From the Cancer Research Institute, Catholic Research
Institutes of Medical Sciences and the
Department of Natural
Sciences, College of Medicine, The Catholic University of Korea, Seoul
137-701, the ** Department of Biochemistry, College of
Natural Science, Biotechnology Research Institute, Chungbuk National
University, Cheongju 361-763, the
Cancer
Metastasis Research Center, Yonsei University College of Medicine,
Seoul 120-752, and the §§ Mogam Biotechnology
Research Institute, Yongin 449-910, Korea
Received for publication, December 4, 2002, and in revised form, January 3, 2003
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ABSTRACT |
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Urokinase plasminogen activator
(uPA) belongs to a family of proteins that contains kringle domain and
plays an important role in inflammation, tissue remodeling,
angiogenesis, and tumor metastasis by pericellular plasminogen
activation. Kringle domains of plasminogen have been shown to
demonstrate anti-angiogenic and anti-tumor activities. Here, we report
our investigation of the kringle domain of uPA for anti-angiogenic
activity and a possible cellular mechanism of action. The recombinant
kringle domain of uPA (Asp45-Lys135)
(UK1) inhibited endothelial cell proliferation stimulated by basic
fibroblast growth factor, vascular endothelial growth factor (VEGF), or
epidermal growth factor. It also inhibited migration of endothelial
cells induced by VEGF or uPA, and in vivo angiogenesis on
the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by
translocation from cytosol to nucleus was determined to be specific to
endothelial cells. It also elicited a transient increase of
Ca2+ flux of more than 2-fold within 2 min of exposure in
an endothelial cell-specific manner. These results suggest that the
kringle domain of uPA exhibits anti-angiogenic activity and that its
anti-angiogenic activity may occur through a different mechanism from
inhibition of uPA-uPA receptor interaction or uPA proteolytic activity
and may be associated with endothelial-cell specific internalization not mediated by the uPA receptor.
Urokinase-type plasminogen activator (uPA or
urokinase)1 has been
implicated in inflammation, angiogenesis, tissue remodeling, and the
progression and metastasis of numerous solid tumors (1-6). uPA is a
multidomain protein composed of a carboxyl-terminal protease domain and
an amino-terminal fragment, which can be further subdivided into a
growth factor-like domain (aa 4-43) and a kringle domain (aa 45-135)
(7). Urokinase is a highly specific serine protease that converts
plasminogen into plasmin. It also binds to a specific glycolipid-anchored receptor (uPA receptor) with high affinity, primarily via its growth factor-like domain, to mediate proteolysis on
the cell surface.
Angiogenesis is a complex, multistep process that occurs during normal
physiology such as wound healing, pregnancy, and development, as well
as under pathological conditions such as diabetic retinopathy and
tumorigenesis (8-11). During angiogenesis, endothelial cells need to
divide, migrate, invade the extracellular matrix, and form capillary
structures from pre-existing blood vessels (12). This complex process
implies the presence of multiple controls, which can be turned on and
off within a short period. There is an increasing body of evidence
showing that inhibition of angiogenesis can lead to the suppression of
tumor growth and metastasis (13-16).
Among the family of endogenous angiogenesis inhibitors, angiostatin has
been demonstrated to be derived from plasminogen (14). Angiostatin was
first isolated from serum and urine of tumor-bearing animals and found
to consist of the first four kringles of plasminogen (14). In
vitro, it specifically inhibits endothelial cell proliferation but
not proliferation of other cell types, including tumor cells (14, 17).
In vivo, angiostatin suppresses neovascularization and
suppresses tumor growth in animals without toxicity (14, 18). However,
its cellular mechanism of action remains unclear.
In general, a kringle is composed of 78-80 amino acids interconnected
by a triple disulfide-linked loop. The triple disulfide bonds are
strictly conserved between kringles (19). The identity of amino acid
sequence between plasminogen kringles averages about 50%.
Interestingly, each kringle of angiostatin has been demonstrated to
display differential effects on endothelial cell proliferation and
migration (17, 20). Besides angiostatin, another plasminogen kringle,
PK5, has also been shown to be a potent inhibitor of endothelial cell
proliferation and migration in vitro (21, 22). Kringles
derived from other molecules such as kringle 2 of prothrombin and
kringles of human hepatocyte growth factor were also found to be
inhibitors of endothelial cell proliferation (23-25), whereas contradictory results have been reported on the anti-endothelial cell
activity of apolipoprotein kringles (26, 27).
Because angiostatin is a cryptic fragment derived from
plasminogen, we were interested to learn whether the urokinase
kringle domain exhibits anti-angiogenic activity, although urokinase
itself promotes angiogenesis. The urokinase kringle also shares a low degree of sequence identity with the plasminogen kringles. In this work
we examined the effects of the recombinant kringle domain of urokinase
in anti-angiogenic activity. To explore the mechanism of action for the
anti-angiogenic activity, we tested whether the kringle affects uPA
proteolytic activity or interacts with uPA receptor. We also
demonstrated that the internalization of the recombinant kringle domain
is specific to endothelial cells, which might explain in part why it
has endothelial cell-specific activity. In addition, we report an
elevation in intracellular Ca2+ concentration elicited by
the kringle domain of urokinase, specifically in endothelial cells.
Materials--
Recombinant human single chain-uPA (sc-uPA)
expressed in Chinese hamster ovary cells was obtained from Mogam
Biotech Research Institute (Kyungki-do, Korea). Two chain-uPA (tc-uPA)
was kindly provided by Green Cross Co. (Kyungki-do, Korea).
Angiostatin, containing kringles 1-4, was prepared by digestion of
human plasminogen with porcine elastase as published previously (28)
and was further purified by size-exclusion chromatography on a Bio-prep
SE 100/17 column. Recombinant human uPA receptor (uPAR, 870-UK/CF),
anti-human uPAR polyclonal antibody (AF807), vascular endothelial
growth factor (VEGF), basic fibroblast growth factor (bFGF), and
epidermal growth factor (EGF) were purchased from R&D system
(Minneapolis, MN). The reagents used for cell culture, M199,
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS),
and trypsin solution were purchased from Invitrogen (Grand Island, NY).
Endothelial cell growth medium (EGM®) and endothelial cell basal
medium (EBM-2®, Mg2+- and Ca2+-free) were
purchased from Clonetics (San Diego, CA). Angiogenin was purified from
cow's milk as described previously (29). Control mouse isotype IgG was
provided kindly by Dr. Jeong-Im Sin (The Catholic University of Daegu,
Korea). Polyclonal antisera were raised against the human recombinant
urokinase kringle domain by injecting BALB/c mice three times
with 100 µg of the recombinant protein. The IgG fraction was then
purified from this antiserum by protein-A chromatography.
Cell Culture--
Human umbilical vein endothelial (HUVE) cells
were isolated from fresh cords by an adaptation of the method described
by Jaffe et al. (30) and maintained in M199 medium
containing 20% FBS, 30 µg/ml endothelial cell growth supplements
(Sigma, St. Louis, MO), 90 µg/ml heparin, 25 mM Hepes,
2.2 g/liter sodium bicarbonate, 2 mM
L-glutamine, and 1% antibiotics. Cells at passage 3, 4, or 5 were used for the experiments. Bovine capillary endothelial (BCE)
cells were obtained from Dr. T. H. Lee (23) and maintained in DMEM
containing 10% FBS, 1% antibiotics, and 3 ng/ml bFGF. NIH 3T3 and
HEK-293 cells were cultured in DMEM supplemented with 10% FBS.
Cultures were kept at 37 °C in a humidified atmosphere of
10% CO2 in air for BCE cells and in 5% CO2 in
air for HUVE, NIH 3T3, and HEK-293 cells.
Construction of the Expression Vectors--
Total RNA was
isolated form HUVE cells by using a Qiagen total RNA isolation kit, and
cDNAs were generated using an oligo(dT) primer and the First Strand
cDNA synthesis kit (Roche Molecular Biochemicals, Mannheim,
Germany). cDNA encoding the amino acids spanning from
Ser1 to Lys135 of human uPA was amplified by
PCR with Pfu polymerase (Stratagene, La Jolla, CA) and two
primers, forward primer A
(5'-CAGAATTCCATGGCCAGCAATGAACCTCATCAAGTTCC-3') and reverse primer B
(5'-GCCGCTCGAGTTTTCCATCTGCGCAGTC-3'). The amplified cDNA was
introduced into a blunt vector T-7Blue according to the manual of
Perfectly pT-7Blue blunt cloning kit (Novagen, Madison, WI). The
nucleotide sequence of construct was confirmed by manual sequencing
using the T7 Sequenase version 2.0 DNA sequencing kit (Amersham
Biosciences, Uppsala, Sweden). For the removal of the NcoI
site, PCR-directed mutagenesis was carried out without any change of
amino acid sequence by two rounds of PCR reactions. In the first
reaction, either forward primer C (5'-ATGGCCCGGCCCTGCCTG-3') and
reverse primer B, or forward primer A and primer D
(5'-CAGGCAGGGCCGGCCCATAGTGTCAGTGCTGGCCTT-3') were used as a
primer set, and the above cDNA was used as a template (the change
from G to A is indicated by boldface type). In the second round,
primers A and B were used as a primer set, and two PCR products from
the first round reactions as templates, to produce a single annealed
PCR product. The final PCR product was digested with NcoI
and XhoI and ligated to pET-21d (Novagen) linearized with
NcoI and XhoI and dephosphorylated, resulting in
the recombinant plasmid pET-21d/ATF. For the construction of the
expression vector for the urokinase kringle region (UK1) spanning from
Asp45 to Lys135, insert cDNA was amplified
using two primers, forward primer E (5'-CATAATTCCATGGATAAGTCAAAAACC-3')
and reverse primer B, and plasmid pET-21d/ATF as a template. The
amplified cDNA fragment was subcloned into NcoI and
XhoI sites of pET-21d, resulting in the expression plasmid,
pET-21d/UK1. pET-21d/UK1 was used for the transformation of
Escherichia coli BL21(DE3) for protein expression. This
construct has the following additional amino acids: methionine and
alanine at the amino terminus and leucine and glutamic acid before His
tag at the carboxyl terminus.
Expression and Purification of UK1--
The E. coli
transformant was grown in 2.5 liters of LB medium containing 100 µg/ml ampicillin at 37 °C to an A600 of
about 0.6. To induce the production of recombinant protein, isopropyl thio- Analysis of Protein--
Amino acid sequence was determined with
an Applied Biosystems, Model Procise 491, protein sequencing system at
the Korea Basic Science Institute (Seoul, Korea). Matrix-assisted laser
desorption ionization mass spectrometry was performed using a Voyager
Biospectrometry workstation with a linear mass analyzer (PerSeptive
Biosystems, Framingham, MA) at the Korea Basic Science Institute
(31).
Endothelial Cell Proliferation Assay--
BCE cell proliferation
assay was performed as previously described (14, 32). 12,500 cells/well
were plated into gelatinized 24-well culture plates and were incubated
in DMEM, 10% FBS, 1% antibiotics for 24 h. The media was
replaced with 0.25 ml of DMEM, 5% FBS, 1% antibiotics, and the test
sample applied. After 30 min of incubation, 0.25 ml of DMEM, 5% FBS,
1% antibiotics, 2 ng/ml bFGF was added to each well. After 72 h,
cells were dispersed in trypsin and counted by a trypan blue exclusion method.
For HUVE cells, [3H]thymidine incorporation assay was
performed as the follows: 2 × 104 cells/well were
plated into gelatinized 24-well culture plates and were incubated in
M199, 10% FBS, 90 µg/ml heparin, 1% antibiotics for 24 h. The
media was replaced with 0.25 ml of M199, 5% FBS, 90 µg/ml heparin,
1% antibiotics, and the test sample applied. After 30 min of
incubation, 0.25 ml of M199, 5% FBS, 90 µg/ml heparin, 1%
antibiotics, and a growth factor (6 ng/ml bFGF, 20 ng/ml VEGF, or 20 ng/ml EGF) was added to each well. After 18 h, 1 µCi (0.037 MBq)
of [3H]thymidine (PerkinElmer Life Sciences, Boston, MA)
was added to each well. After further incubation for 6 h, cells
were fixed with methanol, washed with cold 10% trichloroacetic acid
solution three times, and dissolved in 0.25 N NaOH, 1%
SDS. Radioactivity was determined using a liquid scintillation counter.
Each experiment was performed in triplicate.
Endothelial Cell Migration Assay--
To evaluate migration, a
modified Boyden chamber-based assay (33) was performed. Transwell
polycarbonate membrane (8-µm pore size) (Costar, Cambridge, MA) was
coated with 0.1% gelatin. After HUVE cells were trypsinized and
suspended in M199 medium (4 × 105 cells/ml), 100 µl
was added to each well in the upper chamber. The filter was placed over
a bottom chamber containing M199 medium, 2 ng/ml VEGF, 0.1% bovine
serum albumin. For testing inhibitory activity, HUVE cells were
preincubated with inhibitors in M199 medium in the cell culture
incubator for 30 min before being added to the upper chamber, and the
inhibitor was added to the bottom chamber. The assembled chamber was
incubated for 5 h at 37 °C with 5% CO2 to allow
cells to migrate through the gelatin-coated polycarbonate filter. The
filter membrane was removed from the chamber, fixed with methanol, and
stained with hematoxylin Gill No. 2 (Sigma) and eosin. Non-migrated
cells on the upper surface of the filter were removed by scraping with
a cotton swab. The total number of migrated cells with nuclei per well
was counted using a digital photographic system. Each experiment was
performed in triplicate.
The migration of HUVE cells by uPA was determined using a "scratch"
wound assay (34-36). Briefly, HUVE cells were plated in six-well
culture plates at 5 × 103 cells/cm2 and
cultured in EGM. When the cells are confluent, they were washed with
prewarmed PBS and then cultured in EBM-2 supplemented with 1% FBS for
24 h. The cell monolayer was scraped with a cell scraper to create
a cell-free zone. After washing three times with EBM-2, HUVE cells were
treated with uPA in the absence or in the presence of UK1 (10-500
ng/ml) in EBM-2 containing 1% FBS. HUVE cell migration was quantified
by measuring the width of the cell-free zone (distance between the
edges of the injured monolayer) after 24 h under a Leica DM IRB
real-time inverted microscope. Each sample was assayed in duplicate,
and the assays were repeated twice.
Chorioallantoic Membrane Assay--
To determine anti-angiogenic
activity in vivo, a CAM assay was performed as previously
described (37, 38). Fertilized eggs (Pulmuone, Kyungki-do, Korea) were
incubated at 37 °C and 80-90% relative humidity. At day 2, a
portion of albumin was removed and a window was made on day 3. At day
4.5 of incubation, test samples loaded on a quarter size
Thermanox coverslip (Nunc, Roskilde, Denmark) were applied on
the CAM of individual embryos. After another 2-day incubation, a 20%
fat emulsion was injected into the CAM for observation of the
inhibition zone of angiogenesis. The vascular networks in the CAMs were
blindly scored for the presence or absence of an avascular zone larger
than 5 mm in diameter.
Plasminogen Activation Assay--
Human fibrinogen was prepared
by a modification of the method of Mosesson et al. (39). The
ability of the fibrinogen to clot was higher than 95% when thrombin
was added. Fibrin clot plates were prepared from fibrinogen (3 mg/ml)
containing human plasminogen (0.2 mg/ml) in buffer (20 mM
CaCl2, 150 mM NaCl, 50 mM Tris-HCl,
pH 7.6) by adding of human thrombin (Sigma, 0.4 unit/ml) and incubating
for 1 h at 37 °C. Aliquots of PBS containing tc-uPA (10 pmol in
10 µl of PBS) in the presence or absence of an excess molar
concentration (50- or 100-fold) of UK1 were overloaded on the fibrin
clot plate. After incubation at 37 °C for 1 h, the diameter of
the clear zone was photographed and measured.
Ten microliters of the substrate
(N-p-tosyl-Gly-Pro-Lys-p-nitroanilide,
Sigma) solutions (3 mM) was mixed with 0.2 ml of HEPES buffer, pH 8.0 (0.1 mM, 0.16 M NaCl, 0.1%
bovine serum albumin), and 10 µl of plasminogen (1 mg/ml), and
prewarmed to 25 °C. Aliquots of PBS containing tc-uPA (1.5 pmol in
10 µl of PBS) in the presence or absence of an excess molar
concentration (50- or 100-fold) of UK1 were preincubated for 10 min at
25 °C. The assays were started by the addition of the aliquots.
After 10 min of incubation at 25 °C, the reaction was stopped by the
addition of 50 µl of acetic acid. The digestion of the substrate was
determined by measuring increased absorbance of free
p-nitroaniline at 405 nm.
Binding Assay by a BIAcore Biosensor--
An assay of UK1 and
uPA binding was performed by real-time interaction analysis using a
BIAcore 2000TM apparatus (BIAcore AB, Uppsala, Sweden). All
experiments were performed at 20 °C using PBS buffer containing
0.005% (v/v) surfactant P20 (running buffer). Recombinant uPAR (20 µg/ml in 10 mM sodium acetate buffer, pH 5.0) was
immobilized on a CM-5 type sensor chip using the reagents and
procedures supplied with the Amine Coupling Kit (BIAcore AB). The
samples of UK1, sc-uPA, and tc-uPA were diluted in the running buffer
before injection. For general binding assay, each sample was injected
over the human uPAR at a flow rate of 10 µl/min. The kinetics of
binding was measured at a buffer flow of 30 µl/min, including an
injection phase of 3 min followed by dissociation for 5 min. Sensor
chips were regenerated at the end of each run by the injection of 0.05 M acetic acid, 0.25 M NaCl. The
BIAevaluationTM software was used for the
subtraction of bulk effects (flow cell 1) and a blind curve obtained
with buffer alone to compensate for drift. The sensorgram was analyzed
by non-linear curve fitting using the BIAevaluationTM
software, version 3.0.2, assuming single-site association and dissociation models (1:1 binding with drift base line). Optimal sections of each curve were selected, and the applicability of the
chosen model was ascertained by evaluating the resulting fitted curves,
using the residual plot function of the software (requiring a random
point scattering for the fit to be accepted) as well as calculation of
Cellular Localization of Fluorescein-labeled UK1 in HUVE
Cells--
Fluorescein-labeled UK1 was prepared by reaction of the
fluorescein succidimidyl ester (Pan Vera, Madison, WI) according
to the manufacturer's protocol. HUVE cells were trypsinized, seeded at
5 × 103 cells/cm2 on a 18- × 18-mm cover
glass placed in six-well culture plates, and cultured in EGM for
24 h. After washing with prewarmed PBS, HUVE cells were cultured
in EBM-2 supplemented with 1% FBS for 24 h. The cells were washed
three times with prewarmed (37 °C) EBM-2. A washed cover glass was
mounted in a modified chamber positioned on the platform of a inverted
laser confocal microscope (Carl Zeiss). To incubate cells in prewarmed
(37 °C) EBM-2 with fluorescein-labeled UK1 (1 µg/ml), exchange of
the 0.5-ml chamber volume was achieved with manual volume replacement
by pipetting from the above. After 30 min, the chamber was washed with
prewarmed (37 °C) EBM-2, and then fluorescence was observed with a
confocal laser scanning microscope (Carl Zeiss LSM510). The cells were incubated with 10 µg/ml Hoechst 33342 for 20 min, washed with EBM-2,
and then fluorescence was observed with a fluorescence microscope (Carl Zeiss).
Effect of UK1 on the Concentrations of Ca2+ in the
HUVE, NIH 3T3, and HEK-293 Cells--
Cells were trypsinized and
seeded, at 5 × 103 cells/cm2, on a 18- × 18-mm cover glass placed in six-well plates. HUVE cells were cultured
in EGM for 24 h, and NIH 3T3 and HEK-293 cells were cultured in
DMEM supplemented with 10% FBS for 24 h. After washing with
prewarmed PBS, HUVE cells were cultured in EBM-2 supplemented with 1%
FBS for 24 h, and NIH 3T3 and HEK-293 cells were cultured in DMEM
for 24 h. HUVE cells were washed three times with prewarmed (37 °C) EBM-2, and NIH 3T3 and HEK-293 cells were washed with prewarmed (37 °C) DMEM. The cells on the cover glass were incubated in the medium containing 4.4 µM fluo-3AM (Molecular
Probes, Eugene, OR) for 40-60 min. After washing the cells three times
with prewarmed (37 °C) EBM-2 or DMEM, the washed cover glass was
mounted in a modified chamber positioned on the platform of a ZEISS
inverted laser confocal microscope. The cells in prewarmed (37 °C)
EBM-2 or DMEM were incubated with UK1 (5 µg/ml) as described in the above, and the fluo-3AM fluorescence images were measured at a given
time by a confocal laser scanning microscope (Carl Zeiss LSM510).
Production and Purification of UK1--
Because the kringle domain
(UK1) of urokinase has no glycosylation site (7), we expressed it in
bacteria. The recombinant plasmid constructed for UK1 expression
contains 6 histidine residues at the carboxyl-terminal end. Most of the
expressed protein appeared in inclusion bodies (IB) as an insoluble
form. The solubilized IB protein was purified using a
Ni+-chelate column, refolded in vitro, and
purified to homogeneity by size exclusion chromatography as described
under "Experimental Procedures" (Fig.
1A). The yield of purified
protein was about 20% of the isolated IB. The purified UK1 migrated on
SDS-PAGE as a single band of about 13-14 kDa under reducing
conditions. Under non-reducing conditions, it migrated a little faster,
corresponding well with the expected molecular weight (Fig.
1B). Mass spectrometric analysis indicated a molecular
mass of 11,593 Da, in agreement with the calculated mass of
11,595 Da (6 Da have been subtracted due to the three disulfide bonds
formed). From the amino-terminal sequencing, the sequence of the first
6 amino acids, MDKSKT, matched exactly the predicted sequence of the
recombinant protein.
Dose-dependent Inhibition of Endothelial Cell
Proliferation by UK1--
Purified UK1 was assayed for its inhibitory
activity on BCE cell growth stimulated by bFGF (1 ng/ml). UK1 exerted a
potent growth inhibitory effect on bFGF-stimulated BCE cells in a
dose-dependent manner (Fig.
2A). The concentration of UK1
required to reach 50% inhibition (ED50) was about 80 nM, similar to that for angiostatin. On the other hand, uPA
itself did not affect the proliferation of BCE cells. BCE cells exposed
to UK1 for 30 min and washed prior to bFGF addition resulted in a
dose-dependent inhibition of cell proliferation similar to
the cell proliferation inhibition pattern observed without UK1 removal
(data not shown). The proliferation of HUVE cells induced by bFGF,
VEGF, or EGF was also inhibited in a dose-dependent manner
(Fig. 2B). The anti-endothelial proliferative activity of
UK1 does not seem to depend on a specific growth factor. UK1 produced
as a soluble protein from a Pichia expression system also
showed a similar potency in inhibition of HUVE cell proliferation induced by several growth
factors.2 However, the
addition of high levels of UK1 (up to 640 nM) did not
affect the proliferation of NIH 3T3 fibroblast and HEK-293 epithelial
cells under the same culture conditions (data not shown).
As shown in Fig. 2C, the inhibitory activity of UK1 on
endothelial cell proliferation was blocked by an anti-UK1 antibody, whereas neither anti-uPAR antibody nor control IgG blocked its inhibitory activity. We confirmed the interaction of UK1 and the anti-UK1 antibody by an immunoprecipitation method.
Inhibition of VEGF- or uPA-induced Endothelial Cell Migration by
UK1--
Because the migration response of the endothelial cells is
better with VEGF than bFGF (40), VEGF-induced migration was employed to
evaluate anti-migratory activity of UK1. VEGF (2 ng/ml) was used in the
low chamber to stimulate the migration of HUVE cells subcultured to
passage P3-P4. In this assay, angiostatin inhibited the VEGF-induced
HUVE cell migration in a dose-dependent manner (data not
shown). As shown in Fig. 3, UK1 also
displayed a dose-dependent inhibition in the concentration
range of 10-10,000 ng/ml, with an IC50 value of about 1 nM. In the absence of VEGF, we could not detect any effect
of UK1 (at up to 1,000 ng/ml concentration) on migration of HUVE
cells.
Because uPA itself is able to stimulate endothelial cell motility at
nanomolar concentrations (41), we tested whether UK1 can block the
migration of endothelial cells induced by uPA. As in the modified
Boyden chamber method using VEGF, UK1 blocked the migration of HUVE
cells induced by uPA in a dose-dependent manner in wound
migration assay (Fig. 3B).
Anti-angiogenic Activity of UK1 in the Chick Embryo--
To
evaluate the possible ability of UK1 to inhibit in vivo
angiogenesis, we used the chick chorioallantoic membrane assay (37,
38). Over a concentration range of 1-100 µg/embryo, UK1 inhibited
angiogenesis in a dose-dependent manner (Fig.
4). With repeated doses of 20 µg/embryo, we confirmed that the inhibition is reproducible. No
toxicity was observed in the embryos tested.
The Effects of UK1 on Plasminogen Activation by uPA--
Movement
of endothelial cells involves proteolysis of the extracellular matrix.
Two families of matrix degradation proteases, the plasminogen activator
and the matrix proteinase system, can, in concert, degrade most
extracellular proteins (2). Therefore, we examined whether UK1 can
inhibit plasminogen activation by activated uPA. In a fibrin clot lysis
analysis, the presence of a 50- or 100-fold molar excess of UK1 did not
change any activity of tc-uPA for plasminogen activation (Fig.
5). In a more sensitive chromogenic
substrate assay, UK1 also did not inhibit the activity of tc-uPA at
all. The velocities (nanomoles of p-nitroaniline released/min) of amidolytic activity of plasmin generated by tc-uPA were almost the same in the absence of UK1 (1.34 ± 0.05), and the
presence of a 50-fold molar excess of UK1 (1.37 ± 0.19) or a
100-fold molar excess of UK1 (1.35 ± 0.02). The concentration of
tc-uPA (7.5 nM) used in this assay was chosen in the
concentration range (0.5-10 nM) showing the linear
relationship between enzyme concentration and final cleaved chromogenic
substrate amount in the presence of excess amount of plasminogen and
its chromogenic substrate. From these results, we concluded that UK1
does not inhibit the proteolytic activity of uPA and, thereby, does not affect plasminogen activation by activated uPA.
Analysis of the Interaction of UK1 with Immobilized
uPAR--
Although the amino-terminal growth factor-like domain of
urokinase (aa 4-43) is known as a major binding site for uPAR (42), the possible presence of more than one kind of interaction was supported by some reports (43, 44). In an effort to re-evaluate any
interaction of UK1 with uPAR, an in vitro study was carried out using BIAcore. Recombinant human uPAR was immobilized directly to a
carboxylated dextran matrix chip through amine groups on uPAR, and then
UK1, or uPA was injected over the uPAR surface. Representative
sensorgrams are shown in Fig. 6
(A and B). sc-uPA was observed to bind with high
affinity (ka = 1.66 ± 0.2 × 106 M Endothelial Cell-specific Internalization and Nuclear Localization
of UK1--
To investigate the pattern of interaction of UK1 with
endothelial cells, HUVE cells were incubated with fluorescein-labeled UK1 at 37 °C for 30 min. When cell-associated UK1 was detected by a
confocal microscope, UK1 appeared to have been taken up and distributed
in both nucleus and cytosol of these cells (Fig.
7A). Therefore, we addressed
whether internalization of UK1 is specific to endothelial cells. After
treatment of NIH 3T3 and HEK-293 cells with the fluorescein-labeled
UK1, and we observed its movement for 30 min. Internalization of UK1
was not detected in NIH 3T3 and HEK-293 cells during a period of 30 min, in contrast to HUVE cells (Fig. 7, B and C,
respectively).
UK1-induced Transient Elevation of Ca2+ Concentration
in Endothelial Cells--
Because intercellular Ca2+
concentration ([Ca2+]i) regulates cell
migration (45, 46), progression through the cell cycle (47, 48), and
apoptosis (49, 50), we examined if any change in
[Ca2+]i occurs upon exposure to UK1. HUVE cells
were first equilibrated with 4.4 µM fluo 3-AM for 40-60
min, washed with serum-free medium, and then incubated with UK1 (5 µg/ml). As shown in Fig. 8A,
UK1 elicited two- to 3-fold transient increase of Ca2+
levels in cytosol and nucleus within 2 min and returned to the base
line in 3 min. The second lower transient mount of Ca2+
increase was followed after 5 min. The transient Ca2+ flux
by UK1 was not observed in non-endothelial cells tested, i.e. NIH 3T3 and HEK-293 cells (Fig. 8B).
uPA has one kringle domain, which shares some homology with the
kringles of plasminogen, but is distinct in not being capable of
binding lysine residue. The kringles 1-4 (angiostatin), or kringle 5 of plasminogen, are known for their anti-angiogenic activity by
selectively inhibiting endothelial cell growth (14, 21). In this study,
our data demonstrate the potent anti-angiogenic activity of the kringle
domain of urokinase, although urokinase itself is pro-angiogenic (2,
3). Recombinant UK1 selectively inhibited endothelial cell
proliferation and suppressed the migration of VEGF- or uPA-stimulated
HUVE cells. Its anti-angiogenic activity was also shown in CAM assays
in vivo. More importantly, UK1 was selectively and rapidly
internalized and elicited a transient Ca2+ flux in
endothelial cells.
Kringle domains are independent structural and functional folding units
found in several proteins involved in blood coagulation and
fibrinolysis (51). In an effort to elucidate the structure and
function of kringle domains, several kringle domains, including plasminogen kringles 2, 3, and 2-3 and tissue-type plasminogen activator kringle 2 have been successfully produced by E. coli expression and a refolding process (52-55). The structures
of these recombinant kringles have been proven to be close to native
kringle fragments by identification of disulfide bonds, determination of lysine-binding capacity, NMR spectroscopy, or x-ray crystallography. The recombinant kringles 1, 2, 2-3, or 4 of plasminogen obtained from
a refolding process were also proven to be functional in angiogenesis
inhibition (17). In our work, we were able to produce the recombinant
kringle domain of urokinase as a functional form by a similar refolding
process, and this protein was as functional as the UK1 produced in a
soluble form by Pichia. A successful refolding of the
functional recombinant kringle domain of urokinase was also reported by
another group, who showed that the monoclonal antibody interacting with
the kringle domain of uPA recognizes the intact recombinant kringle
domain but not its reduced form (56).
Amino acid sequence alignment of the individual kringle domain of human
plasminogen shows that PK1, PK2, PK3, PK4, and PK5 display considerable
sequence similarity to each other (46-57%) (17, 21). UK1 from
urokinase, although similar, has somewhat lower sequence identity with
the plasminogen kringles: e.g. 36.5% to PK1, 32.9% to PK2,
30.4% to PK3, 37.8% to PK4, and 40.2% to PK5 (Fig.
9). One stretch of amino acids, between
Val and Leu (underlined in Fig. 9) is distinct in UK1,
although well conserved between human and bovine species (79%
identity). Despite these differences, UK1 exhibits anti-angiogenic
activity, as demonstrated by our current data.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside was added to a final
concentration of 1.0 mM. Cells were incubated for an
additional 3 h at 37 °C and finally harvested by centrifugation
for 15 min (8,000 × g). Refolding and purification of
UK1 were carried out as follows. The cell pellet was suspended in a
lysis buffer (20 mM Tris-Cl, pH. 8.0, 50 mM
NaCl, 1 mM MgCl2, 0.1 mM
CaCl2, 20 µg/ml RNase A, 50 µg/ml DNase I) and
subjected to sonication for 15 min. The soluble and insoluble fractions
were separated by centrifugation for 30 min at 20,000 × g. The insoluble fraction was dissolved in buffer A (20 mM Tris-Cl, pH 7.9, 0.5 M NaCl, 6 M
urea). After centrifugation, the supernatant solution was applied to a
ProBondTM resin His tag affinity column (Invitrogen,
Carlsbad, CA). The column was washed with buffer A containing 20 mM imidazole, and protein was eluted with buffer A
containing 1 M imidazole. The eluant was stepwise diluted
with 0.1 M Tris-Cl, 0.15 M NaCl, pH 8.0, followed by addition of reduced (1 mM) and oxidized (0.1 mM) glutathione and incubation at room temperature
overnight. The solution was concentrated by ultrafiltration and
dialyzed against 20 mM sodium acetate buffer (pH 5.5) and
distilled water. The dialyzed solution was lyophilized, dissolved in 20 mM sodium phosphate, 150 mM NaCl, pH 7.4, and
applied to a Bio-prep SE 100/17 column for the final purification. The
endotoxin content of purified UK1 used for cell assays did not exceed
0.6 endotoxin unit/mg of protein.
2 (requiring a
2 value below 10 in both
the association and dissociation phase for acceptance). Calculated
values for the association constant (ka) and the
dissociation rate constant (kd) were used for
deriving the equilibrium dissociation constant (Kd = kd/ka).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification and SDS-PAGE analysis of
UK1. A, samples obtained from the purification steps
were subjected to electrophoresis on a 14% SDS-polyacrylamide-gel in
Tris-glycine buffer under reducing conditions; the gel was stained with
Coomassie Blue. Lane 1, total protein of E. coli;
lane 2, soluble fraction of E. coli lysate;
lane 3, insoluble fraction from E. coli lysate;
lane 4, flow-through fraction from the His tag
Ni+ affinity column; lane 5, sample eluted from
the His tag affinity column; lane 6, purified UK1 after the
size exclusion column. B, purified UK1 was analyzed under
reducing condition (lane 1) and non-reducing condition
(lane 2).
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Fig. 2.
Inhibition of endothelial cell proliferation
by UK1. A, inhibition of BCE cell proliferation induced
by bFGF. Anti-proliferative activity was assayed with BCE cells in the
presence of 1 ng/ml bFGF for 72 h as described under
"Experimental Procedures." The effects of UK1 (closed
circles) were compared with those of angiostatin (open
circles) and sc-uPA (closed triangles). Values, given
as percentages of inhibition, represent the mean of three
determinations (±S.E.). B, inhibition of HUVE cell
proliferation by UK1. The cells were cultured with 3 ng/ml bFGF, 10 ng/ml VEGF, or 10 ng/ml EGF with or without various amounts of UK1 for
18 h, followed by incubation with [3H]thymidine as
described under "Experimental Procedures." C represents
a condition of no growth factor and no inhibitor. Each value represents
the mean ± S.E. C, effects of anti-UK1 and anti-uPAR
antibodies on UK1 inhibitory activity. The cells were cultured in the
absence or presence of 3 ng/ml bFGF with or without 320 nM
UK1 preincubated with control antibody (100 µg/ml) or anti-UK1
antibody (100 µg/ml). In the case of anti-uPAR antibody, it was added
directly to the cells 45 min before addition of UK1. A
[3H]thymidine incorporation assay was followed. Results
are expressed as the percentages of DNA synthesis in the absence of
inhibitor UK1 (as 100%).
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Fig. 3.
Inhibition of endothelial cell migration by
UK1. A, effect of UK1 on VEGF-induced migration.
Anti-migratory activity of UK1 was examined at several doses in a
modified Boyden chamber-based assay. HUVE cells were preincubated with
the test sample at each concentration for 30 min, and VEGF (2 ng/ml)
was used as a chemoattractant to elicit the migration of cells. Control
cells (C) were not treated with UK1 and VEGF. B,
effect of UK1 on uPA-induced endothelial cell migration. HUVE cells
were plated in six-well plates, and cell migration was quantified by
the scratch method as described under "Experimental Procedures."
After scratch, the cells were treated with EBM-2 supplemented with 1%
FBS (C), or 1 µg/ml angiogenin (A), 200 ng/ml
uPA, 200 ng/ml uPA plus 10 ng/ml UK1, or 100 ng/ml UK1, or 500 ng/ml
UK1 in EBM-2 supplemented with 1% FBS.
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Fig. 4.
Inhibitory effect of UK1 on angiogenesis in
chick CAMs. Thermanox coverslips containing a
variable amount of UK1 were applied to the CAMs of 4.5-day-old chick
embryos. After 48 h, the avascular zone ( 5 mm in diameter) of
each CAM was observed. Inhibition (%) of the treated samples was
evaluated by the number of CAMs with avascular zones over the total
number of CAMs (indicated above each column). The
representative appearance of chick CAMs implanted with a coverslip
containing 20 µg of albumin (upper) or UK1
(lower) are shown. The marked black line
indicates the location of the implanted coverslip. An avascular zone
around the coverslip was observed only with UK1 (lower
panel).
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Fig. 5.
Effect of UK1 on uPA-mediated fibrin clot
lysis. tc-uPA (10 pmol) in the presence or absence of an excess
molar concentration (50- or 100-fold) of UK1 was added in the clots
prepared by adding thrombin to human fibrinogen. After incubation at
37 °C for 1 h, the clear zones generated were
photographed.
1s
1,
kd = 3.05 ± 0.3 × 10
3
s
1, and Kd = 1.84 nM) as
expected, whereas UK1 did not show any binding under identical
conditions over a concentration range of 15.6-250 nM.
Furthermore, it also did not block the binding of uPA to the
immobilized receptor when added at various molar ratios of uPA and UK1
(1:1, 1:2, or 1:4) (Fig. 6C). Thus, the observed UK1
inhibitory effects on in vitro endothelial proliferation, migration, and in vivo angiogenesis do not seem to be
related to the uPA receptor.
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Fig. 6.
Real-time interaction analysis of UK1 binding
to immobilized recombinant uPA receptor. Ligand binding curves
were obtained by automated measurements of surface plasmon resonance
(BIAcore Technology). Recombinant uPAR was covalently coupled to a
carboxymethyl-type sensor chip as described under "Experimental
Procedures." Immobilization of uPAR was performed in flowcell 2, while flowcell 1 was immobilized with bovine serum albumin. Samples of
uPA (A and C) and UK1 (B and
C) were subjected to real-time interaction analysis with the
coupled sensor chips, using a BIAcore 2000 instrument. For the
competition analysis, the concentration of tc-uPA was fixed at 150 nM, and the concentration of UK1 was increased from 150 to
600 nM (C). A different level of immobilization
was applied for the kinetics (Rmax = 780, A and B) and for the competition analysis
(C).
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Fig. 7.
Internalization and nuclear translocation of
UK1 is specific for endothelial cells. Fluorescein-labeled UK1 (1 µg/ml) was added to HUVE (A), NIH 3T3 (B), and
HEK-293 (C) cells, respectively, and incubated for 30 min.
Internalization and localization of the fluorescein-labeled UK1 was
detected by a confocal laser scanning microscope (Carl Zeiss LSM 510).
The nuclei were stained with Hoechst 33342, and their location was
detected by using a Carl Zeiss fluorescence microscope. a,
Hoechst fluorescence; b, fluorescein fluorescence;
c, transmission; d, merged images.
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Fig. 8.
Effect of UK1 on concentrations of
Ca2+ in HUVE, NIH 3T3, and HEK-293 cells.
Fluo-3-AM-loaded cells were incubated with 5 µg/ml UK1 for 10 min.
The fluo-3-AM fluorescence intensities in the HUVE (A), NIH
3T3 (B), and HEK-293 (B) were measured at a given
time by a confocal laser scanning microscope (Carl Zeiss LSM510).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Comparison of the amino acid sequences of
plasminogen kringles (PK1-PK5) and UK1. The sequences were
aligned according to their six conserved cysteine residues (indicated
by stars) and by reference to the homologous 80-residue PK5.
Amino acid residues that are conserved between UK1 and one or more
plasminogen kringles are shaded.
The individual kringles of plasminogen have been shown to exhibit differential effects on endothelial cell proliferation and migration, probably related to their molecular diversity (17, 20). For example, plasminogen kringles 1, 2, 4, and 5, but not 3, have lysine binding capability (17, 21). Kringle 4 shows a marked functional difference in its anti-endothelial cell proliferative and migratory activities from other kringles. It alone is comparatively inefficient in the suppression of endothelial cell growth (17) but potent in inhibiting endothelial cell migration (20). The lack of lysine binding ability of UK1 does not appear to influence its anti-proliferative activity, which agrees with earlier studies that showed no influence of lysine on plasminogen kringles in anti-proliferation of endothelial cells (17, 32). Interestingly, UK1 is also potent in inhibiting the migration of endothelial cells induced by VEGF. These results taken together strongly support the conclusion that lysine-binding capability has no relationship with anti-endothelial cell activities, even though it seems to be important in the fibrinolysis process. Rather, we presume that other structural characteristics may be involved in anti-endothelial cell activity and specificity.
It is unlikely that the anti-angiogenic activity of UK1 is related to uPAR interaction, because no UK1 binding to immobilized uPA receptor and no competitive inhibition of uPA binding were detected in real-time interaction analysis. Rather than that, the specific internalization of UK1 in endothelial cells may imply that another cellular receptor exists in endothelial cells. Rapid internalization and nuclear localization in endothelial cells may explain in part why exposure of just 30 min can exert an inhibition of BCE cell proliferation. It is not certain at present whether its receptor may be directly involved in the mechanism of action of the kringle domain or whether it may only affect cell-specific entry. Further studies will be done to elucidate this specific receptor and its relevance to anti-endothelial cell activities. Recently, another recombinant kringle domain construct (aa 42-166) of urokinase was reported to have a binding site of low affinity on an unidentified cell surface target for muscle cells and to induce migration of these cells (56). Because recombinant UK1 used here only includes aa 45-135, it may not have the same binding pattern as the one reported in that study. In fact, treatment with UK1 alone did not induce migration of HUVE cells in our hands. In addition, the connecting peptide (aa 136-143) has been reported to inhibit cancer cell invasion and endothelial cell migration as a non-competitive antagonist of the uPA-uPAR interaction (57).
Up to now, the molecular mechanism of action of angiostatin on
endothelial cell is not clear. ATP synthase, angiomotin, and integrin
v
3 were independently found to bind to
angiostatin (58-60). Cell cycle arrest and apoptosis resulting from
treatment of angiostatin may occur by additional independent mechanisms (61-63). In this study, we have demonstrated that angiogenesis inhibitor UK1 elicited the 2- to 3-fold increase of transient Ca2+ flux into cytosol and nucleus within a 2-min
period, specifically in endothelial cells. These results suggest
that nuclear localization of UK1 and Ca2+ signaling may be
involved in the cellular function of UK1 in the endothelial cells. This
notion is also supported by the report of Jiang et al. (64)
that potent angiogenesis inhibitors angiostatin and endostatin
transiently elevate [Ca2+]i in primary cultures
of bovine and human endothelial cells. They also reported an
interesting result that endostatin pretreatment of endothelial cells
for 18 h reduced ~70% the magnitude of acute Ca2+
signals elicited subsequently by VEGF and bFGF. This result may explain, in part, why UK1 and uPA can have opposite effects on endothelial cell migration, although both UK1 and uPA increase [Ca2+]i. It can also be presumed that UK1 may
inhibit the alterations of Ca2+-mobilizing properties,
because it was suggested that the alterations of Ca2+ store
site-related Ca2+ mobilizations, i.e.
Ca2+ sequestration, release, and thapsigargin-evoked
Ca2+ entry, may be involved in the cellular mechanisms of
endothelial migration (65). Further studies on how UK1 acts in
endothelial cells with respect to Ca2+ signaling may
provide some insight in more detail into this question.
An increasing number of endogenous angiogenesis inhibitors that become
active as a cryptic fragment of an inactive precursor protein have been
identified in recent years. For example, angiostatin derived from
plasminogen inhibits endothelial cell proliferation and blocks
angiogenesis in vivo (14). However, plasminogen is inactive.
Similar cases were reported for endostatin of collagen XVIII (15) and
the 16-kDa amino-terminal fragment of human prolactin (66). In these
cases, it was suggested that proteolytic processing of a large protein
might change the conformational structure of the original molecular or
expose new epitopes that are anti-angiogenic. At present, we don't
know whether the kringle domain itself circulates in blood or whether
it exists in the extracellular matrix of an angiogenic area, even
though it has three potential cleavage sites, Lys46-Ser47,
Lys135-Lys136, and
Glu143-Lys144, near the kringle domain of
urokinase (7). It remains to be studied further, together with the
mechanism of action of UK1 in its anti-angiogenic activity.
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ACKNOWLEDGEMENTS |
---|
We thank Jun-Yeoung Jeon for technical support for the BIAcore biosensor assay; Hyun-Joo Ku for assistance in the CAM assay; Dr. Jeong-Im Sin, Chung-Kwon Kim, and Hyun-Kyung Kim for assistance in antibody generation; and Dr. Edith C. Wolff, NIDCR, National Institutes of Health, Bethesda, MD, for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by grants from the Korea Health 21 R&D Project, Ministry of Health & Welfare (02-PJ1-PG10-20802-0007), the National Projects of the Korean Ministry of Science and Technology (98-G-08-03-A-26), and the Korea Science and Engineering Foundation through the Cancer Metastasis Research Center at Yonsei University and the Vascular System Research Center at Kangwon National University.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.
§ These authors contributed equally to this work.
¶ To whom correspondence should be addressed: Cancer Research Institute, Catholic Research Institutes of Medical Science, The Catholic University of Korea, Banpo-dong 505, Seocho-ku, Seoul 137-701, Korea. Tel.: 82-2-590-2404; Fax: 82-2-532-0575; E-mail: youngjoe@catholic.ac.kr.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212358200
2 H.-K. Kim, Y.-K. Hong, H.-E. Park, S. H. Hong, and Y. A. Joe, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: uPA, urokinase-type plasminogen activator; sc-uPA, single-chain uPA; tc-uPA, two-chain uPA; uPAR, urokinase-type plasminogen activator receptor; UK1, uPA kringle; PK, plasminogen kringle; HUVE, human umbilical vein endothelial; BCE, bovine capillary endothelial; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; EGF, epidermal growth factor; IB, inclusion bodies; CAM, chorioallantoic membrane; [Ca2+]i, intracellular Ca2+ concentration; aa, amino acid(s); PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; EGM, endothelial cell growth medium; EGM-2, endothelial cell basal medium; M199, medium 199.
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