The Urokinase Receptor Takes Control of Cell Migration by Recruiting Integrins and FPR1 on the Cell Surface
et al. (2014) The Urokinase Receptor Takes Control of Cell Migration by Recruiting Integrins and
FPR1 on the Cell Surface. PLoS ONE 9(1): e86352. doi:10.1371/journal.pone.0086352
The Urokinase Receptor Takes Control of Cell Migration by Recruiting Integrins and FPR1 on the Cell Surface
Anna Gorrasi 0
Anna Li Santi 0
Giuseppina Amodio 0
Daniela Alfano 0
Paolo Remondelli 0
Nunzia Montuori 0
Pia Ragno 0
Neil A. Hotchin, University of Birmingham, United Kingdom
0 1 Department of Chemistry and Biology, University of Salerno , Salerno , Italy , 2 Department of Medicine and Surgery, University of Salerno , Salerno , Italy , 3 Department of Translational Medical Sciences, ''Federico II'' University , Naples , Italy
The receptor (uPAR) of the urokinase-type plasminogen activator (uPA) is crucial in cell migration since it concentrates uPA proteolytic activity at the cell surface, binds vitronectin and associates to integrins. uPAR cross-talk with receptors for the formylated peptide fMLF (fMLF-Rs) has been reported; however, cell-surface uPAR association to fMLF-Rs on the cell membrane has never been explored in detail. We now show that uPAR co-localizes at the cell-surface and coimmunoprecipitates with the high-affinity fMLF-R, FPR1, in uPAR-transfected HEK-293 (uPAR-293) cells. uPAR/b1 integrin and FPR1/b1 integrin co-localization was also observed. Serum or the WKYMVm peptide (W Pep), a FPR1 ligand, strongly increased all observed co-localizations in uPAR-293 cells, including FPR1/b1 integrin co-localization. By contrast, a low FPR1/ b1 integrin co-localization was observed in uPAR-negative vector-transfected HEK-293 (V-293) cells, that was not increased by serum or W Pep stimulations. The role of uPAR interactions in cell migration was then explored. Both uPAR-293 and V293 control cells efficiently migrated toward serum or purified EGF. However, cell treatments impairing uPAR interactions with fMLF-Rs or integrins, or inhibiting specific cell-signaling mediators abrogated uPAR-293 cell migration, without exerting any effect on V-293 control cells. Accordingly, uPAR depletion by a uPAR-targeting siRNA or uPAR blocking with an antiuPAR polyclonal antibody in cells constitutively expressing high uPAR levels totally impaired their migration toward serum. Altogether, these results suggest that both uPAR-positive and uPAR-negative cells are able to migrate toward serum; however, uPAR expression renders cell migration totally and irreversibly uPAR-dependent, since it is completely inhibited by uPAR blocking. We propose that uPAR takes control of cell migration by recruiting fMLF-Rs and b1 integrins, thus promoting their co-localization at the cell-surface and driving pro-migratory signaling pathways.
Funding: Associazione Italiana per la Ricerca sul Cancro(AIRC, IG 4714) University of Salerno,FARB 2011 (ORSA113852). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Current address: Institute of Genetics and Biophysics Adriano Buzzati-Traverso, Consiglio Nazionale delle Ricerche (CNR), Naples, Italy
To reach their final destination or their workplace, cells must
move through the extracellular matrix (ECM) and, sometimes, also
between each other. Cell migration is essential for many biologic
and pathologic processes and is the result of highly coordinated
events which involve cell polarization, actin-driven protrusion,
formation and turn-over of cell adhesions, localized ECM
Since many years, the receptor (uPAR) of the urokinase-type
plasminogen activator (uPA) serine-protease has been considered
crucial in cell migration processes since it concentrates uPA
proteolytic activity at the cell surface, thus allowing localized ECM
degradation . Indeed, uPAR is moderately expressed in various
tissues in the healthy organism, but its expression strongly
increases in organs undergoing extensive tissue remodeling. uPAR
expression is also increased in many pathologic conditions, in
particular in cancer, inflammation and infections .
uPAR is a heavily glycosylated protein formed by three
cysteinerich LY6-like domains (DI, DII, and DIII, from the external
Nterminus) connected by short linker regions. It is anchored to the
cell surface through the glycosyl-phosphatidylinositol (GPI) tail of
the C-terminal DIII. The three uPAR domains define a deep
cavity which accomodates uPA, leaving the whole external surface
available for other potential interactions . Indeed, uPAR acts
also as a high affinity receptor for vitronectin (VN), an ECM
component, particularly abundant in ECM associated to tumor
tissues . Both uPA and VN, which require full-length uPAR for
binding, are able to activate intracellular signaling pathways,
leading to cell proliferation, survival, adhesion and migration, in
spite of the absence of a transmembrane and a cytosolic region in
the uPAR molecule . Thus, cell surface molecules, able to
associate to uPAR and to connect uPAR to intracellular signaling
pathways, have been largely investigated. Integrins seem the most
probable candidates as uPAR signaling partners . In fact,
uPAR-integrin association has been shown by
co-immunolocalization, co-immunoprecipitation, FRET and by in vitro binding
assays between purified uPAR and a5b1 integrin . Despite the
controversy surrounding whether uPAR and integrins interact
physically, a large body of evidence shows that uPAR signaling
requires integrins as co-receptors. uPAR, beside using integrins,
also regulates their activity, with different extents in different cell
The linker region between the N-terminal DI and DII uPAR
domains is extremely sensitive to various proteases, including uPA;
the proteolytic cleavage removes DI and generates a shorter uPAR
form (DIIDIII-uPAR), unable to bind both uPA and VN and to
associate to integrins .
Both full-length and cleaved uPAR can be released by the cell
surface in soluble forms. The soluble form of DIIDIII-uPAR
(sDIIDIII-uPAR) exposing the SRSRY sequence (aa 8892) at its
N-terminus, is unable to bind both uPA and VN, as its
cellmembrane counterpart, nevertheless it acquires a new important
activity; in fact, it is a ligand for the G-protein-coupled receptors
for the fMLF (fMet-Leu-Phe) peptide, a peptide of bacterial origin
Three fMLF receptors (fMLF-Rs) have been identified and
cloned: the high-affinity N-formyl-peptide receptor (FPR1) and its
homologue FPR-like 1 (FPR2) and FPR-like2 (FPR3) receptors.
FPR2 has a much lower affinity for fMLF, but it is efficiently
activated by several other molecules, including lipoxin A4, serum
amyloid A, HIV derived peptides. FPR3 shows a high homology
with the other two fMLF-Rs but does not bind fMLF and shares
few ligands with the other fMLF-Rs . Activation of fMLF-Rs
by their ligands induces cell migration. Thus, s-DIIDIII-uPAR is a
potent chemoattractant for cells expressing FPR1, FPR2 or FPR3
. The SRSRY sequence that, in the soluble uPAR form, is
unmasked only after the removal of DI, is instead exposed in the
cell-anchored receptor, as demonstrated by the observation that
an antibody directed against this specific uPAR region (residues
8495, uPAR8495) reacts with full-length cell-surface uPAR and
does not with the full-length soluble form (suPAR) . It is then
reasonable to hypothesize that also full-length cell-surface uPAR
could be able to interact with fMLF-Rs through the same sequence
of the cleaved suPAR. However, GPI-uPAR co-localization or
association to fMLF-Rs on the cell membrane has never been
explored in detail.
A high number of cell-surface molecules interacting with uPAR
have been reported, including most of integrin families,
growthfactor receptors as the receptors for the epidermal growth factor
(EGFR) and for the platelet derived growth factor receptor
(PDGFR)-beta, and several other molecules . Thus, uPAR
seems to interact with a multitude of different molecules with
disparate functions, using some of them as signaling partners and/
or regulating their activity.
Indeed, it would be reasonable that only few and specific
molecules could really interact with uPAR, thus regulating its
relations with the other neighbour cell-surface molecules. In fact,
we recently showed that uPAR is able to regulate the activity of the
receptor for the stromal derived factor 1 (SDF1) chemokine,
CXCR4, in a fMLF-R- and integrin-dependent manner ; this
finding is greatly in agreement with results by Furlan et al., which
showed that the cleaved form of soluble uPAR can modulate the
ability of monocytes to migrate toward MCP-1 and RANTES
chemokines by binding FPR2 and decreasing chemokine-induced
integrin-dependent rapid cell adhesion . These evidences
would suggest that integrins and fMLF-Rs could represent possible
functional intermediators between uPAR and the other
On this basis, we aimed firstly to assess uPAR/fMLF-R
localization and association at the cell surface and to evaluate
the relationship of this potential complex with integrins. Then, we
intended to explore the role of GPI-uPAR interactions with
fMLFRs and integrins in regulating uPAR cross-talk with other
cellsurface receptors, by evaluating the effects of such interactions on
cell migration to serum, which contains various and different
chemoattractants able to bind various and different cellular
Materials and Methods
The rabbit anti-uPAR polyclonal 399 antibody was from
American Diagnostica (Greenwich, CT); the anti-uPAR
monoclonal antibodies R2 and R4 were kindly provided by Dr. G.
Hoyer-Hansen (Finsen Laboratory, Copenhagen, Denmark).
Mouse monoclonal antibodies against b1 integrins and FPR2,
and rabbit polyclonal antibodies against b1 integrins, FPR1 and
FPR3 were from Santa Cruz Biotechnology (Santa Cruz, CA).
Rabbit anti-b-actin, mouse anti-tubulin antibodies, mouse
antiFLAG M2, uPAR-targeting and control siRNAs, the protease
inhibitor cocktail and the inhibitors of PI-3K and ERKs were from
SIGMA (St. Louis, MO). Inhibitors of the Rac-specific GEF
(guanine nucleotide exchange factor) Trio and Tiam1 and of the
Rho-associated kinase (ROCK) were from Calbiochem
(Darmstadt, Germany). The rabbit antibody recognizing the SRSRY
sequence of uPAR has been developed by PRIMM (Milan, Italy)
by using the uPAR8495 peptide (corresponding to uPAR residues
8495, which include the SRSRY sequence) assembled onto a
branching lysine core (15). EGFP- tagged uPAR, inserted in the
pEGFP-N1 vector, was a kind gift of Dr. N. Sidenius (IFOM,
Milan, Italy), and fMLF-R cDNAs were kindly provided by Dr. M.
Perretti (William Harvey Research Institute, London, United
Kingdom). Cy3 or Alexa 488 conjugated secondary antibodies
were purchased from Jackson Immunoresearch (West Grove, PA)
and the Prolong AntiFade kit and Lipofectamine 2000 from
Invitrogen (Grand Island, NY). Horseradish
peroxidase-conjugated anti-mouse and anti-rabbit IgG were from Bio-Rad (Hercules,
CA). ECL detection kit was from Amersham International
(Amersham, England) and Polyvinylidene fluoride (PVDF) filters
from Millipore (Windsor, MA). Collagen was purchased from
Collaborative Research (Bedford, MA) and the chemotaxis
polyvinylpyrrolidone-free (PVPF) filters from Whatman Int. (Kent,
UK). The W (WKYMVm) and the P-25
(AESTYHHLSLGYMYTLN)  peptides were synthesized by PRIMM (Milan,
uPAR-293 cells are HEK-293 previously transfected with the
entire coding region of uPAR cDNA cloned in the EcoRI site of
the pcDNA3 vector . These cells were transiently transfected
with FPR1, FPR2 or FPR3 cDNAs inserted in a pcDNA3 vector
for co-immunoprecipitation assays or with FPR1 cDNA for
Immunofluorescence analysis. EGFP-tagged uPAR, cloned in
the pEGFP-N1 vector , or the empty vector, were transiently
transfected in HEK-293 cells, which are uPAR negative cells ,
for Immunofluorescence analysis. In both cases, 2.56106 cells,
plated in 60 mm dishes, were transfected with 9 mg of DNA and
Figure 1. FPR1 co-localizes with uPAR on the surface of uPAR-expressing HEK-293 cells. HEK-293 cells stably transfected with uPAR cDNA
(uPAR-293 cells) were seeded on glass coverslips and transiently transfected with FPR1 cDNA. After 24 h, cells were incubated for further 24 h in
culture medium (DMEM) containing 10% serum (Basal) or in DMEM without serum (2FBS). Prior to fixation, some serum-starved samples were
stimulated for 1 h at 37uC, 5% CO2, with 10% FBS in DMEM (+FBS) or with 5 nM WKYMVm peptide (+W Pep). All samples were then fixed, incubated
with the anti-uPAR monoclonal R4 antibody and the anti-FPR1 polyclonal antibody (A) or with the anti-uPAR monoclonal R4 antibody and the rabbit
anti-b1 polyclonal antibody (B), stained with Cy3 or Alexa 488 conjugated secondary antibodies, and analyzed by confocal microscopy. The insets
show a 46magnification for the indicated region in each merge image. C: The degree of co-localization of the fluorescent signals was quantified on a
minimum of 50 different cells by using the LSM 510 software. The number of co-localizing pixels was normalized to the total pixels of each
fluorophore. Thus, the number of yellow pixels, corresponding to co-localizing uPAR-FPR1, has been normalized to green (uPAR) or red (FPR1) pixels
shown in A and reported in 1st and 2nd set of columns, respectively. The number of pixels corresponding to co-localizing uPAR-b1 integrin has been
normalized to green (uPAR) or red (b1 Integrin) pixels shown in B and reported in 3rd and 4th set of columns, respectively. (*) p#0.05, as determined
by the Students t test.
22.5 ml of LipofectAMINE 2000 (Invitrogen) in serum-free
DMEM for 5 h at 37uC (5% CO2). Cells where then lysed after
48 h for co-immunoprecipitation experiments or treated for the
To knock-down uPAR, 26105 PC3 cells were seeded in 35 mm
plates and transfected with 100 nM uPAR-targeting or control
siRNAs in antibiotic-free medium using Oligofectamine,
according to the manufacturers instructions. Cells were incubated for
48 h at 37uC, 5% CO2, and then washed and lysed in 1%
TX100 or loaded in Boyden chamber for migration assays.
Immunofluorescence analysis and co-localization
uPAR-293 cells, grown on glass coverslips and transiently
transfected with FPR1 cDNA, or HEK-293 cells, grown on glass
coverslips and transiently transfected with EGFP-uPAR or the
pEGFP-N1 empty vector, or with the 36FLAG-Frizzled-4(fz4)
receptor cDNA as co-localization control, were washed and fixed
10 min in 4% paraformaldehyde. Then, FPR1-transfected
uPAR293 cells were incubated for 1 h at room temperature with 4 mg/
ml of R4 anti-uPAR monoclonal antibody and 2 mg/ml of
FPR1specific rabbit polyclonal antibody, or with 4 mg/ml of R4 and
4 mg/ml of b1 integrin-specific rabbit polyclonal antibody;
HEK293 cells transfected with EGFP-uPAR or the pEGFP-N1 vector
were incubated for 1 h at room temperature with 2 mg/ml of
FPR1-specific rabbit polyclonal antibody and 4 mg/ml of anti-b1
integrin mouse monoclonal antibody; HEK-293 cells transfected
with 36FLAG-fz4 were incubated for 1 h at room temperature
with 0.5 mg/ml of mouse anti-FLAG M2 and 2 mg/ml of
FPR1specific rabbit polyclonal antibody or with mouse anti-FLAG M2
and 4 mg/ml of anti-b1 integrin rabbit antibody. Then, uPAR-293
cells or 36FLAG-fz4-HEK-293 cells were further incubated with
Cy3 or Alexa 488 conjugated secondary antibodies, and
EGFPuPAR-293 cells or their negative control with Alexa 594 and Cy5
conjugated secondary antibodies.
Coverslips were mounted with the Prolong AntiFade kit. Images
were collected as specified using a laser scanning confocal
microscope (LSM 510; Carl Zeiss MicroImaging) equipped with
plan Apo 636, NA 1.4 oil immersion objective lens. The percent
of co-localization of the fluorescence signals was calculated
multiplying by 100 the Manders co-localizing coefficients, as
measured by the LSM 510 4.0 SP2 software. In detail, 8 bit images
are acquired and subjected to intensity threshold in order to
eliminate background intensities. The threshold was set to 100 for
every image analyzed and the percent of co-localization was
quantified on a minimum of 50 different cells. The percent of
colocalization is relative to the single z-plane stack shown in the
immunofluorescence panels; no significant differences of
colocalization were found in the range of 0.5 mm above or below
the shown in-focus z-plane.
Immunoprecipitation was performed as previously described
. The cells were washed twice with microtubule stabilization
buffer (0.1 M Pipes, pH 6.9, 2 M glycerol, 1 mM EGTA, 1 mM
magnesium acetate) and then extracted in 0.2% Triton X-100 in
the presence of protease inhibitors. The insoluble residue, enriched
in cytoskeleton-associated proteins, was solubilized in RIPA buffer
(150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% deoxycholate,
0.1% SDS, 1% Triton-X 100, and protease inhibitors) and
preincubated with nonimmune serum and 10% protein
ASepharose for 2 h at 4uC. After centrifugation, the protein content
of the supernatants was measured by a colorimetric assay
(BIORAD) and 0.5 mg of protein was incubated for 2 h at 4uC
with 30 mg/ml of rabbit polyclonal antibodies against FPR1 or
FPR3 or of a mouse monoclonal antibody directed to FPR2, or
with 30 mg/ml of nonimmune rabbit or mouse immunoglobulins,
as controls. After 30 min of incubation with 10% protein
ASepharose at room temperature, the immunoprecipitates were
washed, subjected to 10% SDS-PAGE, and analyzed by Western
blot using 1 mg/ml of the R2 anti-uPAR monoclonal antibody for
FPR1 and FPR3 immunoprecipitates, or 2 mg/ml of the 399
rabbit polyclonal antibody for FPR2 immunoprecipitates. Finally,
washed filters were incubated with horseradish
peroxidaseconjugated secondary antibodies and detected by ECL.
Cell migration assay
Cell migration assays were performed in Boyden chambers
using 8 mm pore size PVPF polycarbonate filters coated with
50 mg/ml of collagen. 26105 transfected-HEK-293 cells or 16105
PC3 cells were loaded in the upper chamber in serum-free
medium; 10% FBS-DMEM or 100 ng/ml EGF were added in the
lower chamber as chemoattractants. Transfected-HEK 293 cells
and PC3 cells were allowed to migrate for 4 h and 2 h,
respectively, at 37uC, 5% CO2. Then, the cells on the lower
surface of the filter were fixed in ethanol, stained with
hematoxylin, and counted at 2006 magnification (10 random
fields/filter). When indicated, cells were preincubated with 5 mg/
ml of polyclonal antibodies directed to full-length uPAR or
uPAR8495 region, or with 50 mM of P-25 peptide for 1 h at room
temperature, or with 10 mM of inhibitor of Rho-signaling or
50 mM of inhibitor of Rac-signaling or 10 mM of ERK inhibitor or
20 mM of PI3K inhibitor for 30 min at 37uC, or with 5 nM of W
peptide for 1 h at 37uC, 5% CO2.
Differences between each group of values and its control group
were evaluated by the Students t test using PRISM software
(GraphPad, San Diego, CA). P#0.05 was considered statistically
uPAR and FPR1 co-localize at the surface of
uPARtransfected HEK-293 cells
We and others previously observed a cross-talk between
cellanchored uPAR and fMLF-Rs [18,2022]; however, their
association on the cell membrane has never been explored in
detail. Thus, we firstly investigated uPAR and fMLF-R
colocalization on the cell surface. To this end, we used
uPARnegative HEK-293 cells [5,1718], stably transfected with uPAR
cDNA  and here named uPAR-293. Since HEK-293 cells
mainly express FPR1 , we focused our studies on uPAR
association to this fMLF-R. uPAR-293 cells were transiently
transfected with the FPR1 cDNA, to reinforce FPR1 expression,
and analyzed by confocal microscopy with uPAR- and
FPR1specific antibodies; the analysis confirmed the expression of both
receptors on the cell surface and showed their co-localization
(Fig. 1A, top panel). Quantification of uPAR-FPR1
co-localization, obtained by normalizing the pixels corresponding to
colocalizing uPAR-FPR1 (yellow) to total uPAR pixels (green) or to
total FPR1 pixels (red), showed that, in basal conditions, about
44% of uPAR co-localized with FPR1 and about 41% of FPR1
colocalized with uPAR on the cell surface of transfected cells (Fig. 1C,
first and second set of columns).
To investigate whether external stimuli can increase such
uPAR/FPR1 co-localization, transfected cells were serum-starved
for 24 h and then incubated for 1 h with 10% serum or with a
fMLF-R ligand, the W Peptide (WKYMVm; W Pep) . In the
absence of serum, uPAR/FPR1 co-localization values were lower
than those in basal conditions, however, cell stimulation with
serum or with the W Pep, strongly and significantly increased
uPAR/FPR1 co-localization, up to 69% and 61% for uPAR and
up to 64% and 76% for FPR1 (Fig. 1A and 1C, first and second set
uPAR co-localization and association with b1 integrins has been
largely demonstrated in the past, as well as their strict
collaboration in uPAR cell-signaling , thus we investigated also uPAR/
b1 integrin co-localization in the same cells. Indeed, the results
showed that, in basal conditions, about 55% of uPAR co-localized
with b1 integrins and about 50% of b1 integrins co-localized with
uPAR on the cell surface of transfected cells (Fig. 1B and 1C, third
and fourth set of columns). Also in this case there was a significant
increase of co-localization after serum or W Pep stimulations, up
to 70% and 81% for uPAR and up to 70% and 71% for b1
These results suggest that uPAR and FPR1 co-localize on the
surface of HEK-293 cells to a similar extent as uPAR and b1
integrins; these co-localizations strongly increase after cell
stimulation with serum or with a FPR1 ligand, thus supporting the
possibility of reciprocal interactions among these three receptors.
uPAR recruits FPR1 and integrins at the cell surface
We then investigated whether uPAR can promote
integrinfMLF-R aggregation on the cell membrane. To this end, we used a
different approach for the fluorescence assay, analyzing the
localization of a fluorescent proteintagged uPAR, transiently
transfected in HEK-293 cells; FPR1 cDNA was not co-transfected.
Then, not only uPAR co-localization with endogenously expressed
b1 integrins and FPR1 was examined, but also b1 integrin/FPR1
co-localization. Also with this different approach uPAR/FPR1
and uPAR/b1 integrin co-localization was observed, with a strong
and significant increase after serum or W Pep cell-stimulation
(Fig. 2A and 2B, left panel, first four sets of columns), confirming
previous results (Fig. 1). Indeed, we also evaluated co-localization
with FPR1 and b1 integrins of a control molecule, Frizzled-4 (Fz4),
a G-protein-coupled receptor not involved in uPA-uPAR system;
co-localization values ranged between 2030% in both cases and
did not increase after serum or W Pep cell-stimulation (not shown).
Interestingly, about 77% of FPR1 co-localized with b1 integrins
in uPAR-293 cells and about 42% of b1 integrins co-localized with
FPR1; their co-localization significantly increased after cell
stimulation with serum or with the W Pep (Fig. 2A and 2B, last
two sets of columns). b1 integrin/FPR1 co-localization was finally
evaluated also in HEK-293 cells transiently transfected with the
empty GFP-vector, as a control. Endogenous expression of FPR1
was similar in both uPAR-transfected and vector-transfected
control cells, as assessed by Western blot analysis (Fig. 3A). Low
values of FPR1/b1 integrin co-localization (about 30%) were
observed in uPAR-negative HEK-293 cells; FPR1/b1 integrin
colocalization was not increased by serum or W Pep (Fig. 2C and
These results suggest that uPAR expression on the cell surface
promotes FPR1 co-localization with b1 integrins; FPR1/b1
integrin co-localization is strongly increased by serum or the
FPR1 ligand only in uPAR-expressing cells.
uPAR co-immunoprecipitates with fMLF-Rs
Then, biochemical assays were utilized to investigate whether
FPR1 associates to uPAR on the cell membrane. FPR1 cDNA was
transiently transfected in uPAR-293 cells or in HEK-293 cells
stably transfected with the empty vector (V-293 cells), as control.
Cells were lysed and immunoprecipitated with a FPR1-specific
antibody or with nonimmune immunoglobulins, as control;
Western blot analysis of immunoprecipitates with uPAR-specific
antibodies showed that uPAR co-immunoprecipitated with FPR1
(Fig. 3A). FPR1 immunoprecipitates were also analyzed by
Western blot with anti-b1 integrin antibodies, but, under these
experimental conditions, we cannot detect any specific band at the
correct Mr (130 kDa) (not shown).
Indeed, FPR1 transfection induced only a moderate increase in
FPR1 expression, as shown by Western blot analysis with specific
antibodies of transfected cells (Fig. 3A, right panel); thus, we also
performed co-immunoprecipitation experiments with uPAR-293
cells not transfected with FPR1, showing that uPAR
coimmunoprecipitates also with endogenous FPR1 (Fig. 3B).
To investigate whether uPAR associates also to the other
fMLFRs, uPAR-293 cells were transiently transfected with FPR2 and
FPR3 cDNAs, which are not or poorly expressed by HEK-293
cells , and the corresponding lysates were immunoprecipitated
with FPR2- and FPR3- specific antibodies. Western blot analysis
with uPAR-specific antibodies of the immunoprecipitates showed,
also in this case, a specific band corresponding to uPAR (Fig. 3C
Co-immunoprecipitation assays thus showed that uPAR
associates to all three fMLF-Rs. Since uPAR associates also to integrins
Figure 4. uPAR expression controls cell migration toward serum. uPAR-293 cells (A) or V-293 cells (B) were pre-incubated with nonimmune
immunoglobulins (Ig), anti-uPAR or anti-uPAR8495 polyclonal antibodies, plated in Boyden chambers and allowed to migrate toward 10% FBS.
Migrated cells were fixed, stained with hematoxylin, and counted (left panels). The values are the mean6SD of three experiments performed in
triplicate. (*) p#0.05, as determined by the Students t test. Results of migration assays are also expressed as percentage of cells migrated towards
serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (right panels). (*) p#0.05, as
determined by the Students t test.
, it is reasonable to hypothesize that uPAR could bridge both
molecules at the cell surface.
uPAR expression controls cell migration toward serum
uPAR expression is required for cell migration toward fMLF
and, conversely, fMLF receptors are required for cell migration
toward uPA, suggesting a functional interaction between these
receptors . However, cell-surface uPAR regulates also
migration toward other ligands, such as SDF1 and PDGF, and regulates
the activity of the EGFR ; the involvement of fMLF-Rs has
been investigated and demonstrated only in the SDF1-dependent
We hypothesized that uPAR could be able to regulate cell
migration independently of a specific chemoattractant, through a
mechanism involving fMLF-Rs and integrins, as shown in the
SDF1-dependent migration. To test our hypothesis we evaluated
the migration of uPAR-293 and V-293 control cells toward serum
(which could be considered a mixture of various
chemoattractants), blocking or not uPAR interactions at the cell surface.
Migration assays were performed on filters coated with collagen
(CG), which should not have any type of interaction with uPAR
and thus should not interfere with uPAR-dependent cell
uPAR-293 cells or V-293 cells were allowed to migrate toward
serum in the presence of non-immune immunoglobulins or of
polyclonal antibodies directed against the whole molecule of uPAR
or against the uPAR8495 region (residues 8495 of uPAR), the
latter corresponding to the region of the soluble form of cleaved
uPAR involved in the binding to fMLF-Rs [10,24]. Both
uPAR293 cells and V-293 control cells efficiently migrated toward
serum; anti-uPAR antibodies did not exert any effect on their basal
migration (in the absence of chemoattractant) (Fig. 4A and 4B, left
panels). However, anti-uPAR antibodies totally inhibited
seruminduced migration of uPAR-293 cells, without exerting any effect
on serum-induced migration of V-293 control cells (Fig. 4A and
These results suggest that even if HEK-293 cells are able to
migrate independently of uPAR expression, when they express
uPAR, their migration seems to become totally and irreversibly
uPAR-dependent, in fact it is completely inhibited by uPAR
fMLF receptors and b1 integrins are involved in uPAR
capability to control cell migration
Serum-induced uPAR-dependent cell migration was inhibited
by a polyclonal antibody directed against the whole uPAR
molecule which, presumably, blocks all uPAR interactions at the
cell-surface, and by an antibody recognizing the uPAR region
involved in the interaction with fMLF-Rs (Fig. 4). We then
investigated whether fMLF-Rs and/or integrins, which interact
with uPAR, are involved in uPAR capability to control cell
fMLF receptors can be desensitized by pre-treating cells with
their ligands before migration . We then performed migration
Figure 5. fMLF receptors and b1 integrins are involved in uPAR capability to control cell migration. uPAR-293 cells (A and C) or V-293
cells (B and D) were pre-incubated with diluent (-) or W Peptide (W Pep) (A and B), or with diluent (-) or P-25 peptide (C and D). Cells were then
plated in Boyden chambers and allowed to migrate toward 10% FBS. Migrated cells were fixed, stained with hematoxylin, and counted (left panels).
The values are the mean6SD of three experiments performed in triplicate. (*) p#0.05, as determined by the Students t test. Results of migration
assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration
in the absence of chemoattractants (right panels). (*) p#0.05, as determined by the Students t test.
assays with uPAR-293 cells and V-293 cells after pre-incubation
with or without W Pep, a ligand of fMLF-Rs. Desensitization of
fMLF-Rs did not affect basal migration of both uPAR-293 and
V293 cells (Fig. 5A and 5B, left panels) but totally impaired
uPAR293 cell migration toward serum without exerting any significant
effect on serum-induced migration of V-293 control cells (Fig. 5A
We then investigated whether uPAR capability to control cell
migration could depend also on its interaction with integrins. To
this end, migration assays were performed in the presence or in the
absence of the P-25 peptide, which has been shown to disrupt
uPAR interactions with b1 or b2 integrins . Treatment of
uPAR-293 cells with the P-25 peptide inhibited their migration
toward serum, whereas treatment of V-293 control cells with the
same peptide did not exert any effect (Fig. 5C and 5D). Also in this
case basal migration of both cell types was not affected (Fig. 5C
and 5D, left panels).
Altogether, these results suggest that, when expressed, uPAR
takes control of cell migration by interacting with b1 integrins and
uPAR-dependent cell migration is mediated by signaling
mediators not involved in uPAR-independent cell
We then investigated whether uPAR-dependent and
uPARindependent cell migrations toward serum were mediated by same
It has been previously shown that small GTPases as Rac1,
RhoA, Cdc42 and RhoB mediate uPA- induced cell migration
[6,25], we thus performed migration assays with uPAR-293 cells
and V-293 control cells in the presence or in the absence of
inhibitors of the Rac-specific GEF (guanine nucleotide exchange
factor) Trio and Tiam1 and of the Rho-associated kinase (ROCK).
We found Rac1 and Rho involvement in uPAR-controlled
migration, since inhibition of their downstream signaling pathways
significantly impaired migration toward serum of
uPAR-expressing cells (Fig. 6A). By contrast, both mediators were not involved
in serum-induced migration of uPAR-negative cells (Fig. 6B).
uPAR-dependent signaling pathways can also include PI3K and
lead to the activation of ERK MAPKs . Indeed, specific
inhibitors for PI3K and ERKs inhibited serum-induced migration
of both uPAR-293 cells and V-293 control cells, indicating that
these two mediators are used by both uPAR-expressing and
uPAR-negative cells for migration (Fig. 6C and 6D, respectively).
Figure 6. uPAR-dependent cell migration is mediated by signaling mediators not involved in uPAR-independent cell migration.
uPAR-293 cells (A and C) or V-293 cells (B and D) were pre-incubated with diluents (-) or inhibitors of Rho- or Rac1-dependent signaling pathways
(A and B), or with diluents (-) or inhibitors of PI3K or ERK-MAPKs (C and D). Cells were then plated in Boyden chambers and allowed to migrate
toward 10% FBS. Migrated cells were fixed, stained with hematoxylin, and counted (left panels). The values are the mean6SD of three experiments
performed in triplicate. (*) p#0.05, as determined by the Students t test. Results of migration assays are also expressed as percentage of cells
migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (right
panels). (*) p#0.05, as determined by the Students t test.
These results suggest that uPAR-controlled migration, which is
allowed by uPAR interactions with fMLF-Rs and b1 integrins,
involves specifically small Rho GTPases as signaling mediators.
uPAR expression controls cell migration toward EGF
All previous migration assays were performed using serum as
chemoattractant, which, indeed, is a mixture of various
chemoattractants, including also uPAR ligands, such as uPA or VN. Thus,
it was possible to hypothesize that, in uPAR-expressing cells, the
effect of uPAR ligands prevailed on that of other serum
chemoattractants, inducing activation of new signaling pathways
beside the ones activated in V-293 control cells. We then
performed migration assays using purified EGF as
chemoattractant. EGF is a growth factor largely present in serum and does not
bind uPAR, even if its receptor, EGFR, is involved in uPAR
EGF induced migration of both uPAR-293 and V-293 cells with
a similar efficiency (Fig. 7). We then assessed whether uPAR
expression and/or interactions influenced HEK-293 cell migration
toward EGF, as it occurs in cell migration toward serum. Indeed,
polyclonal antibodies against the whole uPAR molecule or the
uPAR8495 region, P-25 peptide and fMLF-R desensitization
significantly inhibited EGF-induced migration of uPAR-293 cells
(Fig. 7A and 7C), without affecting basal migration (not shown),
whereas they did not exert any significant effect on EGF-induced
migration of V-293 control cells (Fig. 7B and 7D).
Then, we evaluated the effect of signaling inhibitors on
EGFinduced migration of uPAR-293 and V-293 cells, showing that,
also in this case, migration of uPAR-expressing cells involved both
Rho and Rac1 small GTPases, unlike migration of uPAR-negative
cells, even a very low but significant effect was observed with the
Rac-1 inhibitor also in V-293 cells (Fig. 7E and 7F, respectively).
Thus, the results obtained in chemotaxis assays using a purified
chemoattractant are very similar to those obtained in chemotaxis
assays using serum as chemoattractant, suggesting that uPAR
controls the migration mechanism of the cell rather than the
migration toward specific factor/s.
uPAR depletion or blocking impairs migration of
Since uPAR over-expression in uPAR-negative cells takes
control of their migration, we assessed whether uPAR depletion
or blocking in cells which constitutively express uPAR, as prostate
carcinoma (PC3) cells , impair their migration. PC3 cells were
transfected with a uPAR-targeting siRNA or a control siRNA,
then, cells were partly lysed for Western blot analysis and partly
Figure 7. uPAR expression controls cell migration toward EGF. Stably-transfected uPAR-293 cells (A, C, E) or V-293 cells (B, D, F) were
preincubated with nonimmune Ig (-) or anti-uPAR or anti-uPAR8495 polyclonal antibodies (A and B), with diluents (-) or P-25 or W (W Pep) peptides (C
and D), with diluents (-) or inhibitors of Rho- or Rac1-dependent signaling pathways (E and F, left panels), with diluents (-) or inhibitors of PI3K or
ERK-MAPKs (E and F, right panels). Cells were then plated in Boyden chambers and allowed to migrate toward 100 ng/ml EGF. Migrated cells were
fixed, stained with hematoxylin, and counted; results are expressed as percentage of cells migrated towards EGF over the cells migrated without EGF;
100% values represent cell migration in the absence of chemoattractants. The values are the mean6SD of three experiments performed in triplicate.
(*) p#0.05, as determined by the Students t test.
used for chemotaxis assays. Western blot analysis with
uPARspecific antibodies of transfected-cell lysates showed that control
cells expressed high uPAR levels, which were strongly reduced in
PC3 cells transfected with the uPAR-targeting siRNA (Fig. 8A,
left); uPAR-depletion impaired PC3 cell migration toward serum
(Fig. 8A, right). Accordingly, PC3 cell migration toward serum was
completely blocked by anti-uPAR polyclonal antibodies (Fig. 8B).
These results suggest that uPAR takes control of cell migration
also in cells which constitutively express it; in fact inhibition of its
expression or interactions abrogate cell ability to migrate.
In the last decade we and others have observed a role for the
fMLF-Rs in uPAR activities. We started from the finding of Blasis
group that the cleaved form of soluble uPAR, exposing the
residues 8892 at its N-terminus, is a ligand for the low-affinity
receptor for fMLF (FPR2) and is able to activate it, thus inducing
cell migration [10,24]. We subsequently observed that the peptide
covering this uPAR region (uPAR8495) was able to induce cell
migration by stimulating also the other two fMLF-Rs, FPR1 and
fMLF-Rs also cross-talk with cell-surface uPAR. In fact, we and
others showed that fMLF-induced cell migration requires
cellsurface uPAR expression [18,2021]; on the other hand,
uPAinduced cell migration requires not only cell-surface uPAR
expression but also fMLF-R expression . Cell-surface uPAR
exerts its regulatory effect on the fMLF-induced migration both in
the cleaved form exposing the uPAR8495 region and in the
fulllength form, which, unlike full-length suPAR, exposes this specific
region [12,18]. Further, it has been reported that mutations in the
uPAR8495 region of cell-surface uPAR interfere with
uPAdependent signals and regulate FPR1 activation [21,29].
Altogether, these findings strongly suggested an interaction
between cell-surface uPAR and fMLF-Rs, nevertheless, their
association has never been explored in detail. We now show that,
in uPAR-negative HEK-293 cells stably transfected with
uPARcDNA, or transiently transfected with an EGFP-uPAR cDNA, a
fraction of uPAR co-localizes with FPR1 and, as expected, with b1
integrins, and viceversa, a fraction of FPR1 and b1 integrins
colocalizes with uPAR. Interestingly, cell stimulation with a generic
stimulus as serum, or with a specific stimulus as a fMLF-R ligand,
strongly promotes not only uPAR/b1 integrin and uPAR/FPR1
co-localizations, but also co-localization of FPR1 with b1 integrins.
These observations suggest that uPAR is able to recruit a large
Figure 8. uPAR depletion or blocking impairs migration of uPAR-expressing cells. A: Prostate carcinoma (PC3) cells were transfected with
a uPAR-targeting siRNA or a non-targeting control siRNA; then, cells were partly lysed for Western blot analysis with a uPAR-specific antibody (left)
and partly loaded in Boyden chamber and allowed to migrate toward 10% FBS. Migrated cells were fixed, stained with hematoxylin, and counted
(middle panel). The values are the mean6SD of a representative experiment performed in triplicate. Results of the migration assay are also expressed
as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of
chemoattractant (right). (*) p#0.05, as determined by the Students t test. B: PC3 cells were pre-incubated with nonimmune immunoglobulins (Ig) or
anti-uPAR polyclonal antibodies, plated in Boyden chambers and allowed to migrate toward 10% FBS. Migrated cells were fixed, stained with
hematoxylin, and counted (left). Results of the migration assay are also expressed as percentage of cells migrated towards serum over the cells
migrated without serum; 100% values represent cell migration in the absence of chemoattractant (right). The values are the mean6SD of three
experiments. (*) p#0.05, as determined by the Students t test.
fraction of FPR1 and b1 integrins and strongly promotes their
colocalization when cells are stimulated, suggesting a functional
meaning for these events occurring at the cell surface.
Indeed, uPAR-FPR1 closeness allows their
co-immunoprecipitation; uPAR co-immunoprecipitates also with FPR2 and FPR3,
in agreement with the fact that cell-surface uPAR functionally
interacts with all three fMLF-Rs and that cleaved suPAR activates
all of them [2,9]. Since it has been largely demonstrated that
uPAR contains also binding sites for b1 integrins, not localized in
the uPAR 8495 region , it is reasonable to hypothesize that
uPAR contemporaneously associates to both these molecules, as
suggested by fluorescence assays, thus bridging them at the cell
We then explore the functional meaning of these specific uPAR
interactions. We previously demonstrated that uPAR is able to
regulate the activity of CXCR4, the receptor for the SDF1
chemokine, through a mechanism involving integrins and
fMLFRs . We now investigate whether this uPAR regulatory
capability is restricted to CXCR4 or relies on a general
mechanism, involving integrins and fMLF-Rs, by which uPAR
can regulate cell migration, independently of the specific
chemoattractant. To this end, we performed cell migration assays
with uPAR-expressing and uPAR-negative HEK-293 cells, using
serum as chemoattractant, which is a mixture of various
chemoattractants, and observed the effect of the impairment of
uPAR interactions on cell migration. uPAR-negative cells
migrated toward serum with a similar efficiency as compared to
uPAR-expressing cells. Nevertheless, uPAR expression rendered
cell migration totally uPAR-dependent, since blocking uPAR
interactions blocked uPAR-293 cell migration, unlike migration of
uPAR-negative control cells. This conclusion was confirmed in
prostate carcinoma (PC3) cells which constitutively express uPAR;
in fact, uPAR depletion by a uPAR-specific siRNA or uPAR
blocking by a uPAR-specific antibody completely impaired PC3
cell migration toward serum.
Analysis of signaling mediators involved in cell migration
showed the selective involvement of Rho and Rac-1 small
GTPases in serum-induced migration of uPAR-293 cells,
suggesting that, when formed, the uPAR/FPR1/integrin complex
Figure 9. uPAR expression controls cell migration. uPAR-293 and V-293 cells efficiently migrate toward serum growth factors or EGF (GF).
However, uPAR, when expressed, recruits and bridges fMLFRs and b1 integrin at the cell surface, thus driving pro-migratory signaling (upper panels).
In fact, anti-uPAR antibodies (anti-uPAR), FPR1 desensitization by the W peptide (W Pep), inhibition of uPAR/b1 integrin interaction by P-25 peptide
(P-25) or of specific cell signalling mediators block migration of uPAR-293 cells without affecting migration of uPAR-negative V-293 control cells
activates new signaling pathways, thus probably taking control of
the migration process.
However, we reasoned that the involvement of new signaling
mediators in uPAR-293 cells could be merely due to the specific
uPAR stimulation by a uPAR ligand present in serum, such as
uPA or VN. Thus, we repeated all migration experiments with a
purified chemoattractant, EGF, which does not bind uPAR, is a
serum component and a suitable chemoattractant for epithelial
cells. EGF induced migration of both uPAR-293 and V-293 cells;
treatments blocking uPAR interactions at the cell surface impaired
EGF-induced migration of uPAR-expressing cells without
interfering with migration of uPAR-negative cells; uPAR-293 cell
migration involved all tested signaling mediators, unlike V-293 cell
migration. Thus, all results obtained with a purified serum
chemoattractant were comparable to those obtained with total
serum. We have to underline that uPAR has been shown to
associate and to activate the EGFR, which has been proposed as a
possible component of the uPAR-signaling machinery .
Nevertheless, more recent reports showed transactivation of the
EGFR by FPR1 in glioblastoma cells and in monocytes, in which
FPR1 modulates the activation of EGFR and TrkA, the NGF
receptor ; thus, we cannot exclude that the
uPARmediated activation of EGFR observed in previous studies 
involved also FPR1.
Indeed, integrins can directly activate growth factor receptors in
the absence of any growth factor ligand ; in fact, a hierarchy is
established where cell adhesion, which induces integrin clustering,
represents the limiting factor - and an alternative priming event
for growth factor receptor activation. Specifically, the tyrosine
kinase receptors for EGF, PDGFb, VEGF, hepatocyte growth
factor (HGF), and macrophage-stimulating protein (MSP) are all
transactivated after integrin engagement . The simple integrin
clustering can regulate integrin activity, as well as inside-out
signaling, when external stimuli initiate intracellular signals that
alter the affinity state of the integrins . In fact, fMLF-Rs
activation can regulate the activity of various integrins .
In this context, our results, depicted in Fig. 9, suggest that
uPAR, which can associate to b1 integrins and fMLF-Rs (ref.7 and
Fig. 3), could act as a docking cell-surface molecule for both
receptor types, recruiting and bridging them on the cell surface.
Thus, cell-surface uPAR could bind and activate fMLF-Rs, as the
cleaved soluble form of uPAR is able to do ; stimulated
fMLFRs, in turn, could activate signaling pathways able to modulate
activation status and/or signaling of uPAR-recruited b1-integrins,
which are crucial for the activity of various growth-factor receptors
. It is noteworthy that a fraction of uPAR is associated to
lipid rafts, which are cholesterol-rich membrane platform
concentrating signaling mediators .
In conclusion, we propose that uPAR overexpression controls
the mechanisms of cell directional migration by recruiting
integrins and FPR1 at the cell surface and regulating their
We are grateful to Dr. N. Sidenius (IFOM, Milan, Italy) for providing the
construct encoding EGFP-uPAR, to Dr. G. Hoyer-Hansen (Finsen
Laboratory, Copenhagen, Denmark) for the anti-uPAR monoclonal
antibodies, to Dr. M. Perretti. (William Harvey Research Institute,
London, United Kingdom) for fMLF-R cDNAs, and to Dr. S. Bonatti
(Federico II University, Naples, Italy) for the 36FLAG-FZ4 construct.
Conceived and designed the experiments: PRagno AG ALS. Performed
the experiments: AG ALS GA DA. Analyzed the data: PRagno AG ALS
GA PRemondelli NM. Contributed reagents/materials/analysis tools:
PRemondelli NM DA. Wrote the paper: PRagno. Manuscript revision:
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