 cells and from the stable PAR1 transfectant clone 13, both of
which were thrombin-activated or not. Again, the blotted membranes were
incubated with the mAb of anti-
5 subunit. As we
expected, there was no co-immunoprecipitation in the parental cells,
whether or not they were activated; however, there was a significant
level of co-precipitation of
5 subunit in the PAR1 clone
13, that was greatly increased upon activation by thrombin (Fig.
5c). When, instead of anti-
v
5,
we used anti-
v
3 to probe the same blot,
we found no evidence of the
3 subunit (data not shown).
These results indicate that
v
5 and the
typical signaling molecules, paxillin and FAK, were tightly associated
and thus co-precipitated. We found that this kind of association was
likely to occur within focal adhesions rather than in other cellular compartments, as demonstrated by the induced assembly and signaling of
FACs. Furthermore, this association appeared to be labile and seemed to
occur in response to PAR1 activation, indicating that the
v
5 integrin was present within newly
assembled FACs. Our data do not exclude the possibility that
v
3 is present on the cell surface. Our
data do suggest, however, that the
v
3
integrin probably does not cooperate with PAR1-specific signaling to
induce the cellular responses described here.
View larger version (30K):
[in a new window]
Fig. 5.
Activation of full-length PAR1 but not of
truncated PAR1 led to the co-precipitation of
v
5
with paxillin and FAK and reduced invasiveness in the presence of
anti-
v
5
antibodies. Co-precipitation of paxillin with
v
5 (a) or with
v
3 (b) was measured in cells
lysates of naive SB-2 cells (C) and of a stable PAR1
transfectant clone 13 that was either thrombin-activated (A)
or not (B). c, paxillin and FAK were
immunoprecipitated from cell lysates of SB-2 cells that had been
thrombin-activated (A) or not (B) and from the
stable PAR1 transfectant clone 13 that was either thrombin-activated
(C) or not (D). The blotted membrane was
incubated with anti-
5 subunit mAb. d,
non-invasive MCF7 cells, naturally expressing very low levels of PAR1,
were transfected with cDNA expression vectors coding either for
PAR1 or for truncated PAR1. In lysates of the PAR1 transfectants,
co-precipitation of FAK and paxillin was detected after PAR1 activation
by thrombin (B) but not without activation (A).
In lysates of truncated PAR1 transfectants, no co-precipitation of FAK
with paxillin with
v
5 was observed
regardless of whether the cells were thrombin-activated (D)
or not (C). Tyrosine-phosphorylated paxillin co-precipitated
with
v
5 (lower
panel) in PAR1-transfected cells (A); the level
of this precipitation increased following thrombin activation of PAR1
(B); in truncated PAR1-transfected cells, only minor levels
of phosphorylated paxillin were detected with (D) or without
(C) thrombin activation. e, PAR1 expression
levels were measured by FACS analysis using anti PAR1 mAb (WEDE15,
Immunotech, Cedex, France), followed by incubation with FITC-labeled
anti-mouse IgG. The analysis was carried out on MCF7 cells following
transfection by DNA coding for the full-length PAR1 (A and
C) or for the truncated PAR1 (B and
D). Their levels were compared with non-transfected cells
(first peak, B and A). This
is true also when PAR1 or truncated PAR1 expression was measured
relative to empty vector-transfected cells (first
peak, C and D, respectively).
f, A375SM cells were activated with 1 unit/ml thrombin
(B-D) or not (A). The activated cells were then
treated with 20 µ g/ml either
anti-
v
5-blocking mAbs (C) or
nonspecific IgG (D). The treated cells were then subjected
to a Matrigel invasion assay. The data presented here are the averages
of data from at least three replicate experiments. One hundred percent
invasion by the metatastic cells corresponded to 48 ± 3 invading
cells as compared with 17 ± 1.5 invading cells by SB-2
non-metastatic cells (data not shown).
v
5 following PAR1 activation, we used a
truncated form of PAR1, consisting of the extracellular and seven
transmembrane domains but lacking the entire cytoplasmic portion of the
receptor, and compared its function to the intact receptor. We carried
out these experiments in MCF7 cells, which are non-invasive cells that
naturally express very low levels of PAR1. These parental cells were
transiently transfected with cDNA coding for either the intact PAR1
or truncated PAR1; 48 h after transfection, the transient
transfectants were either activated or not and then subjected to
immunoprecipitation analysis as described above. In lysates of the MCF7
PAR1 transient transfectants, we detected high levels of
co-precipitation of FAK, paxillin, and
v
5
after PAR1 activation by thrombin but not without activation. In
lysates of truncated PAR1 transfectants, we observed no
co-precipitation of FAK with paxillin, and
v
5 regardless of whether the cells were
thrombin activated or not (Fig. 5d, upper
panel). In the PAR1 transfectants, tyrosine phosphorylated
paxillin co-precipitated with
v
5 and the
level of this precipitation increased following thrombin activation of
PAR1; in truncated PAR1-transfected cells, we detected only minor
levels of phosphorylated paxillin with or without thrombin activation
(Fig. 5d, lower panel). As seen by the
results of the flow cytometry (FACS) analysis (Fig. 5e), the
failure to immunoprecipitate FAK by
anti-
v
5 in PAR1-truncated transfectants
did not result from the inability to express properly on the cell
surface. Transfectants of either PAR1 (Fig. 5e,
A) or truncated PAR1 (Fig. 5e, B)
showed cell surface expression of the truncated receptor protein as
determined by flow cytometry (FACS) analysis (Fig. 5e).
Using anti-PAR1 WEDE15 mAbs, we found similar levels of expression in
both the PAR1 (Fig. 5e, A, second peak) and the truncated PAR1 (Fig. 5e,
B, second peak) transfectants, relative to the expression levels in naive cells (Fig. 5e,
A and B, first peaks). We
obtained similar results when we compared the levels of expression of
the transfectants (Fig. 5e, C and D,
second peaks) to those of empty
vector-transfected cells (Fig. 5e, C and
D, first peaks). The results of these
experiments strongly support the notion that following activation the
PAR1 cytoplasmic tail recruits and activates the
v
5 integrin. That the PAR1 molecule participates in other signaling activities is supported by our finding
that, although Shc was phosphorylated in the presence of the
full-length activated PAR1, this was not the case in the presence of
the activated truncated PAR1 (data not shown). We conclude that,
although, like the full-length PAR1, the truncated PAR1 is expressed
and assembled on the cell surface, unlike the full-length PAR1, the
truncated PAR1 is incapable of carrying out PAR1 signaling. To
ascertain that in fact the
v
5 integrin may cooperate with PAR1 during tumor invasion, we asked whether neutralizing the activity of the
v
5
integrin would affect the invasive properties of the highly invasive
A375 SM cells. A375 SM cells were activated or not with 1 unit/ml
thrombin; the activated cells were then either not treated at all or
treated with anti-
v
5-blocking mAbs
antibodies (clone P1F6) or with nonspecific IgG. The cells were then
subjected to a Matrigel invasion assay. As one can see, PAR1 activation
further induced the invasive properties of the cells by 60%
while the addition of anti-
v
5 antibodies attenuated this induction; the addition of a non-related IgG led to no
significant effect (Fig. 5f).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
5 integrin, through cytoskeletal
reorganization, and through distinct signaling at FACs. The fact that
PAR1 alters the invasive properties of tumor cells reinforces our
initial observations that PAR1 expression correlates with the invasive
potential of both the malignant invasion processes of breast carcinoma
(3) and the physiological invasion processes of placenta trophoblast
implantation (3)3 emphasizing the central role of PAR1
during invasion. The on-going process of invasion by cells is
characterized by extensive proteolytic remodeling, in part by serine
proteases, of the tumor microenvironment (1, 2). Serine
proteases also serve as ligands for several cell-surface receptors,
among which is uPAR, which, through binding uPA, efficiently converts
plasminogen to plasmin (11). TF is another protease cell surface
receptor that binds factor VII, thereby initiating the coagulation
pathway during perivascular hemostasis (43). It is interesting that, in
addition to their involvement in hemostasis, these receptors are also
implicated as central players in tumor progression and metastasis
(8-10). The extracellular proteolytic activation of factor VII by TF
is also responsible for the generation of thrombin from circulating plasma prothrombin (44, 45). In fact, thrombin production is probably
the direct result of disseminated overactivation of the coagulation
system, a widely described pathology among cancer patients (46). The
abundant localization of either soluble or immobilized thrombin in the
vicinity of the tumor milieu enables the excessive activation of PAR1
and the subsequent cellular response during invasion. In fact, although
the repertoire of signaling pathways is limited, it can be harnessed to
integrate the information obtained from multiple receptors for a wide
range of cellular responses. Here we have presented evidence showing
that the overexpression of PAR1 increases the invasiveness of melanoma
cells (Figs. 1b and 5f) and is also associated
with an increase in the adhesion properties of the cell (Fig. 2,
a-c). The activation of PAR1 resulted in the
phosphorylation of the focal adhesion proteins FAK and paxillin that
are typical of integrin signaling (Fig. 3a). Although the
levels of the cell surface integrins were not affected (Fig. 4a), there was notable change in their mode of distribution.
In particular, in response to PAR1 activation, the integrins
v
3 and
5
1
remained diffusely distributed but, in contrast, we found that the
integrin
v
5 was uniquely recruited to
the sites of focal adhesion contacts (Fig. 4b).
3
1 in keratinocytes (51), with the
2 integrin Mac-1 in leukocytes (52), and with
v
5 in breast cancer (53). The interaction
of uPAR with integrin
1 has also been shown to involve
the functional cooperation of integrins with cell surface receptors via
caveolin in a manner dependent on the conformational state of the
receptors (54, 55). Alternatively, the activation state of integrins
can be modified in an "inside-out" manner. Internal signals
conveyed by intersecting cascades react with the cytoplasmic domain of
the integrin
subunit and thereby increase the affinity to their
ligands of the extracellular portion of the integrins. Activation of
G-protein-coupled receptor initiates a signal transmission through the
C-terminal cytoplasmic domain of the receptor that leads to the
assembly of adaptor proteins, non-receptor tyrosine kinases, and small
G-proteins. Signals that are transduced in forking pathways, like
Ras-Raf-mitogen-activated protein kinase and phosphatidylinositol
3-kinase-Akt/PKB, are also largely shared by integrin and thymidine
kinase receptors. In endothelial cells (56), astrocyte cell rounding
(57), and nuerite retraction (58), cytoskeletal responses to thrombin are known to involve the activation of the Ras-dependent
ERK1/2 mitogen-activated protein kinase pathway during gap formation. These responses have been found to be Rho-dependent and
require Rho-specific guanine nucleotide exchange factors (57, 59). More
specifically, the Rho-dependent pathway controls barrier maintenance and stress fiber formation while Rac induction and myosin
light chain kinase activation are both implicated in barrier dysfunction (56). Together, these facts imply that integrin-related signaling can be intersected by PAR1 signaling at the intracellular level.
v
5 may occur at the
extracellular level. Using a truncated PAR1 construct that lacks the
entire cytoplasmic tail domain, we demonstrated here that it was the cytoplasmic portion of the PAR1 molecule that was responsible for
cooperation with the
v
5 integrin. We
found that the truncated PAR1 was unable to transmit intracellular
signals, and therefore, was unable to recruit
v
5 and to initiate the typical
integrin-associated signals. The other vitronectin receptor,
v
3, has been widely implicated in both
angiogenesis and melanoma cell invasion and metastasis (60, 61).
Nevertheless, many tumor cells that lack
v
3 can still readily metastasize (62). In
cells that express both
v
5 and
v
3,
v
3 is
constitutively capable of inducing cell spreading and migration, while
v
5 cannot promote cell spreading and
migration without an additional exogenous soluble factor (60, 63).
Based on our results, we propose that during the invasion process
v
5 is the dominant integrin involved in
PAR1-ECM signaling interactions. This is consistent with previous
suggestions (64) that
v
5 has a role in
mediating human keratinocyte locomotion. Filardo et al. (36)
also showed that
v
5, as the sole integrin expressed in melanoma cells, could promote cell spreading and migration
in cooperation with insulin-like growth factor signaling. It has also
been postulated that
v
5-mediated cell
migration is protein kinase C
-dependent (66). Whether,
as has been shown for endothelial cell migration (67), PAR1-mediated
association of
v
5 during tumor invasion
is under the regulation of protein kinase C
associated to TAP20
(theta-associated protein) remains to be determined.
v
5 integrin (65). The convergence point
of the PAR1 and the
v
5 signaling pathways
is not yet known and is currently under study in our laboratory.
Nevertheless, the data that we have presented here suggest that this
unique mode of cooperation specifically promotes the invasive
properties of tumor cells. We believe that the PAR1 and the
v
5 signaling pathways that we have
studied here may prove to be crucial for other PAR1 functions in
vascular biology and embryonic development.
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ACKNOWLEDGEMENTS |
---|
We thank Prof. Israel Vlodavsky for helpful discussions, Dr. Xiao-Ping (Merck Research Laboratory, West Point, PA) for providing us with the full-length PAR1, and F. R. Warshaw-Dadon for editorial revisions of the text.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Ministry of Health, the Ministry of Science and the Arts, the Joint German and Israeli Research Program, the Middle East Cancer Constridium, the Israel Cancer Association, and the Israel Science Foundation of Science and Humanities (to R. B.-S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom all correspondence should be addressed. Permanent address: Dept. of Oncology, Sharett Inst., Hadassah University Hospital, P.O. Box 12000, Jerusalem 91120, Israel. Tel.: 972-2-677-7563; Fax: 972-2-642-2794; E-mail: barshav@md2.huji.ac.il. Current address (during the academic year 2000-2001): Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-3971/3972; Fax: 617-432-3969; E-mail: rachel_barshavit@hms.harvard.edu.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M007027200
2 E. Pokroy, B. Uziely, S. Even-Ram Cohen, M. Maoz, I. Cohen, S. Ochayon, R. Reich, J. Pe'er, O. Drize, M. Lotem, and R. Bar-Shavit, submitted for publication.
3 S. Even-Ram Cohen, S. Grisaru-Granovsky, M. Maoz, S. Zaidoun, Y.-J. Yin, and R. Bar-Shavit, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PAR, protease activated receptor; FAC, focal adhesion complex; FAK, focal adhesion kinase; ECM, extracellular matrix; TRAP, thrombin receptor-activating peptide; AS, antisense; FACS, fluorescence-activated cell sorting; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; mAb, monoclonal antibodies; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TF, tissue factor; uPA, urokinase; uPAR, urokinase receptor.
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