Density enhanced phosphatase-1 (DEP-1) down-regulates
urokinase receptor (uPAR) surface expression in confluent
Short title: DEP-1 down-regulates uPAR
Patrick M. Brunner,1,2 Patricia C. Heier,1 Judit Mihaly-Bison,1 Ute Priglinger,1
Bernd R. Binder1,‡ , and Gerald W. Prager1,3
1Department of Vascular Biology and Thrombosis Research, Center for
Biomolecular Medicine and Pharmacology, Medical University Vienna, Austria
2Current address: Department of Dermatology, DIAID, Medical University Vienna,
‡Deceased August 28th, 2010
3Clinical Division of Oncology, Department of Medicine I and Cancer Center,
Medical University Vienna, Vienna, Austria;
Correspondence: Gerald W. Prager, Clinical Division of Oncology, Department of
Medicine I and Cancer Center, Medical University Vienna A-1090 Vienna, Austria
Phone: +43 1 40400 4450, FAX: +43 1 40400 4451
Scientific heading: Vascular Biology
Blood First Edition Paper, prepublished online February 8, 2011; DOI 10.1182/blood-2010-09-307694
Copyright © 2011 American Society of Hematology
VEGF165, the major angiogenic growth factor, is known to activate various steps
in proangiogenic endothelial cell behavior, such as endothelial cell migration and
invasion, or endothelial cell survival. Thereby, the urokinase-type plasminogen
activator (uPA)-system has been shown to play an essential role not only by its
proteolytic capacities, but also by induction of intracellular signal transduction.
Therefore, expression of its cell surface receptor uPAR is thought to be an
essential regulatory mechanism in angiogenesis. We found that uPAR
expression on the surface of confluent endothelial cells was down-regulated
when compared to sub-confluent proliferating endothelial cells. Regulation of
uPAR expression was most likely affected by ERK1/2 activation, a downstream
signaling event of the VEGF/VEGF-receptor system. Consistently, the receptor-
like protein tyrosine phosphatase DEP-1 (density enhanced phosphatase-
1/CD148), which is abundantly expressed in confluent endothelial cells, inhibited
the VEGF-dependent activation of ERK1/2, leading to down-regulation of uPAR
expression. Overexpression of active ERK1 rescued the DEP-1 effect on uPAR.
That DEP-1 plays a biological role in angiogenic endothelial cell behavior was
demonstrated in endothelial cell migration, proliferation as well as capillary-like
tube formation assays in vitro.
Angiogenesis is currently in the focus of basic and translational research: a
detailed analysis of the complex mechanisms involved in its regulation led to
various therapeutic concepts1 in oncology for tumor angiogenesis,
ophthalmology as in macular degeneration or choroidal neovascular disease,
rheumatology in pannus formation or dermatology for psoriasis.
We have demonstrated previously that initial steps of angiogenesis are
dependent on VEGF receptor-2 (VEGFR-2) induced pro-urokinase (pro-uPA)
activation.2 Thereby, pro-uPA bound to its GPI-anchored cell surface receptor
uPAR becomes activated, a process which involves integrin-dependent
membrane type-1 matrix metalloproteinase and matrix metalloproteinase-2
activation in a phosphatidylinositol-3 kinase dependent manner.2-3 Active uPA in
turn is blocked by its specific inhibitor PAI-1 (plasminogen activator inhibitor-1).
The so formed ternary complex uPAR-uPA-PAI-1 becomes internalized via an
LDL-receptor like molecule.4-5 Apart from an eminent role of uPAR in cancer cell
biology,6-9 the above described process seems to mediate VEGF-induced
endothelial cell migration in vitro as well as angiogenesis in vivo as shown by a
MatrigelTM plaque angiogenesis assay.2 Furthermore, uPA was shown to mediate
endothelial cell survival via induction of the X-linked inhibitor of apoptosis protein
(XIAP), a process which is also dependent on uPAR.10 These data suggest that
the amount of cell surface uPAR is affecting (i) the initial migratory response of
endothelial cells towards angiogenic stimulation11 and (ii) endothelial cell survival
during angiogenesis. Thereby, it is known that activation of somatic cells,12 and
specifically of endothelial cells13-14 by growth factors, is greatly limited when cells
are grown to confluence.
In this study, we show that uPAR surface expression is dependent on endothelial
cell density. Thereby, DEP-1 negatively regulates ERK1/2 phosphorylation,
which is involved in uPAR protein expression. These data indicate that DEP-1 is
a regulator of uPAR, a central molecule for proangiogenic endothelial cell
Materials and Methods
Human umbilical vein endothelial cells (HUVECs) were cultured at 37°C and 5%
CO2 in M199 (Sigma, St. Louis, MO) supplemented with 20% fetal calf serum
(FCS) and bovine endothelial cell growth supplement ECGS (Technoclone,
Vienna, Austria), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin (referred to as “complete growth medium”) as described
previously.3 Experiments were performed using endothelial cell cultures up to
passage 5 seeded in polystyrene 6- to 96-well plates (Iwaki microplate, Japan)
coated with 1% gelatine.
Human embryonic kidney cells (293cells) were obtained and cultured as
recommended (ATCC, Manassas, VA).
Expression plasmids for full-length human DEP-1 (pSRα DEP-1/HA), the
catalytically inactive mutant, (pSRα DEP-1 C-S/HA, C1239S), the cytoplasmic
deletion mutant (pSRα DEP-1 ΔCyto/HA, terminated at aa1051), the extracellular
deletion mutant (pSRα DEP-1 Myr) and the empty vector (pSRα) were prepared
by Ute Priglinger in Thomas O. Daniel’s laboratory, Vanderbilt University School
of Medicine, Nashville, TN.15 pEGFP-N1 plasmid was purchased from Clontech
(Mountain View, CA). pEGFP-ERK1 plasmid was kindly provided by Michael
Freissmuth, Medical University Vienna.
LipofectAMINE transfection: HUVECs were seeded in 6-well plates and
transfected after 24 hours at 50% confluence. Transient transfections were
performed using the LipofectAMINE Plus reagent (Invitrogen, Carlsbad, CA)
according to the manufacturer’s protocol. Cells were incubated with a
transfection mixture containing 1.5µg of total DNA, 6µl of Plus reagent, and 4µl of
LipofectAMINE in a total volume of 1ml M199 for 130 min. Cells were then
incubated with full growth medium and harvested after 12 hours.
Amaxa® nucleofection: Amaxa® nucleofection was used for transient
transfection with the HUVEC Nucleofector® Kit according to manufacturer’s
instructions. Briefly, 5x105 cells were resuspended in Nucleofector® solution
previously mixed with plasmids outlined in the respective figure legends, and
incubated in full growth medium. After 48 hours, cells were harvested for the
Calcium-phosphate transfection: 293 cells were transiently transfected for
luciferase reporter assays using calcium–phosphate16 and 1.5μg of DNA as
Human embryonic kidney (293) cells or HUVECs were transfected with the
constructs outlined in the figure legends as well as with luciferase reporter
constructs (Elk-1) and SV40-renilla vector as an internal control. Luciferase and
renilla assays were performed 24 hrs after transfection of the cells with a Dual
Luciferase Assay Kit (Promega, Madison, WI) as described.18 Luciferase activity
was normalized to the respective renilla values.
RNA isolation and relative quantitative reverse transcriptase-polymerase
chain reaction (Q-PCR)
RNA from HUVECs was extracted using Trizol reagent. Total RNA (900ng) was
reverse transcribed with MuLV-reverse transcriptase using the Gene Amp RNA
PCR kit (Applied Biosystems) and oligo d(T)16 primers. The mRNA sequences for
the genes to be analyzed were obtained from GenBank. The primers were
designed using the PRIMER3 software (Whitehead Institute for Biomedical
Research, Cambridge, MA). The following forward (F) and reverse (R) primers
were used for uPAR: F, 5'-GAG AAG AGC TGG AGC TGG TG-3'; R, 5'-CTT
CGG GAA TAG GTG ACA GC-3'; DEP-1: F, 5´-AGC AGG CTC AGG ACT ATG
GA-3´; R, 5´-AAC GAG GTA CCG GAA GTT GA-3´; DUSP-1: F, 5´-TCA AGA
ATG CTG GAG GAA GG -3´; R, 5´-CAG CCT CTG CCG AAC AGT-3´; DUSP-4:
F, 5´-TAC TCG GCG GTC ATC GTC TA -3´; R, 5´-TCT GGG TAC TCG GAG
GAA AA-3´; Q-PCR was performed by LightCycler technology using the Fast
Start SYBR Green I kit for amplification and detection (Roche Diagnostics, Risch,
CH). In all assays, cDNA was amplified using a standardized program (10'
denaturing step and 55 cycles of 5' at 95°C; 15' at 65°C, and 15' at 72°C; melting
point analysis in 0.1°C steps; final cooling step). Each LightCycler capillary was
loaded with 1.5µL DNA Master Mix; 1.8µL MgCl2 (25mM); 10.1µL H2O; and
0.4µL of each primer (10µM). The final amount of cDNA per reaction
corresponded to 2.5ng total RNA used for reverse transcription. Relative
quantification of target gene expression was performed using a mathematical
model described by Pfaffl.19 The expression of the target molecule was
normalized to the expression of porphobilinogen deaminase (PBGD) as
Monolayers of HUVECs were treated as indicated, thereafter harvested with
0.05% EDTA in PBS and fixed with 4% paraformaldehyde in PBS (15 min), and
aliquots were permeabilized using 0.2% Tween 20 (Amersham Pharmacia, GE
Healthcare, Buckinghamshire, UK) for 30 min at room temperature. Primary
antibodies as well as fluorochrome conjugated secondary antibodies were
incubated for at least 30 min at 4°C in concentrations ranging from 2 to 10μg/ml.
Samples were analyzed with LSR II flow cytometer (Becton-Dickinson). Antigen
amount was calculated from geometric mean fluorescence values. Primary
antibodies: monoclonal mouse anti human uPAR (domain 2+3) IgG1 #3937 and
polyclonal rabbit anti human uPAR IgG1 #399R (both from American
Diagnostica, Greenwich, CT); monoclonal mouse anti human DEP-1 (CD148)
IgG1 #AHT4802 (from Biosource, Invitrogen, Carlsbad, CA); secondary
antibodies: PE-conjugated goat anti-mouse IgG #P9670 (Sigma, St. Louis, MO);
Alexa Fluor 488-conjugated goat anti-mouse IgG1 (Molecular Probes, Leiden,
Netherlands); Total uPAR was measured in permeabilized cells, while surface
uPAR was measured on non-permeabilized cells.
HUVECs were washed with phosphate-buffered saline (PBS) and lysed in
Laemmli buffer or in RIPA buffer at 4°C. Samples were subjected to 10% SDS-
polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride
membrane (Millipore, Bedford, MA). Membranes were blocked in PBS containing
0.1% Tween 20 and 5% non-fat dry milk and incubated with polyclonal rabbit anti
pERK1/2 antibody, 1:1000, polyclonal rabbit anti pan-ERK1/2 antibody, 1:1000
(both Cell Signaling, Danvers, MA), monoclonal mouse anti human DEP-1
(CD148) IgG1 #AHT4802 (from Biosource, Invitrogen, Carlsbad, CA) and
monoclonal R2 mouse anti human uPAR (a kind gift of Gunilla Hoyer-Hansen,
Finsen Laboratory, DK) for 1 hour at room temperature. Incubation with the
secondary HRP labeled antibody (Amersham Biosciences, UK) was done 1:2500
at room temperature for 45 minutes. Detection of bands was performed with ECL
Plus (Amersham Biosciences, UK) according to the manufacturer’s protocol.
Bands were visualized and quantified with FluorChem HD2 (Alpha Innotech, San
Transfection of siRNA into HUVECs was done using polyethylenimin (Sigma, St
Louis, MO). Sparsely seeded HUVECs (5 000 cells/cm2) were transfected after
24 hours of culture in complete growth medium. SiRNA against DEP-1 was
obtained from Ambion (Foster City, CA): 5′-GCAGUACAGCAGAAUCCUUdTdT-
3′; 5’-AAGGAUUCUGCUGUACUGCdTdT-3’; For one 6-well, 8µl of vortexed
polyethylenimin (35µM, pH 7.0) was mixed with 93µl of 2xHeBS buffer (280mM
NaCl, 1.5mM Na2HPO4, 50mM Hepes), and 10µl of siRNA (20µM) was mixed
with 93µl of 2xHeBS buffer. The two solutions were unified and incubated in the
dark at room temperature for 20min. Afterwards, 200µl of transfection mix was
added to 800µl of serum free medium (M199) per 6-well and HUVECs were
incubated for 4 hours at 37°C and 5% CO2. The transfection solution was then
replaced by complete growth medium and cells were harvested for analysis after
grown to confluence.
Cell proliferation assay
Amaxa® nucleofected HUVECs were seeded subconfluently in 96-well plates
and incubated for 24 hours with M199 containing 10% FCS. Afterwards, 20µl
M199 containing 1 mCi (37x103 Bq) of 3H-thymidine (Perkin Elmer; Waltham,
MA) were added per well. Cultures were harvested 20 hours after labelling using
the Harvester 96 (Tomtec, Hamden, CT), and 3H-thymidine incorporation into
DNA was measured using the 1205 BetaplateTM Liquid Scintillation Counter
(Wallac, Perkin Elmer). Results of triplicate cultures are shown as mean cpm
(counts per minute) ±SEM.
Endothelial cell trans-migration assay
Trans-migration was performed in a modified Boyden chamber system by using
transwell membranes (8 µm) coated with 1% gelatine. Amaxa® nucleofected
HUVECs were seeded to the top of the membrane, while VEGF165 (50mg/ml)
was added to the lower chamber. After 5 hours of incubation filters were washed
with PBS x 1, fixed with 10% buffered formalin and stained with 4'-6-diamidino-2-
phenylindole (DAPI) for nuclear staining. Migrated cells were counted using an
AX70 Olympus microscope.
In vitro MatrigelTM tube-formation assay
Matrigel™ tube formation assays were performed as described before.20 Briefly,
Amaxa® nucleofected HUVECs were seeded on Matrigel™ in 48-well plates and
incubated in M199 supplemented with 1% FCS. Photographs were taken after 24
hours and the length capillary-like structures were quantified using ImageJ
software (NIH, Bethesda, MD).
Experimental values are expressed as mean ± SEM if not otherwise indicated.
Statistical significances were determined by unpaired Student’s t-test and one-
way ANOVA. Significance was assigned to a value of *p<0.05 and **p<0.01. All
results were reproduced at least in three independent experiments.
Confluence affects uPAR expression in endothelial cells
In an initial experiment, we analyzed possible effects of endothelial cell density
on uPAR expression. By the use of fluorocytometric analyses we found that
confluent endothelial cells express markedly reduced amounts of uPAR
compared to sparsely seeded endothelial cells, showing a consistent density
dependence (Figure 1A). As surface and total uPAR expression were
concomitantly decreased, we further analyzed uPAR mRNA expression and
found a concordant decrease. Consistently, the degree of cell density was
inversely correlated with uPAR mRNA expression (Figure 1B). Although it has
been shown that both forms of uPAR - an intact approximately 44 kDa21
(comprising three homologous domains D1, D2 and D3) as well as the cleaved
form of uPAR (30 kDa, comprising only D2 and D3) can be detected on cell
surface of particular cultured cells,22 in endothelial cells mainly intact uPAR was
detected as shown by Western blot analysis (Figure 1C). This expression pattern
was unaffected by cell density (data not shown), suggesting that regulation of
uncleaved uPAR might play a functional role in cell surface bound proteolysis
and ECM interaction.
ERK/MAPK mediates cell density-dependent uPAR expression
The MAPK pathway represents one of the most characterized signaling
cascades in mitogenic stimulation26-27 and differentiation.28 It was previously
shown that confluence of vascular endothelial cells induces cell cycle exit by
inhibition of the ERK1/2 signaling pathway, as shown by a decreased response
to stimulation by different growth factors such as fibroblast growth factor-2 (FGF-
2).14 When we analyzed endothelial cell lysates of experiments shown in Figure
1A, we found that sparsely seeded endothelial cells had markedly higher
amounts of basal pERK1/2 as compared to confluent and thus more quiescent
endothelial cells (Figure 2A).
Previous findings29 suggest that uPAR expression is regulated via the MAPK
pathway, and uPAR expression can be down-regulated by the MAPKK-MEK1/2
inhibitors PD 098059 or U0126.30 Consistently, overexpression of active ERK1
led to an increase in uPAR expression in resting endothelial cells (Figure 2B).
Increasing endothelial cell confluence up-regulates phosphatases and
blunts activation of ERK/MAPK induced by VEGF165
To determine whether confluence affects endothelial cell responses towards
VEGF, HUVECs seeded at different densities were stimulated with VEGF165 and
ERK1/2 phosphorylation was analyzed. Confluent endothelial cells were much
less responsive to VEGF165 stimulation compared to sub-confluent endothelial
cells (Figure 3A). Identical activation patterns of ERK1/2 were observed in sparse
and confluent cells, however, the amplitude of activation was attenuated with
increasing cell density. By evaluating possible mechanisms responsible for the
decrease of pERK1/2 levels, we examined HUVECs for major phosphatases
regulating the MAPK signaling pathway31-32 including dual-specificity
phosphatase-1 (DUSP-1 or MAPK Phosphatase-1, MKP-1), dual-specificity
phosphatase-4 (DUSP-4 or MAPK Phosphatase-2, MKP-2), and density
enhanced phosphatase-1 (DEP-1).
Expression of the nuclear ERK/MAPK regulator phosphatases DUSP-1 and
DUSP-4 was not significantly affected by cell density. With increase of cell
density, the mRNA levels of the transmembrane tyrosine phosphatase DEP-1
were significantly up-regulated (Figure 3B) which pointed us to further investigate
effects of DEP-1 on uPAR expression.
DEP-1 decreases uPAR surface expression via regulation of the ERK/MAPK
Expression of DEP-1 is regulated by cell confluence; therefore the role of DEP-1
in confluence-related changes of uPAR expression was investigated.
Subconfluent HUVECs were transfected with plasmids either encoding wild type
DEP-1 or mock. We observed a significant decrease in uPAR surface expression
down to 64.8% ± 2.2 (Figure 4A) when DEP-1 was overexpressed. The
expression of CD59, another GPI-anchored protein, was unaltered in the
presence or absence of DEP-1 (data not shown). Consistently, inhibition of DEP-
1 expression by siRNA revealed an increase in total uPAR expression by 3.03 ±
0.46 fold (Figure 4B). As a next step, we investigated the influence of DEP-1 on
the MAPK signaling pathway. Elk-1 is a transcription factor downstream of
ERK1/233 and thus a potential surrogate for uPAR transcription. Due to high
transfection efficacy we have first overexpressed ERK-1 in the human embryonic
kidney cell line 293 (Figure 4C), which resulted in a marked increase of Elk-1
activity. Co-transfection of DEP-1 strongly and dose dependently decreased
ERK1-induced Elk-1 activation (Figure 4C). These results could be confirmed in
primary endothelial cells (HUVECs) as shown in Figure 4D. These data are
consistent with the observation that DEP-1 has an inhibitory effect on the MAPK
signaling cascade13 via direct de-phosphorylation of ERK1/2.32
Phosphatase activity of DEP-1 is required for confluence dependent
regulation of uPAR expression
DEP-1 is a receptor protein tyrosine phosphatase (RPTP) that consists of an
extracellular domain including eight fibronectin III domains, a transmembrane
segment and an intracellular tail with a single protein tyrosine phosphatase
domain.34-36 In order to reveal the structural and functional involvement of DEP-1
in the regulation of uPAR expression, mutated variants of the protein were
generated. We introduced the mutated constructs of DEP-1 either lacking
phosphatase activity (C=>S) or the entire intracellular (DeltaCyto) or the
extracellular (Myr) domain into HEK293 cells (Figure 5A). These cells were
transiently transfected with elements of the Elk-1 reporter system. We found that
the cytoplasmic domain of DEP-1 is required and sufficient to regulate Elk-1
activity. Activation of Elk-1 is affected by the DEP-1 phosphatase domain,
because mutants of DEP-1 lacking phosphatase activity were not capable of
reducing basal Elk-1 activity. These results could be confirmed in primary human
endothelial cells (Figure 5B). Finally, we were interested which domain of DEP-1
is involved in the regulation of uPAR expression. Therefore, the mutated DEP-1
variants were introduced by transient transfection in HUVECs and uPAR protein
levels were investigated. The cytoplasmic domain of DEP-1 was required and
sufficient to reduce uPAR protein levels. The down-modulating effect of DEP-1
on uPAR could be prevented by co-expression of ERK1 and was again
dependent on phosphatase activity, as mutants of DEP-1 lacking phosphatase
activity were not able to decrease uPAR expression (Figure 5C). From these
data we conclude that DEP-1 regulates uPAR expression via the MAPK signaling
DEP-1 inhibits key steps of angiogenesis in vitro
As shown before,2 uPAR plays a central role in VEGF-induced endothelial cell
migration. Therefore, we analyzed the effect of DEP-1-overexpression on
VEGF165-induced endothelial cell migration. As expected, DEP-1 significantly
inhibited trans-migration of HUVECs when compared to mock transfected cells
(Figure 6A). Another prerequisite for efficient angiogenesis is endothelial cell
proliferation,1 which was also significantly reduced whenever DEP-1 was
overexpressed. Notably, this effect was dependent on intact phosphatase activity
of DEP-1 (Figure 6B). Finally, we assessed capillary-like tube formation on
MatrigelTM, another surrogate of angiogenic cell behavior.20 Capillary-like
structures were significantly reduced in HUVECs transfected with wild-type DEP-
1 compared to endothelial cells overexpressing either a phosphatase inactive
mutant (C=>S) or mock (Figure 6C).
From these in vitro angiogenesis assays we conclude that DEP-1 affects
endothelial cell behavior.
A variety of pro-angiogenic growth factors and chemokines activate endothelial
cells (EC) to migrate and invade surrounding tissue, an essential step in
angiogenesis. For invasion, the coordinated formation of a localized proteolytic
machinery is essential. Focusing uPAR towards the leading edge of migrating
cells provides such armor2,37, while inhibition of uPA binding to its receptor
prevents invasion of endothelial cells.38 The functionality of uPAR critically
depends on the presence of its intact form, as cleavage of uPAR disrupts its
ability to bind uPA or the ECM protein vitronectin.39 VEGF induces endothelial
cell migration and invasion by activating pro-uPA and mediating uptake of uPA-
uPAR-PAI-1 complexes via LRP-like proteins.5 These initial steps of VEGF-
induced endothelial cell migration are independent of transcriptional activity.2
Therefore, the amount of surface uPAR and pro-uPA bound to its receptor are
expected to mediate endothelial cell responses towards VEGF. Here we show
that uPAR expression is strongly regulated by cell density and that in confluent
endothelial cells, the amount of uPAR is only approx. 30% compared to sparsely
seeded endothelial cells (Figure 1). Consistently, uPAR was found to be down-
regulated in myoblasts when they reached confluence, cease migration and start
to differentiate.40 Therefore, it can be expected that confluent endothelial cells
respond much less to VEGF-induced endothelial cell migration or survival as
compared to sparsely seeded cells. In fact, different responses towards FGF
(fibroblast growth factor -2) depending on cell density were described before.14
Here we show that in fact the response of confluent endothelial cells towards
VEGF stimulation is dramatically reduced with respect to the activation of the
MAPK signaling pathway (Figure 2A). Therefore, confluent endothelial cells, as
expected to be found in mature vessels, not only showed decreased responses
towards VEGF stimulation but also express less uPAR. Notably, intact uPAR is
the most prominent form expressed on the surface of endothelial cells (Figure
1C). As potential factors responsible for down-regulating ERK1/2 activation as
well as decreased uPAR expression, we suggest DEP-1 as a hitherto
undescribed regulator for uPAR. As indicated by its name, expression of DEP-1
is regulated by cell density.34 Thereby, DEP-1 is up-regulated with increasing
density in endothelial cells35 and inhibits the MAPK pathway13,15,32 in confluent
compared to sparsely seeded endothelial cells (Figure 2A). In fact,
overexpression of DEP-1 not only decreased ERK1/2 activity but also uPAR
expression (Figure 4). This effect of DEP-1 was dependent on the intact
phosphatase-domain since mutants deficient in DEP-1 phosphatase activity were
not active, while a mutant deficient in the extracellular domain showed activity,
but not to the same extent as full length DEP-1 (Figures 4 and 5). Indeed, it was
shown before that certain extracellular matrix proteins increased protein tyrosine
phosphatase activity of DEP-1, however, only when its extracellular domain was
present.41 The remaining activity of the extracellular deficient mutant indicates
that the extracellular domain is not responsible for linking DEP-1 to growth
factors or other extracellular surface molecules guiding DEP-1 phosphatase
activity to intracellular targets. Our results further support the data from
Takahashi et al.,15 who reported that an increase in DEP-1 phosphatase activity
by an activating antibody strongly inhibits angiogenesis in vivo using a murine
corneal pocket model. Consistently, DEP-1 is shown to be important for contact
inhibition of VEGF-induced proliferation13 and during neo-intima formation.42 That
DEP-1 affects cell behavior has been shown for many cell types.13,43-46 For
primary endothelial cells we here report that DEP-1 inhibited trans-migration
(Figure 6A), proliferation (Figure 6B) and formation of capillary-like structures
(Figure 6C). This is consistent with previous findings that disrupted DEP-1 gene
led to early embryonic lethality at midgestation due to impaired vascularisation.44
In this study, we suggest that DEP-1, which is up-regulated during confluence,
not only decreases VEGF-induced MAPK activity but also impairs MAPK
dependent uPAR expression. As it has been shown that uPAR represents a
central player for VEGF-induced pro-angiogenic endothelial cell behavior2-3,10,47
we suggest DEP-1 as a novel target for agents modulating angiogenesis.
This work is dedicated to Bernd R. Binder, who was an important contributor to
this project. He deceased on August 28th, 2010.
The study was supported by the Hans and Blanc Moser Foundation, the
Integrated Project of the 6th European Union (EU) framework program
Cancerdegradome (contract no. LSHC-CT-2003-503297), as well as the Austrian
Science Foundation project (FWF P21301) of G.W.P. We also thank Melanie
Gschaider, Christine Wagner and Marina Poettler for technical advice.
Contribution: P.M.B., P.C.H. and G.W.P. performed experiments; U.P. prepared
plasmids; J.M.B. analyzed results; and P.M.B., G.W.P. and B.R.B. designed
the research project and wrote the paper.
Correspondence: Gerald W. Prager, Clinical Division of Oncology, Department of
Medicine I and Comprehensive Cancer Center, Medical University Vienna,
Vienna, Austria; e-mail: email@example.com.
Conflict of Interest Disclosures
The authors declare no competing financial or conflicting interests.
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Figure 1. Expression of intact uPAR inversely correlates with cell density.
HUVECs were seeded in different densities (indicated as cells per cm²) and
grown in complete growth medium for 4 days. Photographs were taken prior to
harvest. (A) Immunocytofluorimetric detections of cell surface uPAR (non-
permeabilized cells; gray line) and total uPAR (permeabilized cells; black line).
The total amount of uPAR decreases with cell density. Geometric means for
fluorescence intensities were calculated. Filled gray: IgG. Mean ± SEM; (B)
Relative quantitative reverse transcriptase-PCR (Q-PCR) of uPAR mRNA
normalized to PBGD; Mean ± SEM, *p<0.05; (C) Representative Western blot
(WB) for uPAR of lysates of HUVECs. Mainly intact (i.e. full length) uPAR
(approximately 44kDa) was detected in contrast to cleaved uPAR (30kDa).
Figure 2. Density dependent uPAR expression is mediated by ERK/MAPK
(A) Western blots (WB) for phospho-(p)-ERK1/2 and pan-ERK1/2 from
endothelial cell lysates. All samples were lysed in the same volume and prepared
as described in Materials and Methods irrespective of cell density, and the same
volume of samples was loaded. Consequently, the amount of loaded protein is
higher in the 20 000 cells/cm² sample than the 5 000 cells/cm² sample, reflected
by the stronger signal in pan-ERK. Proteins were separated on an SDS-10%
polyacrylamide gel, and chemiluminescence of phosphorylated and pan-ERK1/2
was quantified with FluorTech HD2 from Alpha Innotech.
(B) Immunocytofluorimetric detections of cell surface uPAR (non-permeabilized
cells; gray) and total (permeabilized cells; black) uPAR in HUVECs over-
expressing either EGFP tagged ERK1 or EGFP alone. Transfected HUVECs
were gated via EGFP fluorescence; Mean ± SEM, **p<0.01;
Figure 3. Influence of cell density on modulators of the MAPK signaling
pathway in endothelial cells.
(A) Western blots for phospho-(p)-ERK1/2 and pan-ERK1/2 from endothelial cell
lysates. Confluent (20 000), subconfluent (10 000) and sparse (5 000) HUVECs
were grown for 4 days in complete growth medium and afterwards rendered
quiescent for 4h by serum deprivation (4% BSA in M199). Cells were then
stimulated with 20 ng/ml VEGF165 for the time indicated. All samples were lysed
in same volumes and obtained as described in Materials and Methods.
Quantification of pERK1/2 chemiluminescence normalized to pan-ERK1/2 at 10’
of VEGF165 stimulation (RLU): 0.36/1 (5 000), 0.16/0.38 (10 000), 0.05/0.1 (20
000); ERK1 = p44mapk, ERK2 = p42mapk. (B) Relative quantitative reverse
transcriptase-PCR of HUVECs seeded in different densities (indicated as cells
per cm²) and grown in complete growth medium for 4 days. DUSP-1 (MAPK
phosphatase-1): Dual specificity phosphatase-1; DUSP-4 (MAPK phosphatase-
2): Dual specificity phosphatase-4; DEP-1: Density enhanced phosphatase-1;
Among major phosphatases impacting the MAPK signaling pathway, only DEP-1
mRNA levels increased significantly; values normalized to PBGD; Mean ± SEM,
Figure 4. DEP-1 decreases uPAR expression.
(A) Immunocytofluorimetric detections of cell surface uPAR in non-permeabilized
HUVECs over-expressing DEP-1 (gray line) compared to mock transfected
HUVECs (black line). DEP-1 positive cells were detected by monoclonal mouse
anti DEP-1 antibody. Filled gray: IgG; Mean ± SEM; (B) Western blot for uPAR
from endothelial cell lysates. HUVECs were seeded sparsely (5 000 cells per
cm2) and were allowed to grow for 24 hours in complete growth medium.
Consequently, cells were transfected with DEP-1 siRNA, which resulted in an
efficient and sustained reduction of DEP-1 expression compared to transfection
with scrambled-(scr)-RNA (data not shown). Cells were then incubated in
complete growth medium until all samples had reached confluence. All samples
were lysed and obtained as described in Materials and Methods. Knock down of
DEP-1 yielded a 3.03±0.46 fold increase in uPAR protein expression in confluent
endothelial cells. (C) HEK 293 or (D) HUVECs were transfected for reporter gene
analysis (i.e. Elk-1 activity, a downstream target of ERK1/2) with DEP-1 and/or
ERK1 over-expressing plasmids (indicated as amounts of DNA in ng/ml). Cells
were harvested 24 hours after transfection and analyzed for luciferase activity;
pSRα was taken as empty plasmid (control); Mean ± SEM; *p<0.05
Figure 5. MAPK-mediated uPAR expression is regulated by DEP-1
(A) HEK 293 cells and (B) HUVECs were transfected for reporter gene analysis
(Elk-1 activity) with plasmids over-expressing either wild type DEP-1 or its
mutated forms. Cells were harvested after 24 hours and analyzed for luciferase
activity; Mean ± SEM, *p<0.05 **p<0.01; Only catalytically active DEP-1 was able
to reduce Elk-1 activity; (C) Immunocytofluorimetric detections of cell surface
(non-permeabilized cells; gray) and total (permeabilized cells; black) uPAR in
HUVECs over-expressing different DEP-1 mutants and either EGFP or EGFP-
ERK1 gated for EGFP fluorescence; *p<0.05; Mean ± SEM; values calculated as
percent over EGFP control; Only catalytically active mutants of DEP-1 were able
to decrease uPAR expression. This effect could be rescued by ERK1
coexpression. (DEP-1: wild type; C=>S: Inactivated tyrosine phosphatase due to
cysteine to serine mutation in the catalytic domain; DeltaCyto: deletion of the
intracellular domain (including phosphatase domain); Myr: deletion of the
Figure 6. Dep-1 inhibits proliferation, trans-migration and capillary-like tube
formation of EC
(A) Endothelial cell migration of transfected HUVECs was assessed in a modified
Boyden chamber assay in the presence of 50ng/ml VEGF165. Migrated cells were
fixed, stained, and quantified by microscopic counting. Overexpression of DEP-1
significantly decreased EC migration towards VEGF165 compared to mock
transfected cells; mean ± SEM, ** p < 0.01. (B) 3H-thymidine incorporation in
HUVECs transfected with plasmids overexpressing either wild-type DEP-1, a
mutant lacking phosphatase activity (C=>S), or mock. After 24 hours of culture in
full growth medium, 3H-thymidine (1µCi/well) uptake of proliferating cells was
measured (20 hours). Overexpression of DEP-1 significantly inhibited cell growth.
Mean (cpm) ± SEM, **p<0.01; (C) Capillary-like tube formation of HUVECs
transfected with plasmids either over-expressing wild type DEP-1, the C=>S
mutated form lacking phosphatase activity, or mock. Transfected cells were
seeded on MatrigelTM in the presence of 1% FCS and analyzed 24 hours after
seeding. Tubular-like structures were quantified as described in Materials and
Methods. DEP-1 inhibited capillary-like tube formation dependent on
phosphatase activity. Mean ± SEM, *p<0.05 **p<0.01 (DEP-1: wild type; C=>S:
Inactivated tyrosine phosphatase due to cysteine to serine mutation in the
*103seeded cells per cm²
uPAR relative mRNA
0 20 406080100 120
log fluorescence intensity
mean fluorescence intensity
VEGF stimulation (min.)
*103seeded cells per cm²
relative increase in
5 10205 10205 1020
*103seeded cells per cm²
uPAR surface expression
log fluorescence intensity
uPAR mean fluorescence
percent of EGFP-control
lenght of capillary-like tubular
structures (arbitrary units)
3H-thymidine uptake (cpm)