Báo cáo khoa học: Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade pptx

Eur. J. Biochem. 269, 3945–3957 (2002) Ó FEBS 2002

doi:10.1046/j.1432-1033.2002.03068.x

Suppression of urokinase receptor expression by bikunin
is associated with inhibition of upstream targets of extracellular
signal-regulated kinase-dependent cascade
Hiroshi Kobayashi1, Mika Suzuki1, Naohiro Kanayama1, Takashi Nishida2, Masaharu Takigawa2 and
Toshihiko Terao1
1

Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan; 2Department
of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

Our laboratory showed that bikunin, a Kunitz-type protease
inhibitor, suppresses 4b-phorbol 12-myristate 13-acetate
(PMA)- or tumor necrosis factor-alpha (TNFa)-induced
urokinase-type plasminogen activator (uPA) expression in
different cell types. In addition to its effects on protease
inhibition, bikunin could be modulating other cellular events
associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress
the expression of uPA receptor (uPAR) mRNA and protein
in a human chondrosarcoma cell line, HCS-2/8, and two
human ovarian cancer cell lines, HOC-I and HRA. The
present study showed that (a) bikunin suppresses the
expression of constitutive and PMA-induced uPAR mRNA
and protein in a variety of cell types; (b) an extracellular
signal-regulated kinase (ERK) activation system is necessary
for the PMA-induced increase in uPAR expression, as
PD098059 and U0126, which prevent the activation of
MEK1, reduce the uPAR expression; (c) bikunin markedly

suppresses PMA-induced phosphorylation of ERK1/2 at
the concentration that prevents uPAR expression, but does
not reduce total ERK1/2 antigen level; (d) bikunin has no
ability to inhibit overexpression of uPAR in cells treated with
sodium vanadate; and (e) we further studied the inhibition of
uPAR expression by stable transfection of HRA cells with
bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that
bikunin downregulates constitutive and PMA-stimulated
uPAR mRNA and protein possibly through suppression of
upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or
transfected with bikunin gene.

Tumor cell invasiveness is a complex, multistep process that
involves cell attachment, the proteolysis of matrix components, and the migration of cells through the disrupted matrix
[1]. Activation of receptor-bound uPA on the cell surface
appears to play an important role in cancer cell invasion and

metastasis [2]. In a number of cancers, the expression of the
uPA and uPAR is required for the invasive phenotype [3,4].
uPAR binds uPA with high affinity with a Kd of  0.5 nM
[5,6]. It has been shown previously that increased levels of
uPA and uPAR correlate well with higher invasive phenotype
[7]. Most uPAR protein is concentrated at invasive foci [8]; it
accelerates plasmin formation at the cell surface. Overexpression of a human uPAR cDNA increased the ability of tumor
cells to penetrate a barrier of reconstituted basement
membrane. For colon cancer, a high uPAR expression was
strongly correlated with the poor prognosis in colon cancer
[9]. In contrast, exposure to anti-uPAR Ig [10], soluble uPAR
[11,12], or stable transfection with antisense uPAR cDNA

[13] rescues the invasiveness of tumor cells.
The uPAR protein is inducible by epidermal growth
factor (EGF), transforming growth factor-beta (TGF-b)
[14,15], hepatocyte growth factor (HGF) [16], vascular
endothelial growth factor (VEGF) [17], interferon gamma
(IFN-c), tumor necrosis factor-alpha (TNF-a) [18], and by
the tumor promoter, phorbol ester [5,19]. Activation of the
protein kinase C pathway by PMA has been reported to
increase uPAR mRNA in certain cell types [20]. An increase
in mRNA stability in response to PMA have been also
reported [21].
Bikunin [also known as urinary trypsin inhibitor (UTI)],
a light chain of the interalpha-inhibitor (IaI) family, is a

Correspondence to H. Kobayashi, Department of Obstetrics
and Gynecology, Hamamatsu University School of Medicine,
Hamamatsu, Shizuoka, Japan.
Fax: + 81 53 435 2309, Tel.: + 81 53 435 2308,
E-mail:
Abbreviations: ATF, N-terminal fragment of uPA; DIP, diisopropyl
fluorophosphate; DMEM, Dulbecco’s minimum Eagle’s medium;
EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; GraPDH,
glyceroaldehyde-3-phosphate dehydrogenase; HGF, hepatocyte
growth factor; HI-8, a C-terminal domain of bikunin; IaI, interalphainhibitor; IFN-c, interferon-gamma; PMA, 4b-phorbol 12-myristate
13-acetate; TGFb, tumor growth factor-beta; TNFa, tumor necrosis
factor-alpha; uPA, urokinase-type plasminogen activator; uPAR,
uPA receptor; UTI, urinary trypsin inhibitor; VEGF, vascular endothelial cell growth factor.
(Received 20 December 2001, revised 20 May 2002,
accepted 21 June 2002)

Keywords: bikunin; extracellular signal-regulated kinase;
urokinase-type plasminogen activator; uPA receptor; tumor
invasion.


Ó FEBS 2002

3946 H. Kobayashi et al. (Eur. J. Biochem. 269)

Kunitz-type protease inhibitor [22] and an effective
inhibitor of calcium influx in cell transporter system [23].
Bikunin also inhibits TNF-a-mediated translocation and
activation of PKC [24] as well as PMA-dependent
activation of PKC and MAP kinase cascade [25]. We
reported that it inhibits tumor invasion and metastasis
possibly through suppression of cell-associated plasmin
activity and expression of uPA mRNA and protein.
However, little is known concerning the potential role of
bikunin in the regulation of uPAR mRNA and its protein.
In this paper, the positive modulation of uPAR mRNA
and protein by PMA and the negative regulatory effects
by bikunin on uPAR gene expression in human cancer
cells (ovarian cancer cell lines HOC-I and HRA as well as
chondrosarcoma cell line HCS-2/8) are reported. We
undertook the present study to determine the role of
exogenously added bikunin on regulation of extracellular
signal-regulated kinase (ERK)-dependent uPAR expression. Further, bikunin transfection experiments were
carried out to determine whether endogenously produced
bikunin is necessary for inhibition of uPAR expression
possibly through suppression of an upstream target(s) of

ERK phosphorylation.

MATERIALS AND METHODS
Materials
4b-Phorbol 12-myristate 13-acetate (PMA), calphostin C
and staurosporin were purchased from Sigma, St Louis,
MO, USA. Human urokinase (high molecular mass twochain uPA) was a gift from Yoshitomi Pharmaceutical,
Osaka, Japan. Two-chain uPA was inactivated with
diisopropyl fluorophosphates (DIP) to form DIP-uPA,
as described previously [26]. The N-terminal fragment of
uPA (ATF) was purified, as described previously [27].
a1-antitrypsin was from Kaketsuken, Kumamoto, Japan.
Polyclonal anti-(phospho-ERK1/2) Ig and anti-(c-Jun) Ig
were from Santa Cruz Biotechnology. Polyclonal antiMEK1/2 Ig was from Transduction Laboratories. Total
ERK was detected using an antibody from Zymed (San
Francisco, CA, USA). A uPA-specific antibody, which
recognizes the N-terminus of uPA (#3471), polyclonal
rabbit anti-uPAR Ig (399R) and a specific ELISA for
uPAR (IMUBIND 893) were obtained from American
Diagnostica (Greenwich, CT, USA). Nonimmune anti(mouse/rabbit IgG) Ig and anti-(mouse/rabbit IgG) Ig
conjugated with horseradish peroxidase were from Dako
(Copenhagen, Denmark). Purified bikunin and recombinant C-terminal Kunitz domain II, HI-8 (Thr78-Asn147),
were provided by E. Morishita (Mochida Pharmaceutical
Co., Gotenba, Shizuoka). Bikunin derivatives [O-glycosidelinked N-terminal glycopeptide (bikunin-m1; Ala1-Lys21),
N-glycoside-linked C-terminal tandem Kunitz-domains
(bikunin-m2; Lys22-Leu143), bikunin klacking O-glycoside (bikunin-c; Ala1–Leu143), asialo bikunin (bikunin-a;
Ala1–Leu143), bikunin lacking N-glycoside (bikunin-n;
Ala1–Leu143)] were prepared as described previously
[28]. The MEK inhibitors, PD098059 and U0126, were
purchased from Calbiochem. [32P]dATP random prime

labeling Mega Prime kit was purchased from
Amersham Japan. All reagents used were of analytical
grade.

Cell culture
The human ovarian cancer cell lines HOC-I [29] and HRA
[30], as well as the human chondrosarcoma cell line HCS-2/8
[31], have been described previously. The HRA cells were
provided by Y. Kikuch and the HCS-2/8 cells were a gift
from M. Takigawa, both at Okayama University, Okayama, Japan. All cell lines stained negative for mycoplasma
contamination. Cells were maintained in RPMI-1640
medium (HOC-I and HRA) or Dulbecco’s minimum
Eagle’s medium (DMEM) (HCS-2/8) supplemented with
penicillin (100 mL)1), streptomycin (100 lgỈmL)1) and
10% heat-inactivated fetal bovine serum (Life Technologies,
Inc.; Rockville, MD, USA) at 37 °C in 5%CO2/air atmosphere. Before stimulation, cells were washed three times
with NaCl/Pi and incubated overnight in complete medium
containing 1% fetal bovine serum. The test drugs were
added and incubation was continued for different time
lapses. After culture, medium was aspirated and cells were
harvested and washed extensively. Immediately before
harvest, cell viability was consistently found to be > 90%.
The ELISA procedure we propose measures native IaI and
bikunin in human plasma (H. Kobayashi, M. Suzuki,
Y. Hirashima & T. Terao, unpublished data). EDTA-plasma
from healthy individuals revealed a mean level of  250 and
 10 lgỈmL)1 of IaI and bikunin, respectively. Therefore,
the bikunin concentration of culture medium containing 1%
fetal bovine serum may correspond to about  0.1 lgỈmL)1.
Plasmid construction and transfection into the

HRA cell line
The a1 microglobulin-bikunin cDNA was cloned by PCR
using the human liver cDNA library (Clontech, Palo Alto,
CA, USA) as a template with primers (P1: 5¢-ACCG
AGCCTCGAGGATATACCAAGGCAGAGGAGC-3¢,
P2: 5¢-ACTTGCAGAGCGGCCGCTTGTCAGTTGGA
GAAGC-3¢). Cloned cDNA was inserted into the XhoI–
NotI site of the pCIneo vector (Clontech). In order to remove
the internal sequence of 500 bases from this vector, an
inverted PCR was performed with primers (P3: 5¢-GAGAG
TCAGCGCTGCTGTGCTACCCCAAGA-3¢, P4: 5¢-ACT
TGGATGTTGTCGGGCGGCGTTGGCACA-3¢). The
resulting PCR product was digested with Eco47III and selfligated. Finally, using the vector as a template, bikunin
cDNA was cloned by PCR with primers (P1, P2), and
inserted into the SmaI site of pCMV (cytomegalovirus
promoter)-IRES (internal ribosome entry site)-bsr (blasticidin S hydrochloride resistant gene) vector [32]. The bikunin
expression vector pCMV-bikunin-IRES-bsr and the control vector pCMV-luciferase-IRES-bsr [32] encoding LUC
(luciferase) were transfected into HRA cells by the standard
calcium phosphate precipitation method [33]. The cells were
selected in the presence of 10 mgỈmL)1 blasticidin S hydrochloride (Funakoshi Co. Ltd, Tokyo, Japan). Resistant
clones were obtained after four weeks, and bik+ clones and
luc+ clones were obtained. The cells were subsequently
maintained in the presence of 10 mgỈmL)1 blasticidin S
hydrochloride.
The initial bik+ mass cultures were subjected to at least
two rounds of subcloning in order to obtain stable bik+
clones. DNA sequencing verified the correct insertion of the
bik cDNA. Finally, HRA bik+ tumor cell clones with bik

Ó FEBS 2002

overexpression were confirmed by immunocytochemical
staining and Western blot analysis [34]. cDNA synthesized
from luciferase transfected tumor cells (luc+ clones) was
used as a control.
Northern blot analysis
Northern blot analysis was performed using standard
methods. Ten micrograms of RNA were separated in
1.2% agarose gels and blotted onto Hybond N+ membranes. Prehybridization and hybridization were performed
in 50% formamide at 42 °C with 5 · 106 c.p.m.ỈmL uPAR
cDNA probe, as described previously [35], and filters were
reprobed with the cDNA for glyceraldehyde-3-phosphate
dehydrogenase to correct for the amount of RNA loaded
onto the filters. A 1.1 kb XbaI–EcoRI fragment of uPAR
cDNA [36] was radiolabeled with [32P]dATP via random
hexamer primer extension and used as hybridization probe.
After each hybridization, the membranes were washed and
exposed on Kodak BioMax MS-1 film at )70 °C. Filters
were quantitated by scanning densitometry using a Bio-Rad
model 620Video Densitometer with a ID ANALYST software
package for Macintosh.
Western blot
uPAR, phosphorylated and total ERK were detected by
immunoblot analysis. To detect uPAR protein, conditioned
media were individually harvested and the remaining
monolayers were scraped and lysed in 50 mM Hepes, 0.5 M
NaCl, 0.05% Tween-20, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, 10 lgỈmL)1 E-64, 10 lgỈmL)1
leupeptin to prepare cell lysates. For the detection of ERK
proteins, the cells were extracted with 1.0% Nobidet P-40,

50 mM Hepes, 100 mM NaCl, 2 mM EDTA, 1 lgỈmL)1
leupeptin, 0.4 mgỈmL)1 sodium vanadate, 0.4 mgỈmL)1
sodium fluoride, 5 mgỈmL)1 dithiothreitol, pH 7.4. Samples
were stored at )20 °C and used only once after thawing.
Equal amount of cellular protein (50 or 20 lg per lane) were
subjected to 12% SDS/PAGE and transferred to poly(vinylidene difluoride) membranes. Filters were probed with
primary antibodies and revealed with a biotinylated anti(rabbit/mouse IgG) Ig and avidin-peroxidase. Peroxidase
was detected by enhanced chemiluminescence.
uPAR protein assay: measurement of ligand binding
DIP-uPA was radioiodinated with carrier-free Na-125I, as
described previously [37]. DIP-uPA was labeled with 125I,
resulting in a specific radioactivity of 4900 c.p.m.Ỉng)1,
without loss of latent protease activity. Binding assays were
performed at 4 °C as described previously [37]. In brief,
confluent monolayers of HRA were grown in 24-well plate
wells using complete medium supplemented with 10% fetal
bovine serum. The cells were washed twice each with
medium supplemented with 1% fetal bovine serum. The
cells were maintained overnight medium supplemented with
1% fetal bovine serum. Monolayers were cooled to 4 °C
and washed twice with Tyrode’s-Hepes solution containing
1% fetal bovine serum (washing buffers). Prior to performing binding studies, confluent cultures were subjected to a
mild acid wash (50 mM glycine-HCl, pH 3.0, 0.1 M NaCl)
to dissociate uPAR-associated ligands. The cells were then

Suppression of uPAR by bikunin (Eur. J. Biochem. 269) 3947

incubated at 4 °C for 2 h in binding buffer (150 mM NaCl,
10 mM Hepes, 2 mM CaCl2, 1 mM MgCl2, 1% fetal bovine
serum) supplemented with 10 nM [125I]DIP-uPA and

washed four times. To determine nonspecific binding,
50-fold higher concentrations of unlabeled DIP-uPA were
added to the incubate. Unbound [125I]DIP-uPA was
removed and the contents of the wells were removed using
1 M NaOH and radioactivity of the cell lysates was
measured using a gamma-counter. The cells bound
[125I]DIP-uPA in a specific and saturable manner at 10 nM
[125I]DIP-uPA. Each experimental point was performed in
at least triplicate wells. The nonspecific binding, determined
as the percent of input counts bound in the presence of
0.5 lM unlabeled uPA, was approximately 9% and was
subtracted from all raw data to give the specific bound
counts.
Invasion assay
Invasion assays were performed essentially as described
previously [25]. The effects of agents that alter the activity of
uPA/uPAR expression, including neutralizing monoclonal
antibodies against uPA and uPAR, on the invasiveness of
HRA cells were determined by measuring the ability of cells
treated with these agents to pass through a layer of the
extracellular matrix extract Matrigel coating a filter using
chemoinvasion chambers.
Statistical analysis
Data are presented as mean ± SD. All statistical analysis
was performed using STATVIEW for Macintosh. The Mann–
Whitney U-test was used for the comparisons between
different groups. P < 0.05 was considered significant.

RESULTS
Induction of uPAR mRNA by PMA in HRA cells

Unstimulated cells (HRA, HOC-I and HCS-2/8) expressed
different levels of 1.1 kb uPAR transcripts (Fig. 1A). uPAR
mRNA in HRA cells incubated with PMA (100 nM) for 3 h
was increased 7.5-fold as compared with the unstimulated
cells, which appeared at 1 h, peaked at 3 h and declined at
24 h (Fig. 1B). The effect of PMA on uPAR expression was
dose-dependent at PMA concentrations of 10–100 nM, with
a maximum increase seen after treatment of HRA cells with
100 nM of PMA (Fig. 1C). Similar PMA effects on uPAR
mRNA were found in the two other cancer cell lines (data
not shown).
Suppression by bikunin of uPAR mRNA accumulation
by PMA
We showed previously that bikunin plays an important
role in signaling of PMA [24] and TNFa [25]. It is of
interest to determine whether bikunin also affects the
expression of PMA-induced uPAR mRNA (Fig. 2). When
concentrations of bikunin (0.1–1 lM) known to inhibit
the uPA production [25] were added in the presence of
100 nM PMA (data not shown) to HRA cells, there was a
dose-dependent inhibition of  50 and  70% of the
uPAR mRNA level at concentrations of 0.1 and 1 lM,


Ó FEBS 2002

3948 H. Kobayashi et al. (Eur. J. Biochem. 269)

Fig. 1. Induction of uPAR mRNA by PMA in tumor cells. (A) Relative levels of uPAR mRNA in HRA, HOC-I, and HCS-2/8 cells. Cells were
grown to 90% confluence. Total cellular RNA was extracted and separated on 1.2% agarose/formaldehyde gel and transferred to Hybond N+

membrane. Filters were hybridized with 32P-labeled uPAR cDNA or with 32P-labeled GAPDH cDNA probe. Top, representative autoradiograms;
Bottom, Levels of uPAR mRNA expression as quantified by densitometric scanning. (B) Stimulation of uPAR gene expression in HRA cells. HRA
cells were grown to 90% confluence and then stimulated with PMA (100 nM) for the indicated periods of time. C, PMA stimulates uPAR gene
expression in a dose-dependent manner. HRA cells were incubated for 3 h with different doses of PMA. Results are the mean ± SD of four different
determinations, with unlike superscripts (a–d) are different (P < 0.05).

We examined the capacity of truncated proteins [deglycosylated bikunin (bikunin-c) and HI-8] and a related protein
(a1-antitrypsin) to suppress PMA-stimulated uPAR mRNA
expression. We showed that bikunin might inhibit uPAR
expression by mechanisms different from direct protease
inhibition. HI-8 is the C-terminal domain of bikunin, which
is active fragment for protease inhibitor but is not recognized by the cell-associated bikunin binding sites. We could
not see any effects of deglycosylated bikunin, HI-8, and
a1-antitrypsin on suppressing uPAR expression. Similar
effects on bikunin specificity were found in the other cell
lines (data not shown).
Suppression by bikunin of unstimulated
and PMA-induced uPAR protein expression

Fig. 2. Bikunin specifically suppresses PMA-stimulated uPAR gene
expression. HRA cells were incubated for 3 h with or without 100 nM
PMA in the presence or absence of bikunin, its truncated proteins
[deglycosylated bikunin (bikunin-c) and HI-8] and its related protein
(a1-antitrypsin). Total cellular RNA was extracted and analyzed
for uPAR mRNA expression by Northern blot analysis and compared with untreated control cells (Ctr). Top, representative autoradiograms; Bottom, Levels of uPAR mRNA expression as quantified
by densitometric scanning. Results are the mean ± SD of three different determinations, with unlike superscripts (a–e) are different
(P < 0.05).

respectively, as determined by scanning densitometry.
After treatment with 1 lM bikunin alone (lane 2), 30%

reduction of uPAR mRNA was observed. Similar effects
of bikunin inhibition were found in the other cell lines
(data not shown).

We further investigated whether bikunin could inhibit
unstimulated and PMA-stimulated [125I]DIP-uPA binding
capacity on the cell surface. It has been established that
PMA treatment resulted in an increase in the number of
binding sites and a decrease of the affinity of the uPAR
[38,39]. The quantification of uPAR expression by measurement of the amount of uPA bound is thus not ideal.
Actually, it could be underestimating the level of upregulation. Notwithstanding these limitations, in this study,
uPAR expression was evaluated by measuring cell-associated [125I]DIP-uPA binding capacity. The dose-dependent
ability of bikunin to inhibit expression of uPAR by cells is
clearly demonstrated using the [125I]DIP-uPA binding assay
(Fig. 3-A). HRA cells bound [125I]DIP-uPA in a specific
and saturable manner with a maximum effect at a concentration of 10 nM (data not shown). Equivalent Kd value was
determined ( 1.6 nM), which is consistent with the known
binding affinity of uPA for uPAR [5]. In cells treated with
PMA, cell-associated uPA-binding capacity was significantly
decreased in the presence of 100 nM bikunin. The maximal


Ó FEBS 2002

Suppression of uPAR by bikunin (Eur. J. Biochem. 269) 3949

Fig. 3. Effects of PMA and bikunin on functional uPAR protein expression in HRA cells measured by the [125I]DIP-uPA binding assay. (A) HRA cells
pretreated with bikunin (0, 10, 100, 1000, and 5000 nM) or HI-8 (5000 nM) for 2 h were incubated with or without 100 nM PMA for 12 h.
B, Competitive inhibition of solid-phase [125I]DIP-uPA binding to HRA monolayer cells by unlabeled competitors. The PMA-stimulated cells
(100 nM, 12 h) were treated with 10 nM [125I]DIP-uPA in the presence of 1000 nM unlabeled competitors [uPA, amino-terminal fragment of uPA

(ATF), bikunin, deglycosylated bikunin (bikunin-c) and HI-8]. Levels of uPAR expression as quantified by [125I]DIP-uPA binding assay. The
percent fractions bound on the surface of the cells treated with or without 100 nM PMA correspond to  4 or  1.4%, respectively, of [125I]DIPuPA added. Results are the mean ± SD of three different determinations, with unlike superscripts (a–f) are different (P < 0.05).

suppression of PMA-induced uPAR expression was obtained at 1000 nM bikunin. Constitutive uPAR expression
without stimulation by PMA was also affected by 100–
1000 nM bikunin. Contrary to bikunin, HI-8 failed to
suppress PMA-stimulated [125I]DIP-uPA binding at concentrations of HI-8 as high as 5000 nM. Similar PMA and
bikunin effects on uPAR protein were found in the two
other cancer cell lines (data not shown).
We examined whether bikunin directly inhibits [125I]DIPuPA binding to the cells. The stimulated cells were treated
with [125I]DIP-uPA in the presence of several unlabeled
competitors [uPA, ATF, bikunin, deglycosylated bikunin
(bikunin-c), and HI-8]. As shown in Fig. 3B, we found that
bikunin and its derivatives did not directly inhibit [125I]DIPuPA binding to the uPAR on the cell surface.
In a parallel experiment, we measured the uPAR levels in
cells stimulated with or without PMA using a specific
ELISA for uPAR (data not shown). The levels of uPAR
protein in unstimulated and PMA-stimulated HRA cells
were 4.5 ± 0.53 and 18.0 ± 2.48 ng per 106 cells, respectively, demonstrating that, after stimulation, uPAR protein
levels increased about fourfold. The levels of uPAR protein
in PMA-stimulated cells treated with bikunin (100 and
1000 nM) were 12.3 ± 0.89 and 10.0 ± 0.97 ng per 106
cells, respectively. Thus, the dose-dependent ability of
bikunin to inhibit expression of uPAR protein by cells
was also demonstrated using ELISA.
Results obtained after exposing the cells to bikunin before
and after stimulation by PMA are presented in Fig. 4.
Preincubation of the cells with bikunin during 2 h before
100 nM PMA stimulation results in a concentration-dependent inhibition of the induction of [125I]DIP-uPA binding
(which is associated with uPAR protein expression). At

concentrations of 100 and 1000 nM, [125I]DIP-uPA binding
is inhibited by  35 and  50%, respectively. In contrast, no
significant decrease of uPAR expression is observed when
bikunin is added to the medium 2 h after stimulation by
PMA. More than 90% of the control value still remains at
the highest bikunin concentration (1000 nM) tested. Cell

viability, monitored by LDH leakage in the culture medium
and trypan blue dye exclusion test, is not altered under the
different exposure conditions (data not shown). These
experiments demonstrated that a marked decrease of uPAR
expression is observed when bikunin is added to the medium
before stimulation by PMA.
The effects of bikunin and its derivatives on uPAR
protein expression
To further determine which domains of bikunin are
sufficient to suppress uPAR levels in cells, we determined

Fig. 4. Effect of exposure of cells to bikunin before and after stimulation
by PMA. Levels of uPAR expression as quantified by [125I]DIP-uPA
binding assay. Values represent means ± SD of three experiments.
(A) 10 nM bikunin; (B) 100 nM bikunin; and (C) 1000 nM bikunin. a–f,
means ± SD with unlike superscripts are different (P < 0.05). Results
are representative of two separate experiments.


Ó FEBS 2002

3950 H. Kobayashi et al. (Eur. J. Biochem. 269)

Table 1. Dose-dependent suppression of uPAR expression and ERK phosphorylation stimulated by PMA after intact and truncated bikunin treatment
of HRA cells. The relative amount of PMA-induced cell-associated uPAR expression suppressed in response to increasing concentrations of intact
and truncated bikunins is shown. The amount of uPAR accumulated in the cells treated without or with 100 nM PMA in the absence of competitor
corresponds to 4.5 ± 0.53 and 18.0 ± 2.48 ng per 106 cells, respectively. The amount of ERK phosphorylation in cell lysates treated with 100 nM
PMA in the absence of competitor corresponds to 100% as judged by a densitometric scan. The data of antitryptic activity, cell binding and uPA
suppression are from [28,] These data are representative of two independent experiments.
Bikunin
Molecular mass (kDa)
Antitryptic activity (IC50; lgỈmL)
Cell binding (IC50) nM
uPA suppression
Cell lysate (IC50; nM)
Conditioned medium (IC50; nM)
uPAR suppression
ERK suppression

Bikunin-m1

Bikunin-m2

Bikunin-c

Bikunin-a

Bikunin-n

HI-8

40
1.6

12

10–30
>30
30

21
1.1
350

25
1.4
15

39
1.5
15

35
1.9
9

8
0.9
>1000

20
28
 200
 100

>1000
>1000
>1000
>1000

>1000
>1000
>1000
>1000

>1000
>1000
>1000
>1000

30
35
 200
 100

5
40
 200
 100

>1000
>1000
>1000
>1000

whether the downregulation observed with intact bikunin
could be induced by bikunin derivatives. Dose-dependent
suppression of uPAR in cells treated with PMA in response
to treatment with intact bikunin or bikunin derivatives is
shown in Table 1. The levels of uPAR protein in cell lysates
were determined using a specific ELISA for uPAR.
Bikunin-a and bikunin-n effectively suppressed uPAR
levels. Bikunin-a and bikunin-n were essentially equipotent
to intact bikunin with respect to the inhibition of uPAR
levels as judged by ELISA, with ID50 values of  200 nM.
Other bikunin derivatives had no discernible effect on
uPAR levels in cells.

When HRA cells were preincubated with bikunin for 2 h,
we could detect suppression of phosphorylation of ERK1/2
in a dose-dependent manner. The results, based on densitometric scanning, show that 1 lM bikunin inhibits constitutive and PMA-triggered phosphorylation of ERK1/2, by
 50 and  70%, respectively. However, 5 lM HI-8 failed
to change significantly the expression of phosphorylated

Suppression by bikunin of ERK1/2 activity in the
unstimulated and PMA-stimulated HRA cells
Recent studies demonstrated that activation of a PMAdependent signal pathway involves a relay of phosphorylation of several proteins making up the MAP kinase pathway
[40]. uPAR gene expression is modulated by multiple signal
transduction pathways. EGF and PMA cause increased
uPAR transcription [15]. An increased level of phosphorylated ERK is associated with increased level of cell-surface
uPAR, which results in enhanced invasion [10]. To determine the role of bikunin in the regulation of ERK1/2
phosphorylation, the unstimulated and PMA-stimulated
HRA cells were analyzed for the phosphorylation of ERK1/2
(Fig. 5). Immunoblotting of cell extracts with anti-ERK1/2

Ig indicated the presence of immunoreactive proteins
(44 kDa ERK1 and 42 kDa ERK2). The HRA cells
express primarily ERK1 and some ERK2, as has been
previously reported for HRA cells [24]. Anti-(phosphoERK) Ig showed a strong kinase activity in the PMAstimulated cells corresponding in size to ERK1, while
unstimulated cells contained low ERK1 activity. We found
that, in the stimulated cells, phosphorylation of ERK1/2 is
modified within 30 min of induction by PMA and then
returned to the uninduced state after 5 h (data not shown).
We further demonstrated that the high level of uPAR
expression may have aided in the use of HRA cell line as a
model system, since levels of HRA cell ERK1/2 activation
were typically high. Compared to HRA cells, lower levels of
phosphorylated ERK1/2 were found in HOC-I and HCS-2/8
cells (data not shown).

Fig. 5. PMA-stimulated cells demonstrate increased levels of activated
ERK1/2All cells were extracted when 90% confluent. Cells pretreated
with bikunin or HI-8 for 2 h were incubated with PMA for 30 min and
then extracted to assess ERK phosphorylation. Cells were solubilized
in lysis buffer supplemented with protease inhibitors. Equal amounts
of cellular protein (50 lgỈlane)1) were loaded in each lane, subjected to
SDS/PAGE, and electrotransferred to PVDF membrane for detection
with antiphospho-ERK1/2 (active) and anti-ERK1/2 Ig (total).
Immunoblot analysis was performed to detect phosphorylated and
total ERK1/2 in unstimulated and PMA-stimulated cells. Results
are representative of two separate experiments. Top, representative
immunoblotting; Bottom, Levels of phosphorylated ERK1 expression
as quantified by densitometric scanning.

Ó FEBS 2002

Suppression of uPAR by bikunin (Eur. J. Biochem. 269) 3951

proteins. Bikunin did not reduce total ERK1/2 antigen
level. Therefore, these results show that bikunin inhibits
both constitutive and PMA-induced phosphorylation of
ERK1/2 at the concentration (0.1–1 lM) that prevents
uPAR expression. Again, similar effects on ERK phosphorylation were found in the two other cell lines (data not
shown).
Domain specificity of bikunin on ERK activation
Dose-dependent suppression of ERK phosphorylation in
the stimulated cells in response to pretreatment with intact
bikunin or bikunin derivatives is shown in Fig. 6 and
Table 1. Exposure of cells to bikunin, bikunin-a and
bikunin-n resulted in decrease in the amount of ERK
phosphorylation in cell lysates as judged by immunoblotting, indicating that ID50 was about 200 nM. Other bikunin
derivatives had no significant effect on suppression of ERK
phosphorylation. The inhibition curves for bikunin-induced
suppression of uPAR expression and ERK activation are
shifted towards higher bikunin concentrations in relation to
that for bikunin-induced suppression of uPA expression.
ERK activation is associated with uPAR expression
In order to determine whether the ERK activation system is
necessary for the increase in uPAR mRNA expression, we
cultured these cells in the presence of PD098059 or an
alternative MEK1 inhibitor U0126, which prevents the
activation of MEK1 [41,42]. HRA cells were treated for 12

Fig. 6. Dose-dependent inhibition by bikunin and its derivatives of ERK

phosphorylation in the PMA-stimulated HRA cells. Cells pretreated
with bikunin or its derivatives for 2 h were incubated with 100 nM
PMA for 30 min and then extracted (20 lgỈlane)1) to assess ERK
phosphorylation. Experiments were performed twice with similar
results.

and 3 h with varying concentrations of the inhibitors and
100 nM PMA, respectively. PD098059 (Fig. 7A, lanes 2–4)
and U0126 (Fig. 7C, lane 4) reduced the uPAR mRNA
expression in a dose-dependent manner. Bikunin showed no
additive effect on PD098059-mediated suppression of uPAR
expression (not shown). The reduced amount of uPAR
mRNA reflected a diminished amount of uPAR protein as
determined by the [125I]DIP-uPA binding assay and ELISA

Fig. 7. Levels of uPAR mRNA expression in unstimulated or PMA-stimulated cells treated with bikunin and/or several inhibitors. A, Cells were
pretreated with PD098059 (20, 40, or 60 lM), sodium vanadate (0.1 mM), or bikunin (1000 nM) or vehicle for 9 h before the addition of 100 nM
PMA. After cells were incubated for 3 h, total cellular RNA was extracted and analyzed for uPAR mRNA expression by Northern blot analysis.
(B) Unstimulated cells were treated with PD098059 (60 lM) or vehicle for 12 h. C, Cells were pretreated with staurosporin (50 nM), PD098059
(60 lM), U0126 (20 lM), or with a combination of each agent 9 h before the addition of 100 nM PMA. Top, representative autoradiograms;
Bottom, Levels of uPAR mRNA expression as quantified by densitometric scanning. A, Results are the mean ± SD of three different determinations with unlike superscripts (a–e) are different (P < 0.05). (B) and (C), experiments were performed twice with similar results.


3952 H. Kobayashi et al. (Eur. J. Biochem. 269)

(data not shown). The expression of uPAR mRNA in
unstimulated cells was slightly inhibited by treatment of the
cells with PD098059 (Fig. 7B) or U0126 alone (not shown).
These results suggest that, in unstimulated cells, MEK1
inhibition in itself does not significantly affect uPAR

mRNA levels, although the PMA-stimulated ERK activity
downregulation by bikunin is suggested to be the major
mechanism through which bikunin works.
The overexpression of uPAR mRNA in the PMAstimulated cells was also inhibited by treatment of the cells
with PKC inhibitors, calphostin C (data not shown) and
staurosporin, as measured by Northern blotting (Fig. 7C).
However, MEK1 inhibitors showed no additive effect on
PKC inhibitor-mediated suppression of uPAR overexpression. We next examined whether stimulation of ERK1
activity was associated with increased uPAR mRNA
expression. For this, HRA cells were treated with a protein
tyrosine phosphatase inhibitor (sodium vanadate) [43].
0.1 mM sodium vanadate significantly increased uPAR
mRNA 2.3-fold (Fig. 7A, lane 5). Even in the presence of
bikunin, however, the level of ERK phosphorylation was
not decreased back to the basal level observed without
sodium vanadate, suggesting that bikunin has no ability to
inhibit overexpression of uPAR mRNA in HRA cells
treated with sodium vanadate. We showed that effects of
these inhibitors do not reflect a decrease in cell viability over
the time frame of these experiments (data not shown).
cDNA transfection of bikunin
In order to determine whether bikunin expression in cells
transfected with bikunin gene is necessary for the decrease in
ERK phosphorylation, followed by suppression of uPAR
mRNA expression, HRA cells were transfected to express
bikunin gene. As shown in Fig. 8, bikunin was not detected
in extracts (data not shown) and conditioned media of HRA
and luc+ clone, by immunoblot analysis, but was easily
identified in conditioned medium of bik+ clone.
We examined whether cDNA transfection of bikunin

does not alter levels of representative signaling proteins in
bik+ clone, luc+ clone, and HRA cells. Total antigenic levels
of MEK1/2, ERK1/2, and c-Jun were equivalent in the
different cell types (data not shown), suggesting that bikunin
transfection did not nonspecifically inhibit cellular expression of proteins which impact on MAP kinase cascade.
Down-regulation of uPAR mRNA level
in bikunin-transfected cells
Figure 9A shows that the intensity of the uPAR mRNA
band was much lower in bik+ clone than in HRA and luc+
clone. Scanning autoradiograms of the hybridization signals with a laser densitometer and normalization with
the glyceraldehyde-3-phosphate signal showed a fourfold
decrease in uPAR mRNA in bik+ clone as compared with
HRA and luc+ clone.
Immunoreactive uPAR protein was detected in these
cells. The bik+ clone expressed significantly low levels of
immunoreactive uPAR protein with molecular mass of
50 kDa under nonreducing conditions (Fig. 9B). Based on
densitometric acanning, the expression of uPAR at the
protein level in the parental cells was reduced by 50–60% in
bik+ clone.

Ó FEBS 2002

Fig. 8. Expression of bikunin protein in HRA cells transfected with
cDNA coding for human bikunin. Detection of bikunin protein by
Western blot. Equal amounts (10 lLỈlane)1) of conditioned media
derived from the same number of cells of parental HRA (lane 1), luc+
clone (lane 2), and bik+ clone (lane 3) were applied to SDS/12%
polyacrylamide gel electrophoresis followed by Western blot with
antibik antibody. Expression of the 50–80 kDa bik protein is upregulated in bik+ clones. Molecular mass standards are indicated at the

left. Results are representative of two separate experiments.

Expression of uPAR in HRA cells is associated with
an active ERK1/2
AS we showed in this study that exogenous bikunin
suppresses PMA-induced uPAR expression via an inhibition of ERK1/2 phosphorylation, studies were undertaken
to determine whether the level of ERK phosphorylation is
decreased in bik+ clone in the presence or absence of
exogenous PMA stimuli (Fig. 10). Anti-phospho-ERK
antibody showed a strong kinase activity in the parental
and luc+ clone, high uPAR expressor, corresponding in size
to ERK1. On the other hand, bik+ clone, low uPAR
expressor, contained low ERK1/2 kinase activity. It is
unlikely that the large difference in ERK activity between
the parental and bik+ clone is a consequence of different
growth rates, as we were unable to show a reproducible
decrease in proliferation of the bik+ clone (data not shown).
The results show that not only exogenously applied bikunin
(1 lM) but also bikunin gene transfection significantly
inhibits constitutive and PMA-triggered phosphorylation of
ERK1/2 by about 70%. Similar effects of bikunin on
suppression of MAP kinase activation were found in the
two other cancer cell lines (data not shown).
The effect of bikunin and HI-8 as well as neutralizing
antibodies against uPA and uPAR on HRA cell
invasiveness
Our previous experiments showed that treatment with PMA
produced a significant stimulation of the invasiveness of
HRA cells in a dose-dependent manner, with a maximum
stimulation at 100 nM PMA [25]. Figure 11 shows the effect

Ó FEBS 2002

Suppression of uPAR by bikunin (Eur. J. Biochem. 269) 3953

Fig. 9. Downregulation of uPAR level in bikunin-transfected cells. (A) Downregulation of uPAR mRNA level in bikunin-transfected cells. Ten
micrograms of total RNA isolated from HRA cells, bik+ cells, and luc+ cells, shown in the upper panel, was electrophoresed in a 1.2% agarose/
formaldehyde gel and then transferred to Hybond N+ membrane. The membrane was then hybridized with a radiolabeled cDNA probe specific for
uPAR mRNA. The same blot was stripped and hybridized with radiolabeled GAPDH cDNA to check for equality of loading. uPAR mRNA levels
were measured by scanning autoradiograms with a laser densitometer, and relative hybridization signals were calculated by assigning an arbitrary
value of 100 to the highest intense signal seen by Northern blot analysis correlated for mRNA loading inequalities. Results are the mean ± SD of
three different determinations with unlike superscripts (a and b) are different (P < 0.05). (B) Expression of uPAR protein in bikunin transfected
cells. Cell lysates of control and bikunin transfected cell clones treated with or without PMA were used to analyze uPAR expression by Western
blot. Results are representative of two separate experiments.

of adding increasing concentrations of antibodies or bikunin
and HI-8 on the invasiveness of the PMA-stimulated
cells (left panel) and bik+ clone (right panel). The
PMA-stimulated cell invasion was specifically reversed by
concurrent treatment with either neutralizing anti-uPA Ig or
anti-uPAR Ig, as well as with bikunin. These data support
that HRA cells leading to invasion is induced through the
upregulation of the uPA/uPAR system. Bikunin, but not
HI-8, which could induced change in uPA/uPAR expression, could in turn modify the invasive behavior of HRA
cells. However, bikunin had no additive effect on antibodymediated suppression of cell invasiveness. The bik+ clone
invasion was also reversed by concurrent treatment with
either neutralizing anti-uPA Ig or anti-uPAR Ig, but not
with exogenous bikunin.

DISCUSSION

Fig. 10. Bikunin gene transfection inhibits constitutive and PMA-triggered phosphorylation of ERK1/2All cells were extracted when 90%
confluent. Cells were stimulated with PMA for 30 min and then
extracted to assess ERK1/2. Immunoblot analysis to detect phosphorylated and total ERK1/2 in unstimulated and PMA-stimulated
cells. Results are representative of two separate experiments. Top,
representative immunoblotting; Bottom, Levels of phosphorylated
ERK1 expression as quantified by densitometric scanning. Results are
representative of two separate experiments.

Several authors have reported that the serine protease uPA
and its receptor uPAR play a key role in the invasive and
metastatic capacity of tumor cells [3,4,44]. Phorbol ester
and a variety of growth factors including EGF, FGF, and
VEGF, which upregulate uPAR synthesis, also stimulate
ERK activity [15,45–49]. Further, ERK may represent an
essential step in the pathway by which cytokine and
integrins promote cellular motility [50]. It has been established that PMA induces ERK activity in a number of
systems [45], however, the signaling mechanism by which
PMA modulates uPAR expression is not completely
understood. The effect of PMA on the expression of other
genes has been ascribed to activation of the classical
pathway (Ras fi c-Raf1 fi ERK signaling cascade) [51].
An alternative pathway consists of the sequential activation
of Rac1, MEKK1, c-Jun N-terminal kinase kinase (JNKK)


3954 H. Kobayashi et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

Fig. 11. Suppression of invasiveness in PMAstimulated HRA cells and bik+ clone by treatment with antibodies and bikunin. HRA cells
and bik+ clone (5 · 104 cells) were placed in
the top wells of a chemoinvasion chamber
apparatus with the neutralizing antibodies
against uPA (10 lgỈmL)1) and uPAR
(10 lgỈmL)1), bikunin (0.5 lM) and HI-8
(1 lM) in the presence of 100 nM PMA. Each
point represents the mean of measurements
made on two independent wells. This is a
representative experiment selected from two
performed.

and the c-Jun N-terminal kinase (JNK) subset of MAPK
[52]. Recent studies also demonstrated that the PMAdependent stimulation of uPAR gene expression requires a
JNK1-dependent and -independent signaling modules [53].
Tumor dormancy is induced by downregulation of uPAR
[54]. It has been reported thata  70% reduction in the
uPAR level in human carcinoma HEp3 cells induced a
protracted state of tumor dormancy in vivo. Therefore,
treatment of uPAR-rich cells, which maintain high ERK
activity in vivo, with reagents interfering with the uPAR
signal to ERK activation, mimic the in vivo dormancy
induced by downregulation of uPAR. With these in mind,
we investigated the regulation by bikunin of ERK activation
as a signaling molecule in PMA-induced uPAR overexpression in highly invasive human ovarian cancer cell lines and
human chondrosarcoma cell line.
We previously reported that bikunin expression is
associated with a less malignant phenotype [24]. There is
evidence that bikunin significantly prevented pulmonary

metastasis of mouse Lewis lung carcinoma 3LL cells [55].
Our ongoing study shows that transfection of the highly
invasive and metastatic HRA cell line lacking bikunin
expression with bikunin-encoding constructs causes a
marked decrease in their metastatic ability (Suzuki &
Kobayash, unpublished data). We have clearly demonstrated in the recent studies [28,56–59] that bikunin can
suppress PMA-stimulated upregulation of uPA mRNA and
protein via a specific receptor for bikunin, which results
in bikunin-mediated suppression of cell invasiveness. The
precise mechanism by which expression of bikunin and
decrease in the metastatic ability of the bikunin transfectants
might be linked to the expression of uPA has been explored
in our laboratory. However, nothing is known about the
mechanism by which bikunin would modulate the uPAR
expression.
The present study showed that (a) the effect of PMA on
uPAR expression is time- and dose-dependent; (b) exogenously added bikunin is able to suppress the expression of
constitutive and PMA-induced uPAR mRNA and protein;
(c) a marked decrease of uPAR expression is observed when
bikunin is added to the medium before stimulation by
PMA; (d) PMA promotes cellular uPAR expression, at
least, by activating an ERK-dependent signaling pathway:
the ERK activation system is necessary for the increase in
uPAR expression, since PD098059 and U0126 reduce the
uPAR expression; (e) bikunin markedly suppresses PMA-

induced phosphorylation of ERK1/2 at the concentration
that prevents uPAR expression, but does not reduce total
ERK1/2 antigen level; (f) the inhibition curves for bikunininduced suppression of uPAR expression and ERK activation are shifted towards higher bikunin concentrations in
relation to that for bikunin-induced suppression of uPA

expression; (g) the inhibition of ERK1/2 by bikunin
depends on receptor binding, since HI-8 does not inhibit
ERK1/2 phosphorylation: O-glycoside-linked core protein
without N-glycoside, that is the active domain for suppression of uPA expression, is required for bikunin-mediated
suppression of uPAR production and ERK phosphorylation; (h) stimulation of ERK1 activity by sodium vanadate
is associated with increased uPAR expression: bikunin has
no ability to inhibit overexpression of uPAR in cells treated
with sodium vanadate, indicating that bikunin is able to
suppress uPAR mRNA and protein possibly through
inhibition of upstream components of ERK activation in
MAP kinase cascade: a set of consecutive signaling molecules which teleologically alter the program of gene
expression; (i) bikunin transfection experiments demonstrated that uPAR expression is associated with an active
ERK and the bikunin expression is necessary for inhibition
of uPAR expression possibly through suppression of ERK
phosphorylation; (j) not only exogenous bikunin but also
bikunin gene transfection markedly inhibits PMA-triggered
phosphorylation of ERK1/2; and (k) the HRA cells leading
to invasion is mainly through upregulation of uPA/uPAR
expression: suppression by neutralizing antibodies to uPA/
uPAR of cell invasion is almost complete, while inhibition
by bikunin alone is partial. Bikunin showed no additive
effect on neutralizing antibody-mediated suppression of
invasion. Therefore, the present results clearly show that
bikunin (O-glycoside-linked core protein without N-glycoside) specifically inhibits expression of uPAR mRNA and
protein possibly through suppression of an upstream
target(s) of the ERK-dependent cascade.
A recent publication demonstrated that binding of uPA
to uPAR activates ERK1/2, which is required for increased
cellular motility in breast cancer cells [26]. Furthermore,
uPA [60] and uPAR [49] expression are increased in

response to multiple factors that activate ERK1/2. It is
thus possible that uPA might regulate expression of uPA
itself and its own receptor via ERK activation. Our previous
and present results demonstrated that bikunin specifically
inhibits constitutive and inducible gene expression including


Ó FEBS 2002

uPA and uPAR possibly through suppression of upstream
targets of ERK. Thus, one of the functions of bikunin is the
regulation of uPA synthesis and uPA binding by modulating uPAR. It is most likely that the PMA-induced uPAR
expression shares the characteristics of that of uPA expression, since the increased uPAR expression is affected by
PKC inhibitors (calphostin C and staurosporin) and the
uPA expression is also affected by ERK inhibition [61].
However, the effect of bikunin inhibition of uPAR expression and ERK phosphorylation is less potent than that of
uPA suppression, suggesting that PKC and ERK are
involved in the expression of the uPA and uPAR proteins
possibly through the parallel pathways, but not the same
ones. Further, total antigenic levels of MEK1/2, ERK1/2,
and c-Jun were equivalent in the different cell types,
suggesting that exogenous bikunin and bikunin transfection
do not nonspecifically inhibit cellular expression of proteins
which impact on MAP kinase cascade. These results show
that bikunin may be selective in inhibiting gene expression.
The effect of bikunin on the expression of other proteins
regulated through PKC and ERK could give information
on whether bikunin has ubiquitous effects on protein
expression.
We found that the effects of bikunin are not due to

protease inhibition as the single Kunitz-type domain HI-8 is
not inhibitory with respect to uPA and uPAR mRNA
expression, while full-length bikunin is. A further characterization of the bikunin domains responsible for the effect
on uPAR expression and ERK activation was intensively
examined as previously carried out by us with regard to uPA
expression. The binding and signaling properties of intact
bikunin and truncated bikunins are summarized in Table 1.
We conclude that O-glycoside-linked core protein without
N-glycoside is essential to the bikunin-mediated suppression
of uPA/uPAR production and ERK activation. These
signaling properties of the truncated bikunin indicate that
the simultaneous binding of the chondroitin-4-sulfate side
chain of bikunin and the N-terminal Kunitz-domain I are
required for effective signaling properties.
The results from a recent report by us on bikunin
inhibition of CD44 clustering as a mechanism by which it
might inhibit uPA expression will also be investigated with
respect to uPAR expression [62]. In addition, PMA can
enhance uPAR mRNA stability in certain tumor cells [21].
However, we found a relatively similar half-life of uPAR
mRNA of 10–12 h in the parental cells, luc+ clone and bik+
clone (data not shown). It is unlikely that bikunin reduces
uPAR mRNA stability. Again, we found that bikunin had
no measurable effects on cell viability or on the yield of the
total RNA.
We believe that the present study provides new insight
into how bikunin may be involved in the modulation of the
metastatic phenotype: suppression of uPA/uPAR-dependent increased cell adhesion, migration and invasion by
bikunin, at least, via suppression of an upstream target(s) of
ERK activation. Therefore, reagents which modulate ERK

activation may offer fresh insights into the prevention of
uPA/uPAR-dependent tumor invasion and metastasis.

ACKNOWLEDGEMENTS
The authors thank Drs M. Fujie, K. Shibata, T. Noguchi and A Suzuki
(Equipment center and Photo center; Hamamatsu University School of

Suppression of uPAR by bikunin (Eur. J. Biochem. 269) 3955
Medicine) for helping with the biochemical analysis. We are also
thankful to Drs H. Morishita, Y. Kato, K. Kato and H. Sato
(BioResearch Institute, Mochida Pharmaceutical Co., Gotenba,
Shizuoka), Drs Y. Tanaka and T. Kondo (Chugai Pharmaceutical
Co. Ltd, Tokyo), and Drs S. Miyauchi and M. Ikeda (Seikagaku
Kogyo Co. Ltd, Tokyo) for their continuous and generous support of
our work.

REFERENCES
1. Aznavoorian, S., Murphy, A.N., Stetler-Stevenso, W.G. & Liotta,
L.A. (1993) Molecular aspects of tumor cell invasion and metastasis. Cancer 71, 1368–1383.
2. Mignatti, P. & Rifkin, D.B. (1993) Biology and biochemistry of
proteinases in tumor invasion. Physiol. Rev. 73, 161–195.
3. Reuning, U., Magdolen, V., Wilhelm, O., Fischer, K., Lutz, V.,
Graeff, H. & Schmitt, M. (1998) Multifunctional potential of the
plasminogen activation system in tumor invasion and metastasis.
Int. J. Oncol. 13, 893–906.
4. Aguirre Ghiso, J.A., Alonso, D.F., Farias, E.F., Gomez, D.E. &
de Kier Joffe, E.B. (1999) Deregulation of the signaling pathways
controlling urokinase production. Its relationship with the
invasive phenotype. Eur. J. Biochem. 263, 295–304.
5. Stoppelli, M.P., Corti, A., Soffientini, A., Cassani, G., Blasi, F. &

Assoian, R.K. (1985) Differentiation-enhanced binding of the
amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes. Proc. Natl Acad.
Sci. USA 82, 4939–4943.
6. Vassalli, J.D., Baccino, D. & Belin, D. (1985) A cellular binding
site for the Mr 55,000 form of the human plasminogen activator,
urokinase. J. Cell Biol. 100, 86–92.
7. Rabbani, S.A. & Mazar, A.P. (2001) The role of the plasminogen
activation system in angiogenesis and metastasis. Surg. Oncol.
Clin. N. Am. 10, 393–415.
8. Ellis, V., Pyke, C., Eriksen, J., Solberg, H. & Dano, K. (1992) The
urokinase receptor: involvement in cell surface proteolysis and
cancer invasion. Ann. NY Acad. Sci. 667, 13–31.
9. Ganesh, S., Sier, C.F., Heerding, M.M., Griffioen, G., Lamers,
C.B. & Verspaget, H.W. (1994) Urokinase receptor and colorectal
cancer survival. Lancet 344, 401–402.
10. Webb, D.J., Nguyen, D.H. & Gonias, S.L. (2000) Extracellular
signal-regulated kinase functions in the urokinase receptordependent pathway by which neutralization of low density lipoprotein receptor-related protein promotes fibrosarcoma cell
migration and matrigel invasion. J. Cell Sci. 113, 123–134.
11. Kruger, A., Soeltl, R., Lutz, V., Wilhelm, O.G., Magdolen, V.,
Rojo, E.E., Hantzopoulos, P.A., Graeff, H., Gansbacher, B. &
Schmitt, M. (2000) Reduction of breast carcinoma tumor growth
and lung colonization by overexpression of the soluble urokinasetype plasminogen activator receptor (CD87). Cancer Gene Ther. 7,
292–299.
12. Wilhelm, O., Weidle, U., Hohl, S., Rettenberger, P., Schmitt, M.
& Graeff, H. (1994) Recombinant soluble urokinase receptor as
a scavenger for urokinase-type plasminogen activator (uPA).
Inhibition of proliferation and invasion of human ovarian cancer
cells. FEBS Lett. 337, 131–134.
13. Kook, Y.H., Adamski, J., Zelent, A. & Ossowski, L. (1994) The
effect of antisense inhibition of urokinase receptor in human

squamous cell carcinoma on malignancy. EMBO J. 13, 3983–
3891.
14. Lund, L.R., Romer, J., Ronne, E., Ellis, V., Blasi, F. & Dano, K.
(1991) Urokinase-receptor biosynthesis, mRNA level and gene
transcription are increased by transforming growth factor beta 1 in
human A549 lung carcinoma cells. EMBO J. 10, 3399–3407.
15. Lund, L.R., Ellis, V., Ronne, E., Pyke, C. & Dano, K. (1995)
Transcriptional and post-transcriptional regulation of the receptor
for urokinase-type plasminogen activator by cytokines and


3956 H. Kobayashi et al. (Eur. J. Biochem. 269)

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

tumour promoters in the human lung carcinoma cell line A549.
Biochem. J. 310, 345–352.
Pepper, M.S., Matsumoto, K., Nakamura, T., Orci, L. &
Montesano, R. (1992) Hepatocyte growth factor increases
urokinase-type plasminogen activator (u-PA) and u-PA receptor
expression in Madin–Darby canine kidney epithelial cells. J. Biol.
Chem. 267, 20493–20496.
Mandriota, S.J., Seghezzi, G., Vassalli, J.D., Ferrara, N., Wasi, S.,
Mazzieri, R., Mignatti, P. & Pepper, M.S. (1995) Vascular
endothelial growth factor increases urokinase receptor expression
in vascular endothelial cells. J. Biol. Chem. 270, 9709–9716.
Kirchheimer, J.C., Nong, Y.H. & Remold, H.G. (1988) IFNgamma, tumor necrosis factor-alpha, and urokinase regulate
the expression of urokinase receptors on human monocytes.
J. Immunol. 141, 4229–4234.
Nielsen, L.S., Kellerman, G.M., Behrendt, N., Picone, R., Dano,
K. & Blasi, F. (1988) A 55,000–60,000 Mr receptor protein for
urokinase-type plasminogen activator. Identification in human

tumor cell lines and partial purification. J. Biol. Chem. 263, 2358–
2363.
Wang, H., Skibber, J., Juarez, J. & Boyd, D. (1994) Transcriptional activation of the urokinase receptor gene in invasive colon
cancer. Int. J. Cancer 58, 650–657.
Shetty, S., Kumar, A., Johnson, A., Pueblitz, S. & Idell, S. (1995)
Urokinase receptor in human malignant mesothelioma cells: role
in tumor cell mitogenesis and proteolysis. Am. J. Physiol. 268,
L972–L982.
Lindqvist, A., Rouet, P., Salier, J.P. & Akerstrom, B. (1999) The
alpha1-microglobulin/bikunin gene: characterization in mouse
and evolution. Gene 234, 329–336.
Kanayama, N., Halim, A., Maehara, K., Kajiwara, Y., Fujie,
M. & Terao, T. (1995) Kunitz-type trypsin inhibitor prevents
LPS-induced increase of cytosolic free Ca2+ in human neutrophils and HUVEC cells. Biochem. Biophys. Res. Commun. 207,
324–330.
Kobayashi, H., Sugino, D. & Terao, T. (1998) Urinary trypsin
inhibitor, a Kunitz-type protease inhibitor, modulates tumor
necrosis factor-stimulated activation and translocation of protein
kinase C in U937 cells. Int. J. Oncol. 12, 95–105.
Kobayashi, H., Suzuki, M., Tanaka, Y., Hirashima, Y. & Terao,
T. (2001) Suppression of urokinase expression and invasiveness by
urinary trypsin inhibitor is mediated through inhibition of protein
kinase C- and MEK/ERK/c-Jun-dependent signaling pathways.
J. Biol. Chem. 276, 2015–2022.
Nguyen, D.H., Hussaini, I.M. & Gonias, S.L. (1998) Binding of
urokinase-type plasminogen activator to its receptor in MCF-7
cells activates extracellular signal-regulated kinase 1 and 2 which is
required for increased cellular motility. J. Biol. Chem. 273, 8502–
8507.
Kobayashi, H., Schmitt, M., Goretzki, L., Chucholowski, N.,

Calvete, J., Kramer, M., Gunzler, W.A., Janicke, F. & Graeff, H.
(1991) Cathepsin B efficiently activates the soluble and the tumor
cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA). J. Biol. Chem. 266, 5147–5152.
Suzuki, M., Kobayashi, H., Tanaka, Y., Hirashima, Y. & Terao,
T. (2001) Structure and function analysis of urinary trypsin
inhibitor (UTI): identification of binding domains and signaling
property of UTI by analysis of truncated proteins. Biochim.
Biophys. Acta 1547, 26–36.
Fujii, T., Maeda, M. & Kawashima, Y. (1987) A simple and rapid
crystal violet uptake sensitivity test for anticancer agents. Nippon
Sanka Fujinka Gakkai Zasshi 39, 352–358.
Kikuchi, Y., Kizawa, I., Oomori, K., Miyauchi, M., Kita, T.,
Sugita, M., Tenjin, Y. & Kato, K. (1987) Establishment of a
human ovarian cancer cell line capable of forming ascites in nude
mice and effects of tranexamic acid on cell proliferation and ascites
formation. Cancer Res. 47, 592–596.

Ó FEBS 2002
31. Takigawa, M., Tajima, K., Pan, H.O., Enomoto, M., Kinoshita,
A., Suzuki, F., Takano, Y. & Mori, Y. (1989) Establishment of a
clonal human chondrosarcoma cell line with cartilage phenotypes.
Cancer Res. 49, 3996–4002.
32. Urabe, M., Hasumi, Y., Ogasawara, Y., Matsushita, T.,
Kamoshita, N., Nomoto, A., Colosi, P., Kurtzman, G.J., Tobita,
K. & Ozawa, K. (1997) A novel dicistronic AAV vector using a
short IRES segment derived from hepatitis C virus genome. Gene
200, 157–162.
33. Wigler, M., Pellicer, A., Silverstein, S. & Axel, R. (1978)
Biochemical transfer of single-copy eucaryotic genes using total
cellular DNA as donor. Cell 14, 725–731.

34. Chen, Y.T., Holcomb, C. & Moore, H.P. (1993) Expression and
localization of two low molecular weight GTP-binding proteins,
Rab8 and Rab10, by epitope tag. Proc. Natl Acad. Sci. USA 90,
6508–6512.
35. Marschall, C., Lengyel, E., Nobutoh, T., Braungart, E., Douwes,
K., Simon, A., Magdolen, V., Reuning, U. & Degitz, K. (1999)
UVB increases urokinase-type plasminogen activator receptor
(uPAR) expression. J. Invest. Dermatol. 113, 69–76.
36. Roldan, A.L., Cubellis, M.V., Masucci, M.T., Behrendt, N.,
Lund, L.R., Dano, K., Appella, E. & Blasi, F. (1990) Cloning and
expression of the receptor for human urokinase plasminogen
activator, a central molecule in cell surface, plasmin dependent
proteolysis. EMBO J. 9, 467–474.
37. Kobayashi, H., Hirashima, Y., Sun, G.W., Fujie, M., Nishida, T.,
Takigawa, M. & Terao, T. (2000) Identity of urinary trypsin
inhibitor-binding protein to link protein. J. Biol. Chem. 275,
21185–21191.
38. Picone, R., Kajtaniak, E.L., Nielsen, L.S., Behrendt, N.,
Mastronicola, M.R., Cubellis, M.V., Stoppelli, M.P., Pedersen, S.,
Dano, K. & Blasi, F. (1989) Regulation of urokinase receptors in
monocytelike U937 cells by phorbol ester phorbol myristate
acetate. J. Cell Biol. 108, 693–702.
39. Nykjaer, A., Moller, B., Todd, R.F., 3rd, Christensen, T.,
Andreasen, P.A., Gliemann, J. & Petersen, C.M. (1994) Urokinase
receptor. An activation antigen in human T lymphocytes.
J. Immunol. 152, 505–516.
40. Chevalier, D., Thorin, E. & Allen, B.G. (2000) Simultaneous
measurement of ERK, p38, and JNK MAP kinase cascades in
vascular smooth muscle cells. J. Pharmacol. Toxicol. Methods 44,
429–439.

41. Dudley, D.T., Pang, L., Decker, S.J., Bridges, A.J. & Saltiel, A.R.
(1995) A synthetic inhibitor of the mitogen-activated protein
kinase cascade. Proc. Natl Acad. Sci. USA 92, 7686–7689.
42. Alessi, D.R., Cuenda, A., Cohen, P., Dudley, D.T. & Saltiel, A.R.
(1995) PD 098059 is a specific inhibitor of the activation of
mitogen-activated protein kinase kinase in vitro and in vivo.
J. Biol. Chem. 270, 27489–27494.
43. Zhao, Z., Tan, Z., Diltz, C.D., You, M. & Fischer, E.H. (1996)
Activation of mitogen-activated protein (MAP) kinase pathway
by pervanadate, a potent inhibitor of tyrosine phosphatases.
J. Biol. Chem. 271, 22251–22255.
44. Dano, K., Andreasen, P.A., Grondahl-Hansen, J., Kristensen, P.,
Nielsen, L.S. & Skriver, L. (1985) Plasminogen activators, tissue
degradation, and cancer. Adv. Cancer Res. 44, 139–266.
45. Jones, L.G., Ella, K.M., Bradshaw, C.D., Gause, K.C., Dey, M.,
Wisehart-Johnson, A.E., Spivey, E.C. & Meier, K.E. (1994)
Activations of mitogen-activated protein kinases and phospholipase D in A7r5 vascular smooth muscle cells. J. Biol. Chem. 269,
23790–23799.
46. Zohn, I.E., Yu H., Li, X., Cox, A.D. & Earp, H.S. (1995)
Angiotensin II stimulates calcium-dependent activation of c-Jun
N-terminal kinase. Mol. Cell Biol. 15, 6160–6168.
47. Wiese, R.J., Mastick, C.C., Lazar, D.F. & Saltiel, A.R. (1995)
Activation of mitogen-activated protein kinase and phosphatidylinositol 3¢-kinase is not sufficient for the hormonal stimulation of


Ó FEBS 2002

48.

49.

50.

51.

52.

53.

54.

55.

glucose uptake, lipogenesis, or glycogen synthesis in 3T3-L1
adipocytes. J. Biol. Chem. 270, 3442–3446.
Bogoyevitch, M.A., Glennon, P.E., Andersson, M.B., Clerk, A.,
Lazou, A., Marshall, C.J., Parker, P.J. & Sugden, P.H. (1994)
Endothelin-1 and fibroblast growth factors stimulate the mitogenactivated protein kinase signaling cascade in cardiac myocytes.
The potential role of the cascade in the integration of two signaling
pathways leading to myocyte hypertrophy. J. Biol. Chem. 269,
1110–1119.
Campbell, J.S., Wenderoth, M.P., Hauschka, S.D. & Krebs, E.G.
(1995) Differential activation of mitogen-activated protein kinase
in response to basic fibroblast growth factor in skeletal muscle
cells. Proc. Natl Acad. Sci. U S A 92, 870–874.
Klemke, R.L., Cai, S., Giannini, A.L., Gallagher, P.J., de Lanerolle, P. & Cheresh, D.A. (1997) Regulation of cell motility by
mitogen-activated protein kinase. J. Cell Biol. 137, 481–492.
Nguyen, D.H., Catling, A.D., Webb, D.J., Sankovic, M., Walker,
L.A., Somlyo, A.V., Weber, M.J. & Gonias, S.L. (1999) Myosin
light chain kinase functions downstream of Ras/ERK to promote

migration of urokinase-type plasminogen activator-stimulated
cells in an integrin-selective manner. J. Cell Biol. 146, 149–164.
Olson, M.F., Ashworth, A. & Hall, A. (1995) An essential role for
Rho, Rac, and Cdc42 GTPases in cell cycle progression through
G1. Sci. 269, 1270–1272.
Gum, R., Juarez, J., Allgayer, H., Mazar, A., Wang, Y. & Boyd,
D. (1998) Stimulation of urokinase-type plasminogen activator
receptor expression by PMA requires JNK1-dependent and
-independent signaling modules. Oncogene 17, 213–225.
Aguirre Ghiso, J.A., Kovalski, K. & Ossowski, L. (1999) Tumor
dormancy induced by downregulation of urokinase receptor in
human carcinoma involves integrin and MAPK signaling. J. Cell
Biol. 147, 89–104.
Kobayashi, H., Shinohara, H., Fujie, M., Gotoh, J., Itoh, M.,
Takeuchi, K. & Terao, T. (1995) Inhibition of metastasis of Lewis

Suppression of uPAR by bikunin (Eur. J. Biochem. 269) 3957

56.

57.

58.

59.

60.

61.

62.

lung carcinoma by urinary trypsin inhibitor in experimental and
spontaneous metastasis models. Int. J. Cancer 63, 455–462.
Kobayashi, H., Suzuki, M., Sun, G.W., Hirashima, Y. & Terao,
T. (2000) Suppression of urokinase-type plasminogen activator
expression from human ovarian cancer cells by urinary trypsin
inhibitor. Biochim. Biophys. Acta 1481, 310–316.
Kobayashi, H., Sugino, D., She, M.Y., Ohi, H., Hirashima, Y.,
Shinohara, H., Fujie, M., Shibata, K. & Terao, T. (1998) A
bifunctional hybrid molecul e of the amino-terminal fragment of
urokinase and domain II of bikunin efficiently inhibits tumor cell
invasion and metastasis. Eur. J. Biochem. 253, 817–826.
Kobayashi, H., Hirashima, Y., Sun, G.W., Fujie, M., Shibata, K.,
Tamotsu, S., Miura, K., Sugino, D., Tanaka, Y., Kondo, S. &
Terao, T. (1998) Identification and characterization of the cellassociated binding protein for urinary trypsin inhibitor. Biochim.
Biophys. Acta 1383, 253–268.
Kobayashi, H., Gotoh, J. & Terao, T. (1996) Urinary trypsin
inhibitor efficiently inhibits urokinase production in tumor
necrosis factor-stimulated cells. Eur. J. Cell Biol. 71, 380–386.
Silberman, S., Janulis, M. & Schultz, R.M. (1997) Characterization of downstream Ras signals that induce alternative
protease-dependent invasive phenotypes. J. Biol. Chem. 272,
5927–5935.
Ried, S., Jager, C., Jeffers, M., Vande Woude, G.F., Graeff, H.,
Schmitt, M. & Lengyel, E. (1999) Activation mechanisms of the
urokinase-type plasminogen activator promoter by hepatocyte
growth factor. J. Biol. Chem. 274, 16377–16386.
Suzuki, M., Kobayashi, H., Fujie, M., Nishida, T., Takigawa,
M., Kanayama, N. & Terao, T. (2002) Kunitz-type protease
inhibitor bikunin disrupts phorbol ester-induced oligomerization

of CD44 variant isoforms containing epitope v9 and
subsequently suppresses expression of urokinase-type plasminogen activator in human chondrosarcoma cells. J. Biol. Chem. 277,
8022–8032.