From the Department of Obstetrics and
Gynecology and the ¶ Equipment Center, Hamamatsu University School
of Medicine, Handacho 3600, Hamamatsu, Shizuoka, 431-3192 and the
Department of Biochemistry and Molecular Dentistry, Okayama
University Dental School, 2-5-1 Shikata-cho, Okayama, 700-8525, Japan
Received for publication, October 31, 2000, and in revised form, January 23, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Urinary trypsin inhibitor (UTI) forms membrane
complexes with UTI-binding proteins (UTI-BPs) and initiates modulation
of urokinase-type plasminogen activator (uPA) expression, which
results in UTI-mediated suppression of cell invasiveness. It has been
established that suppression of uPA expression and invasiveness by UTI
is mediated through inhibition of protein kinase
C-dependent signaling pathways and that human
chondrosarcoma cell line HCS-2/8 expresses two types of UTI-BPs; a
40-kDa UTI-BP (UTI-BP40), which is identical to link
protein (LP), and a 45-kDa UTI-BP (UTI-BP45). Here we characterize binding properties of UTI-BPs·UTI complexes in the cells. In vitro ligand blot, cell binding and competition
assays, and Scatchard analyses demonstrate that both
UTI-BP40 and UTI-BP45 bind
125I-UTI. A deglycosylated form of UTI
(NG-UTI), from which the chondroitin-sulfate side chain has been
removed, binds only to UTI-BP40. Additional experiments,
using various reagents to block binding of 125I-UTI and
NG-UTI to the UTI-BP40 and UTI-BP45 confirm
that the chondroitin sulfate side chain of UTI is required for its
binding to UTI-BP45. Analysis of binding of
125I-UTI and NG-UTI to the cells suggests that low affinity
binding sites are the UTI-BP40 (which can bind NG-UTI), and
the high affinity sites are the UTI-BP45. In
addition, UTI-induced suppression of phorbol ester stimulated
up-regulation of uPA is inhibited by reagents that were shown to
prevent binding of UTI to the 40- and 45-kDa proteins. We conclude that
UTI must bind to both of the UTI-BPs to suppress uPA up-regulation.
Urinary trypsin inhibitor
(UTI)1 is produced as a light
chain of inter- Our previous publications also indicated that the binding of UTI to its
binding sites on the cell surface has been implicated in inhibition of
protein kinase C (PKC) translocation and subsequently suppression of
urokinase-type plasminogen activator (uPA) expression (4, 8).
Therefore, UTI interacts with tumor cells as a negative modulator of
the invasive cells (9-18). The effect of UTI on tumor necrosis factor
(TNF)- Whereas both of these UTI-BPs appear to mediate binding to UTI, it is
not known how two types of UTI-BPs may interact with UTI or if they
mediate signals for UTI following ligation by the different domains
within the UTI molecule. The purpose of this study was to evaluate
further structure-function relationship of UTI by comparing interaction
of truncated polypeptides with cells. UTI is structurally and
functionally organized into distinct domains; therefore, we utilized
purified proteins encompassing its three major domains, amino-terminal
domain (N-domain), chondroitin-4-sulfate (C4S) side chain, and
carboxyl-terminal domain (C-domain; an active protease inhibitor
domain), to assess interactions between UTI and cell-associated
UTI-BPs. To identify the structural determinants on UTI required for
cell association and regulation of the uPA-dependent signal
transduction, UTI derivatives (non-glycosylated UTI (NG-UTI) and the
carboxyl-terminal fragment of UTI (HI-8) were produced (see Fig. 1). We
describe experiments designed to determine whether the UTI-BPs are
capable of delivering an UTI-dependent modulation of uPA
expression. We have examined two types of experimental situation;
binding capacity of UTI to the cells or its binding sites and responses
to UTI in cells stimulated with phorbol ester. We have used a human
chondrosarcoma cell line HCS-2/8 that stably expresses two proteins
(UTI-BP40 and UTI-BP45) to determine whether interaction of these proteins is important for ligand-UTI-BP function.
We report here that NG-UTI is bound via UTI-BP40 and does
not suppress uPA expression and that ligation of UTI-BP45
by the C4S side chain of UTI in conjunction with the
UTI-BP40 promote suppression of phorbol ester-induced uPA expression.
Materials--
UTI was purified to homogeneity from human urine.
A highly purified preparation of human UTI was kindly supplied by
Mochida Pharmaceutical Co., Tokyo, Japan. The carboxyl-terminal
fragment of UTI (HI-8) was purified as previously described (2-6, 9, 13, 14). The isolation of cartilage-derived LP has been described in
detail elsewhere (6, 19). Chondroitinase ABC was purchased from Sigma.
Hyaluronidase (Streptomyces hyalurolyticus, no.
100740), hyaluronic acid (Mr 1090),
chondroitin-4-sulfate (C4S; Mr 21), chondroitin-0-sulfate (C0S; Mr 6),
dermatan sulfate (DS; Mr 18), and heparin
(Mr 13) were kindly supplied by Seikagaku Kogyo
(Tokyo, Japan). All other chemicals were of reagent grade or better and were purchased from major suppliers.
Treatment of UTI with Chondroitinase ABC; Preparation of
Non-glycosylated UTI (NG-UTI)--
UTI (1 mg/ml) was treated for
2 h at 37 °C with 50 µg/ml chondroitinase ABC in 50 mM phosphate buffer, 10 mM EDTA, 10 mM NaN3, pH 7.2. A Sephadex G-200 molecular
sieve chromatography was used to reduce the contamination of
chondroitinase. Removal of the chondroitin sulfate moiety was checked
by 12% SDS-polyacrylamide gel electrophoresis.
Cells and Culture Conditions--
Human chondrosarcoma cell line
HCS-2/8 (a gift from Prof. Dr. M. Takigawa; Department of Biochemistry
and Molecular Dentistry, Okayama University Dental School, Okayama,
Japan) was grown and cultured as previously described (6, 19).
Purification of UTI-BP--
The UTI-BPs were purified by
UTI-coupled-Sepharose 4B and molecular sieve chromatography as
described previously (3, 5). Affinity chromatography and
reverse-phase high performance liquid chromatography were used to
purify soluble UTI-specific-binding proteins from the lysates
of HCS-2/8 as described previously (3, 5, 6). Two binding sites for UTI
(UTI-BP40 and UTI-BP45) have been purified.
UTI-BP40 was identified as a truncated form of human
cartilage LP (6).
Preparation of Antibodies--
Rabbit polyclonal antibodies
raised against UTI-BP, LP, or LP synthetic peptide
(LPpep-N) were prepared in our laboratory (6). Briefly, to
generate anti-LP peptide antibodies, two synthetic oligopeptide
sequences, 112VFLKGGSDSDAS123 (amino-terminal
fragment of LP) and 231TVPGVRNYGFWDKDKS246
(carboxyl-terminal fragment of LP), corresponding to the amino-terminal domain and the carboxyl-terminal domain of human LP molecule, respectively, were selected. Antisera against LP synthetic
oligopeptides (anti-LPpep-N and anti-LPpep-C)
were obtained from rabbits immunized four times with 0.2 mg of peptide
conjugated with keyhole limpet hemocyanin together with Freund's
adjuvant (6).
Radioactive Iodination of UTI and Its Derivatives--
UTI was
radioiodinated with carrier-free Na125I. Briefly, 5 µg of
UTI was placed in a conical microvial and then dissolved in 20 µl of
50 mM sodium bicarbonate. Tris-HCl buffer (20 µl each of
50 mM and 500 mM, pH 7.4), 1 mCi of
carrier-free Na125I, and 5 µg of lactoperoxidase were
added to the iodination vial. The iodination reaction was initiated by
the addition of 5 µl of H2O2 diluted
1:75,000. After 2 min, the reaction mixture was applied on a Sephadex
G-25 column (PD-10 column; Amersham Pharmacia Biotech), which was
equilibrated and eluted with 50 mM Tris-HCl, pH 7.4. 125I-UTI obtained from the PD-10 column was diluted with 50 mM Tris-HCl buffer containing 0.2% BSA, pH 7.4, filter-sterilized, and then stored at 4 °C. NG-UTI and HI-8 were
also labeled according to the manufacturer's instructions. UTI,
NG-UTI, and HI-8 were labeled with 125I, resulting in a
specific radioactivity of 4,500 cpm/ng (UTI), 5,100 cpm/ng (NG-UTI),
and 2,500 cpm/ng (HI-8), without loss of latent protease inhibitory activity.
Treatment of Cells with Hyaluronidase--
HCS-2/8 cells (1.0 ml, 5 × 106 cells, >95% viable) were incubated at
23 °C for 2 h in RPMI 1640, pH 7.4 containing 10 µg/ml hyaluronidase with gentle shaking. The cells were washed three times
with RPMI 1640 containing 2% BSA. Analysis of the viability showed
that 2 h after incubation of the cells with hyaluronidase, >92%
of the cells were viable. Hyaluronidase-treated cells were used for
further experiments after washing with phosphate-buffered saline.
Cell Lysate Preparation--
Cells (2 × 108) preincubated with serum-free medium overnight were
treated with PBS containing protease inhibitor mixture supplemented with hyaluronidase (10 µg/ml) for 15 min at 37 °C (3, 5, 6). At
the end of the incubation, the supernatant was recovered for the
hyaluronidase-soluble phase. The pelleted cells were extracted in 4 ml
of lysis buffer (10 mM Tris-HCl, pH 8.1, 140 mM
NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride, 0.1 mg/ml leupeptin, and 1.0% Triton X-100) for
detergent-phase extract. Cell extracts or supernatants were cleared at
10,000 × g for 10 min at 4 °C and stored at
Cell Binding and Competition Experiments--
Binding assays
were performed at 4 °C as described previously (3, 5, 6). Confluent
monolayers of HCS-2/8 were grown in 24-well plate wells using complete
medium supplemented with 10% fetal calf serum. The cells were washed
twice each with serum-free medium supplemented with 0.1% BSA. The
cells were maintained overnight in serum-free medium. Monolayers were
cooled to 4 °C and washed twice with Tyrode's Hepes solution
containing 2% BSA (washing buffer). The cells were then incubated at
4 °C for 2 h in binding buffer (150 mM NaCl, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, 2% BSA, pH 7.8) containing various
concentrations of 125I-labeled UTI or NG-UTI (0-10
nM) and washed four times. The binding of
125I-UTI to cells was then determined as previously
described (7). The contents of the wells were removed using 1 N NaOH,
and radioactivity was measured using a Ligand Blotting and Immunoblotting Procedures--
Cell extracts
(detergent phase) and supernatants (hyaluronidase-soluble phase) were
tested for UTI-BP40 and UTI-BP45 by Western blot or by ligand blotting with labeled ligands. For ligand blotting, purified UTI-BPs, LP, hyaluronidase-soluble, or detergent
phase-separated membrane fractions were run on 10% SDS-polyacrylamide
gels under nonreducing conditions, electroblotted to PVDF membranes
(Bio-Rad), and blocked for 2 h at 23 °C in Tris-buffered saline
contining 2% BSA. The filters were then incubated for 16 h at
4 °C in the presence of 1 nM iodinated ligand in binding
buffer, washed three times, and autoradiographed for 18 h to 2 days. Each band was cut from the membrane, and radioactivity was
measured using a
In a parallel experiment, Western blotting with a rabbit
polyclonal antibodies raised against LP was performed as described previously (6, 20).
Determination of Plasminogen Activator Activity--
Plasminogen
activator (PA) activity in the cell-conditioned media (100 µl) was
quantitated utilizing a functional assay for plasmin. Medium (100 µl)
was then incubated for 3 h in buffer A (phosphate-buffered saline
containing plasminogen (0.165 units/ml) and S-2251 (0.5 mM)) as the chromogenic substrate of plasmin. In a parallel
experiment, after the monolayer cells were washed, the medium was
replaced with buffer A and incubated for 1 h to determine the
cell-associated PA activity. The amount of
p-nitroaniline released was determined
spectrophotometrically at 405 nm. Each assay was run with a
plasminogen-free blank.
Statistical Analysis--
The data presented are the mean of
triplicate determinations in one representative experiment unless
stated otherwise. Data are presented as mean ± S.D. All
statistical analysis was performed using StatView for Macintosh. The
Mann-Whitney U test was used for the comparisons between
different groups. p less than 0.05 was considered significant.
Characterization of UTI and Its Derivatives--
UTI (molecular
mass, 40 kDa) is composed of the N-domain, a C4S sugar side chain, and
the C-domain that has been considered to exist as a protease inhibitor
domain (Fig. 1). Previous work suggested
that the N-domain is important for binding of UTI to the cells (3, 5,
6). To gain insight into the ability of these modules within the UTI
molecule to bind to the UTI-binding sites on the cells, deletion
fragments of this protein were produced, purified, and used as
125I-labeled ligands for cell binding and ligand blotting
analyses. As shown in Fig. 1, under nonreducing conditions, NG-UTI and
HI-8 have an apparent molecular mass of 25 and 8 kDa, respectively.
The Kinetics of UTI Binding to UTI-BP40 and
UTI-BP45--
We have previously demonstrated that there
are at least two types of UTI binding sites, the UTI-BP40
and the UTI-BP45, on the surface of certain cells, and
binding of UTI to cells is specifically inhibited by monoclonal
antibodies 4G12 and 8H11, which recognize the N-domain of UTI,
demonstrating that UTI binds to the cells via the N-domain of UTI (3,
5).
We first tested for the presence of UTI-BPs in HCS-2/8 cells by a
ligand-blotting analysis. Affinity-purified UTI-BP40 and UTI-BP45 proteins prepared from HCS-2/8 cell lysate (6)
were used as a positive control in the identification. To identify the
structural determinants on UTI required for cell association, NG-UTI
and HI-8 were produced and used as 125I-labeled ligands. We
incubated 125I-labeled ligands with affinity-purified
UTI-BP40 or UTI-BP45 transferred to PVDF paper
in the presence or absence of an excess amount of unlabeled compounds.
We found that the cells contain, in addition to a 40-kDa
UTI-BP40, also a 45-kDa UTI-BP45 (Fig. 2, lane 1), because
125I-UTI can bind to both of these UTI-BPs (lane
3). The binding of 125I-UTI to UTI-BP40
and UTI-BP45 was completely competed by unlabeled UTI
(lane 6), supporting the identification of these bands as UTI-specific binding proteins.
We next investigated the binding properties of UTI derivatives lacking
the C4S side chain or the N-domain. 125I-UTI bound to
UTI-BP40 and UTI-BP45, whereas
125I-NG-UTI binds to UTI-BP40 only (lane
9). 125I-HI-8 failed to bind any of the UTI-BPs (data
not shown), demonstrating that there was no indication that the
C-domain of UTI was involved in binding of UTI to these UTI-BPs. Of
interest is the observation that the C4S side chain of UTI moiety would
take place in binding of the UTI to UTI-BP45 as judged from
the above observation.
Hyaluronidase-soluble (Fig. 2H, lanes 4, 7, 10, and
13) and the detergent (Fig. 2D, lanes 5, 8, 11, and 14)
phases of cells were phase-separated and analyzed for the presence of
UTI-BPs. The two phases were analyzed by ligand and immunoblotting for binding to 125I-UTI, 125I-NG-UTI, or
125I-HI-8 and to polyclonal antibodies raised against LP
(Fig. 2, anti-LP, lanes 12-14). In the detergent-soluble
extract (lane 5), 125I-UTI bound mainly to a
slowly migrating band comigrating with UTI-BP45; however,
in the hyaluronidase-soluble extract (lane 4), it bound
mainly to a band comigrating with UTI-BP40. This latter
band corresponds to LP on the basis of comigration with LP and reaction
with anti-LP antibody. We cannot explain the reason why a substantial
amount of the 45- and 40-kDa proteins is visible in
hyaluronidase-soluble and detergent phases of cells, respectively. However, cell-binding experiments employing flow cytometry indicated that hyaluronidase-treated cells have no or very little specific binding sites for NG-UTI on their cell surface (data not shown; see
Ref. 7). By Western blot analysis, there is no significant difference
in the intensity of the 45-kDa band obtained from both intact cells and
hyaluronidase-treated cells (data not shown). Therefore, it is unlikely
that a substantial proportion of the UTI-BP40 retained on
the cells after hyaluronidase treatment or a substantial amount of
UTI-BP45 released into the medium by
hyaluronidase treatment.
We asked whether the binding of 125I-UTI or
125I-NG-UTI could depend on its individual binding sites
in an in vitro ligand-binding and competition assay. A
series of experiments were then performed to identify the binding sites
in the UTI that is involved in this binding and for the identity of the
purified binding sites. As shown in Fig.
3, binding of 125I-NG-UTI to
UTI-BP40 (Fig. 3B) was comparable with that of
125I-UTI to UTI-BP40 (Fig. 3A).
Competitive binding experiments using in vitro ligand blot
assay demonstrated that the binding of 125I-UTI to
UTI-BP40 was completely blocked by a 100-fold molar excess of unlabeled UTI, NG-UTI, anti-LPpep-N antibody, whereas
this binding was essentially unaffected by HI-8 and any
glycosaminoglycans including fluid-phase, soluble C4S,
chondroitin-0-sulfate (C0S), hyaluronic acid (HA), dermatan sulfate
(DS), or heparin at concentrations of these compounds as high as 100 µg/ml. In contrast, the binding of 125I-UTI to
UTI-BP45 (Fig. 3C) was inhibited by about 25 or
85% by soluble C4S at concentrations of 10 µg/ml or 100 µg/ml,
respectively, whereas the binding of 125I-UTI to
UTI-BP45 was essentially unaffected by a 500-fold molar excess NG-UTI, HI-8, or anti-LPpep-N antibodies. The
inhibitory effects of anti-LPpep-N antibodies (10 µg/ml)
and soluble C4S (10 µg/ml) were not additive. We confirmed again that
125I-NG-UTI did not bind to UTI-BP45 (Fig.
3D). Taken together, the results strongly suggest that the
N-domain of UTI is important for contact with UTI-BP40 and
that UTI-BP45, a protein that was distinct from
UTI-BP40, is another binding protein (or receptor) for the
C4S side chain within the UTI molecule.
Interaction of UTI and Its Derivatives with HCS-2/8 Cells; Binding
and Competition Studies--
We asked whether the binding of
125I-UTI could depend on its individual binding sites on
the HCS-2/8 cells in an in vitro cell binding assay.
125I-UTI bound to cells in a dose-dependent
manner (not shown here; see Refs. 3, 5, 6, and 21). Binding reached a
plateau by 120 min. We determined the number of binding sites and the affinity constant for UTI and NG-UTI. Binding of 125I-UTI
or 125I-NG-UTI to the cells was blocked by excess unlabeled
UTI (Fig. 4). As shown in Fig.
5A, Scatchard plot analysis
performed on three preparations of HCS-2/8 cells demonstrated the
presence of 40,300 ± 3,500 (mean ± S.D.) high affinity
binding sites/cell (Kd = 10.1 ± 1.8 nmol/liter, n = 3) and of 329,000 ± 5,700 lower
affinity binding sites/cell (Kd = 245 ± 26.4 nmol/liter n = 3). Fig. 5B also shows an
apparent Kd of about 280 nM at 4 °C
when using 125I-NG-UTI. Scatchard analysis at 4 °C,
using 125I-NG-UTI, revealed that non-glycosylated compound
binds to 345,000 ± 3,600 lower affinity binding sites/cell
(Kd = 276 ± 21.3 nmol/liter, n = 3). The removal of the carbohydrate side chain of UTI influenced its
effectivity (affinity) to bind to the cells. These results allow us to
conclude that the high affinity binding sites may correspond to the
UTI-BP45, which may be the specific receptor for the C4S
side chain within the UTI molecule, and that the low affinity binding
sites corresponds to UTI-BP40 (or LP).
As shown in Fig. 6, a 500-fold molar
excess UTI or NG-UTI inhibited the 125I-NG-UTI binding by
>95% (Fig. 6B). Likewise, excess UTI completely competed
against the binding of the 125I-UTI; however, excess NG-UTI
(upon an excess of 5,000 nM) competed for about 50% of the
125I-UTI binding (Fig. 6A). Binding of
125I-UTI and 125I-NG-UTI was independent of its
protease inhibitor site (HI-8). The binding of 125I-NG-UTI
was completely inhibited by treatment of the cells with anti-LPpep-N antibodies, which are considered to block
binding of UTI or NG-UTI to UTI-BP40, confirming that
UTI-BP40 recognizes the N-domain of UTI but not the C4S
side chain of UTI. The binding of 125I-NG-UTI was not
inhibited by pretreatment of the cells with soluble C4S. Binding of
125I-UTI was inhibited ~50% by 10 µg/ml
anti-LPpep-N antibody alone. Moreover, the fact that
anti-LPpep-N antibody at concentrations as high as 100 µg/ml inhibited the binding of 125I-UTI to cells by a
mean of 60% (not shown) and therefore binding of 125I-UTI
to UTI-BP40 could not account for all the binding of
125I-UTI to cells suggest the involvement of a second
cellular site, UTI-BP45. Comparable with this hypothesis,
soluble C4S (100 µg/ml) specifically inhibited the binding of UTI to
cells by ~40%, whereas other glycosaminoglycans had no effect. In
support of this interpretation, soluble C4S was a potent inhibitor of
the binding of 125I-UTI when the cells were preincubated
with anti-LPpep-N antibody. The inhibitory effects of
anti-LPpep-N antibody (10 µg/ml) and soluble C4S (10 µg/ml) were additive and together almost totally blocked
125I-UTI binding to the cells.
Pretreatment with hyaluronidase (10 µg/ml, 30 min, 23 °C) reduced
125I-NG-UTI binding more than 90%, whereas binding of
125I-UTI was reduced only 40-50% in cells pretreated with
hyaluronidase (data not shown). The recognition site for
NG-UTI (UTI-BP40) is considered to be more
readily affected by hyaluronidase treatment of the cells than
UTI-BP45, because most of UTI-BP40 is attached to the cell membrane via a hyaluronic acid anchor (3, 5, 6).
The Favored Pathway for UTI Action; UTI-BP45 Mediates
UTI-dependent Signal Transduction--
As a final
question, we tested whether UTI-mediated suppression of PMA-stimulated
up-regulation of uPA was dependent on either UTI-BP40 or
UTI-BP45. The amount of uPA expressed on the cells stimulated with 1.0 µM PMA was maximal at 6-9 h of
incubation.2 As expected
(Fig. 7), UTI significantly inhibited
PMA-induced uPA expression during a 7-h incubation. Unlike UTI, NG-UTI
failed to abrogate PMA-induced uPA expression, suggesting that the C4S side chain within the UTI molecule may play a role in
UTI-dependent signal transduction.
To test whether UTI binding to UTI-BPs is necessary to generate
UTI-dependent signal, we used the cells pretreated with an excess amount of soluble C4S or anti-LPpep-N antibody, to
which UTI could bind only to cellular UTI-BP40 or to
cellular UTI-BP45, respectively. Of note is that soluble
C4S and anti-LPpep-N antibody themselves did not affect the
PMA-stimulated uPA expression (not shown). UTI-dependent
signal transduction (i.e. suppression by UTI of
PMA-stimulated uPA expression) was completely abrogated either by
anti-LPpep-N antibody or by soluble C4S added separately, suggesting that UTI function can require binding to both
UTI-BP40 and UTI-BP45. Therefore, masking of
cell-associated UTI-BPs either by anti-LPpep-N antibody or
by soluble C4S could abrogate UTI-dependent signals. As a
control, UTI function was not inhibited by rabbit non-immune IgG.
Moreover, other glycosaminoglycans including fluid-phase HA, DS,
heparin, or C0S had no effect (not shown).
A specific binding site for native glycosylated UTI has been found
on several types of human and murine cells (3, 5-7). Previous studies
have shown that there are at least two different binding sites for UTI
on the surface of the cells and that the 40-kDa UTI-BP40
binding represents the essential step for an efficient binding of UTI
(3, 5-7). Whereas the UTI-BP40 was positively identified
as LP by amino acid sequence (3, 5, 6), the definitive identity of the
UTI-BP45 has not been established yet. It has been recently
reported that UTI induces suppression of uPA expression possibly
through inhibition of translocation of PKC (4, 8). However, the
mechanisms underlying UTI- dependent signal transduction
and the respective functions of UTI-BPs issue that are not yet clear.
The molecular characterization of the association between UTI and its
binding sites provides a framework for understanding their role in the
regulation of uPA expression and invasion. The approach we have taken
toward this objective was to prepare and analyze truncated ligands, to
begin to identify the specific domains of UTI that are involved in its
interaction with the cell-associated-binding protein/receptor. In the
present study, we have examined the responsiveness to UTI in human
chondrosarcoma cell line, HCS-2/8, which expresses predominantly the
cell-associated UTI-binding sites. The cell binding and
ligand blotting analyses revealed constitutive expression of the two
types of UTI-BPs (UTI-BP40 and UTI-BP45) on the
cell surface. The UTI-BPs expressed on HCS-2/8 were similar
to those in the previously described cells with regard to size,
affinity, cross-reactivities to antibodies, and ligand specificity (3, 5, 6). Only in the case of human myometrial cells, however, the
molecular mass of LP is considered to be ~45 kDa (7), which corresponds to intact LP.
Our studies of truncated UTI led us to conclude the following: 1) The
amino terminus is a critical domain for UTI binding to
UTI-BP40 or LP, and the C4S side chain of UTI forms the key determinant for the high affinity specific binding of UTI to
UTI-BP45. This demonstrates that UTI-BP40
recognizes the N-domain of UTI, and UTI-BP45 consists of a
binding portion for the C4S side chain within the UTI molecule. 2)
Either the amino-terminal domain or the C4S side chain can bind cells
even in the absence of the other, demonstrating that the interaction
between UTI and UTI-BP40, and the interaction of UTI and
UTI-BP45, can be considered to occur independently of one
another. 3) Because UTI can bind UTI-BP40 (Kd = ~250 nM) and
UTI-BP45 (Kd = ~10 nM), we believe that UTI binds initially to UTI-BP45 and
subsequently to UTI-BP40, and 4) although essential for
binding, either the amino terminus alone or the C4S side chain alone is
not sufficient for UTI-dependent suppression of
PMA-activated up-regulation of uPA. This function is inhibited by
reagents that were shown to prevent binding of UTI to either the
UTI-BP40 or UTI-BP45. These results suggest
that UTI must bind to both of the UTI-BPs to suppress uPA
up-regulation.
It has been established that UTI binding to the cells may result in
inhibition of PKC translocation (4), abrogation of MAP kinase
activation (8), and subsequently suppression of PMA- or TNF- In contrast, UTI-BP45 should be considered as an active
participant in the complexes rather than simply as an UTI-binding molecule. The ability of UTI-BP45 to directly bind the C4S
side chain of UTI and then generate signals indicates that this
molecule might play a key role in UTI-mediated signal transduction.
However, we noted that direct UTI binding to UTI-BP45
without interaction with UTI-BP40 did not generate
UTI-dependent signals. In addition, treatment of cells with
NG-UTI or HI-8 resulted only in the NG-UTI binding to cells, whereas
the UTI-dependent signal was not detected. The role of C4S
in this process was substantiated by demonstrating that NG-UTI lacking
the C4S side chain does not bind to UTI-BP45 and that
soluble C4S blocks coupling between UTI (or UTI·UTI-BP40 complex) and UTI-BP45, resulting in abrogation of
UTI-dependent signals. However, fluid-phase, soluble C4S
itself had no ability to generate signal via UTI-BP45.
Therefore, UTI-BP45 is an indispensable molecule in the UTI
receptor signal transduction complex, necessary to link events on the
plasma membrane level to downstream signaling pathways (i.e.
outside-in signaling), allowing UTI-dependent suppression of PKC translocation (4) and uPA gene expression induced by phorbol
ester and/or cytokine. Taken together, we conclude that UTI-BP40 acts as a ligand sink, and UTI-BP45
plays a key role in UTI-dependent suppression of
PMA-stimulated up-regulation of uPA. We consider that the term
"binding protein" may be an inappropriate designation and propose
to refer to the UTI-BP45 as a "UTI receptor."
Based on these findings, we postulate that UTI-mediated inhibition of
tumor cell invasion and metastasis appears to consist of at least two
pathways; a direct pathway through inhibition of plasmin activity (1,
11) and an alternate pathway through the suppression of uPA expression,
both of which cooperatively play a pivotal role in the inhibition of
tumor cell invasion and metastasis. Nothing is known as yet whether the
expression of functional UTI-BPs is quantitatively and/or qualitatively
altered on the malignant status. It is conceivable that qualitative and quantitative changes in the cell-associated UTI-BPs might be involved in the effectiveness of UTI. Further characterization of these proteins
is needed to understand its physiologic significance.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-inhibitor (I
I) by liver cells (1). UTI is a
40-kDa sulfated glycoprotein comprised of several structurally and
functionally distinct domains (2). We have examined specific binding
sites for UTI in certain cells (3-7). Cell binding experiments
indicated that not only neoplastic cells (human choriocarcinoma
SMT-cc1, Ref. 3; human chondrosarcoma HCS-2/8, Ref. 6; human promyeloid leukemia U937, Refs. 3, 4; and murine Lewis lung carcinoma 3LL cells,
Ref. 3) but also non-neoplastic cells (human neutrophils, Ref. 3),
human umbilical vein endothelial cells (HUVEC) (4), fibroblasts (5),
and myometrial cells (7)) have specific binding sites for UTI on their
cell surface. UTI is bound to specific binding sites that are
incompletely saturated. However, UTI-binding sites on the cell surface
of U937 and neutrophils were completely saturated with endogenous UTI
when these cells were cultured in the presence of 10% fetal calf serum
(3). A growing body of evidence has implicated that at least two types
of binding sites (a 40-kDa UTI-binding protein (UTI-BP40)
and a 45-kDa UTI-BP (UTI-BP45) have been defined on the
surface of the cells (3-7). The UTI-BP40 is considered to
be identical to a truncated form of human cartilage link protein (LP)
with a molecular mass of 40 kDa, which is attached to the cell membrane
via a hyaluronic acid anchor (3-6). Of note, in cultured uterine
myometrial cells obtained from pregnant women; however, LP has been
identified as an intact protein of ~45 kDa (7). The
UTI-BP45, which is immunologically different from the
UTI-BP40, is still an unidentified molecule.
-or phorbol myristate acetate (PMA)-induced stimulation of uPA
in these cells was studied (4). UTI reduced the secretion of uPA in
some cells (SMT-cc1, HCS-2/8, and 3LL cells) treated with PMA (3). On
the other hand, incubation of U937 cells and HUVEC with UTI had no
effect on the ability of PMA to stimulate cell-associated uPA
expression (4). However, induction of uPA expression by TNF-
was
efficiently inhibited when the U937 and HUVEC were incubated with UTI
(4). One may estimate that the disparity is because of the difference
of signaling cascades in cells used in the experiments.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C until use.
-counter. Each experimental
point was performed in at least duplicate wells. The nonspecific
binding, determined as the percent of input counts bound in the
presence of 1 µM unlabeled UTI, was ~9% and was
subtracted from all raw data to give the specific bound counts. In a
parallel experiment, cells were incubated with 125I-UTI (1 nM) for 2 h at 4 °C in the presence or absence of
several competitors and then washed four times.
-counter. The assays were performed with duplicate
samples. The nonspecific binding was subtracted from all raw data to
give the specific bound counts.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
Schematic model of UTI, NG-UTI, and
HI-8. A, UTI contains N-domain, chondroitin-4-sulfate
sugar side chain, and C-domain (protease inhibitor domain).
B, an aliquot (50 ng/lane) was subjected to
SDS-polyacrylamide gel electrophoresis under non-reducing conditions on
an 18% gel followed by Western blot using polyclonal antibodies raised
against UTI. Lane 1, native UTI; lane 2,
non-glycosylated UTI (NG-UTI); and lane 3, the
carboxyl-terminal domain of UTI (HI-8).
View larger version (16K):
[in a new window]
Fig. 2.
Binding specificity of UTI to
UTI-BP40 and UTI-BP45 proteins. 1.0 µg
of affinity-purified UTI-BPs (lane 1) or 0.5 µg of
purified LP (lane 2), hyaluronidase-soluble (H;
lanes 4, 7, 10, and 13), and detergent
(D; lanes 5, 8, 11, and 14) phases (50 µg of protein/lane) prepared from HCS-2/8 cells, were applied to 10%
SDS-polyacrylamide gels and stained with Coomassie Blue (lanes
1 and 2), or electroblotted to PVDF membranes
(lanes 3-14), blocked, and incubated for 16 h at
4 °C with the reagents indicated at the bottom of each
lane at the following concentrations: 1 nM
125I-UTI in the absence (lanes 3-5) or presence
(lanes 6-8) of 5,000 nM UTI; 1 nM
125I-NG-UTI (lanes 9-11); or 1:1,000 dilution
of anti-LP antibody (lanes 12-14; see Ref. 16). After
washing the membranes, bound radioactivity was visualized by
autoradiography, whereas anti-LP antibody was detected by a
peroxidase-coupled anti-rabbit IgG, followed by ECL chemiluminescence.
The experiment shown in this figure was performed in duplicate with
similar results.
View larger version (32K):
[in a new window]
Fig. 3.
Ligand-blotting assay; specific binding of
labeled UTI or labeled NG-UTI to the purified UTI-BPs in the
absence or presence of different competitors. Competition for
binding of 1 nM 125I-UTI (A and
C) or 1 nM 125I-NG-UTI (B
and D) to the purified UTI-BP40 (A
and B) or UTI-BP45 (C and
D) immobilized on PVDF membranes by unlabeled competitors.
Concentrations of competitors: UTI, 5 µM; NG-UTI, 5 µM; HI-8, 5 µM; anti-LP antibody, 50 µg/ml (preincubation for 16 h at 4 °C); C4S, 10 or 100 µg/ml; C0S, HA, DS, or heparin, 100 µg/ml. Each competitor is
indicated on the left side. Each experiment shown in this
figure was performed in triplicate. The bound ligand was quantified in
a -counter. Specific binding was determined as described under
"Experimental Procedures." The mean ± S.D. of one experiment
is shown. Similar results were obtained in two other experiments using
different preparations of unlabeled fluid-phase ligands.
View larger version (22K):
[in a new window]
Fig. 4.
Effect of unlabeled UTI on the binding of
125I-UTI or 125I-NG-UTI by the cells in
vitro. Competitive inhibition of fluid-phase 125I-UTI
or 125I-NG-UTI binding to cells by unlabeled UTI. Cells
were incubated for 2 h at 4 °C in the presence of 1 nM 125I-UTI ( ) or 1 nM
125I-NG-UTI (
) with increasing amounts of unlabeled UTI.
The cell-bound ligand was quantified in a
-counter. Specific binding
was determined as described under "Experimental Procedures." The
ordinate is the concentration ratio of bound/free ligand. F
(pmol/liter) represents the concentrations of unlabeled UTI. Data
represent the mean of two independent experiments. Inset,
actual binding data, i.e. specific binding (B)
versus free ligand (F).
View larger version (8K):
[in a new window]
Fig. 5.
Competition binding of
125I-UTI or 125I-NG-UTI to HCS-2/8 cells.
Scatchard analysis of a representative experiment was shown. Triplicate
samples of the cells were incubated with 1 nM
125I-UTI (A) or 1 nM
125I-NG-UTI (B) alone or in the presence of
increasing amounts of unlabeled ligand for 2 h at 4 °C. The
cell-bound ligand was quantified in a -counter. The data points
represent the average of triplicate determinations with background
subtract. At the end of the incubation, the amount of radioactivity
bound to its binding sites was measured by Scatchard plot. S.D. was
less than 15% of the mean. Experiments were performed in a separate
culture (n = 2) with reproducible results.
View larger version (18K):
[in a new window]
Fig. 6.
Specific binding of labeled UTI or labeled
NG-UTI to the cells in the absence or presence of different
competitors. Competition for binding of 1 nM
125I-UTI (A) or 1 nM
125I-NG-UTI (B) to the cells by unlabeled
competitors. Each experiment shown in this figure was performed in
triplicate. The cell bound ligand was quantified in a -counter. The
mean ± S.D. of three experiments is shown. For concentrations of
competitors, see the Fig. 2 legend.
View larger version (21K):
[in a new window]
Fig. 7.
Anti-LPpep-N antibody or soluble
C4S independently abrogates suppression by UTI of PMA-induced uPA
expression. Cells were incubated with PMA (1.0 µM)
supplemented with ( ) or without UTI (500 nM;
) for
various time intervals at 37 °C in the absence or presence of 50 µg/ml anti-LPpep-N antibody (
) or 100 µg/ml soluble
C4S (
). Cells were also incubated with PMA (1.0 µM)
supplemented with NG-UTI (500 nM;
) for various time
intervals at 37 °C. Cell-associated uPA activity was determined as
described in "Experimental Procedures," and expressed as
OD405 nm. Data are the mean ± S.D. of three
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced
up-regulation of uPA expression (4). In this study, we postulate that
the binding of UTI to UTI-BP40 does not initiate UTI
signaling, because UTI-BP40 lacks the cytoplasmic domain
that is required to recruit appropriate adapter molecules. UTI-BP40 seems to help recognition of UTI and/or
UTI·UTI-BP45 complexes by guiding a favorable
accumulation of UTI into cell-associated extracellular matrix.
Therefore, UTI-BP40 may act as a molecular trap for UTI or
a decoy target for UTI when the C4S side chain is cleaved by certain
enzymes such as hyaluronidase or chondroitinase.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. K. Shibata, T. Noguchi, and A Suzuki (Equipment center, Photo center; Hamamatsu University School of Medicine) for helping with the biochemical analysis. We are also thankful to Drs. Guang W. Sun, and T. Kobayashi (Dept. of Obstetrics and Gynecology, Hamamatsu University School of Medicine), Drs. H. Morishita, Y. Kato, and K. Kato (BioResearch Institute, Mochida Pharmaceutical Co., Tokyo), Drs. Y. Tanaka and T. Kondo (Chugai Pharmaceutical Co. Ltd., Tokyo), Drs. S. Miyauchi and M. Ikeda (Seikagaku Kogyo Co. Ltd., Tokyo), and Drs. T. Sato and A. Ito. (Department of Biochemistry, Tokyo University of Pharmacy and Life Science) for their continuous and generous support of our work.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 81 53 435 2309; Fax: 81 53 435 2308; E-mail: hirame@hama-med.ac.jp.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M009906200
2 H. Kobayashi, S. Mika, and Y. Hirashima, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
UTI, urinary trypsin
inhibitor;
UTI-BP, UTI-binding protein;
C0S, chondroitin-0-sulfate;
C4S, chondroitin-4-sulfate;
HI-8, a
domain II of UTI;
II, inter-
-inhibitor;
NG-UTI, non-glycosylated UTI;
PKC, protein kinase C;
PMA, phorbol
myristate acetate;
uPA, urokinase-type plasminogen activator;
PVDF, polyvinylidene difluoride;
BSA, bovine serum albumin;
LP, link
protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Daveau, M., Rouet, P., Scotte, M., Faye, L., Hiron, M., Lebreton, J. P., and Salier, J. P. (1993) Biochem. J. 292, 485-492[Medline] [Order article via Infotrieve] |
2. | Kobayashi, H., Shibata, K., Fujie, M., Sugino, D., and Terao, T. (1998) Kidney Int. 53, 1727-1735[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Kobayashi, H.,
Gotoh, J.,
Fujie, M.,
and Terao, T.
(1994)
J. Biol. Chem.
269,
20642-20647 |
4. | Kobayashi, H., Gotoh, J., and Terao, T. (1996) Eur. J. Cell Biol. 71, 380-386[Medline] [Order article via Infotrieve] |
5. | Kobayashi, H., Hirashima, Y., Sun, G. W., Fujie, M., Shibata, K., Tamotsu, S., Miura, K., Sugino, D., Tanaka, Y., Kondo, S., and Terao, T. (1998) Biochim. Biophys. Acta 1383, 253-268[Medline] [Order article via Infotrieve] |
6. |
Kobayashi, H.,
Hirashima, Y.,
Sun, G. W.,
Fujie, M.,
Nishida, T.,
Takigawa, M.,
and Terao, T.
(2000)
J. Biol. Chem.
275,
21185-21191 |
7. |
Kobayashi, H.,
Hirashima, Y.,
and Terao, T.
(2000)
Mol. Hum. Reprod.
6,
735-742 |
8. | Kobayashi, H., Suzuki, M., Tanaka, Y., Hirashima, Y., and Terao, T. (2001) J. Biol. Chem. (in press) |
9. | Kobayashi, H., Sugino, D., She, M. Y., Ohi, H., Hirashima, Y., Shinohara, H., Fujie, M., Shibata, K., and Terao, T. (1998) Eur. J. Biochem. 253, 817-826[Abstract] |
10. | Kobayashi, H., Shinohara, H., Takeuchi, K., Itoh, M., Fujie, M., Saitoh, M., and Terao, T. (1994) Cancer Res. 54, 844-849[Abstract] |
11. | Kobayashi, H., Shinohara, H., Ohi, H., Sugimura, M., Terao, T., and Fujie, M. (1994) Clin. Exp. Metast. 12, 117-128[Medline] [Order article via Infotrieve] |
12. | Kobayashi, H., Fujie, M., Shinohara, H., Ohi, H., Sugimura, M., and Terao, T. (1994) Int. J. Cancer 57, 378-384[Medline] [Order article via Infotrieve] |
13. |
Kobayashi, H.,
Gotoh, J.,
Hirashima, Y.,
Fujie, M.,
Sugino, D.,
and Terao, T.
(1995)
J. Biol. Chem.
270,
8361-8366 |
14. | Kobayashi, H., Gotoh, J., Kanayama, N., Hirashima, Y., Terao, T., and Sugino, D. (1995) Cancer Res. 55, 1847-1852[Abstract] |
15. | Kobayashi, H., Shinohara, H., Gotoh, J., Fujie, M., Fujishiro, S., and Terao, T. (1995) Br. J. Cancer 72, 1131-1137[Medline] [Order article via Infotrieve] |
16. | Kobayashi, H., Shinohara, H., Fujie, M., Gotoh, J., Itoh, M., Takeuchi, K., and Terao, T. (1995) Int. J. Cancer 63, 455-462[Medline] [Order article via Infotrieve] |
17. |
Kobayashi, H.,
Gotoh, J.,
Hirashima, Y.,
and Terao, T.
(1996)
J. Biol. Chem.
271,
11362-11367 |
18. | Sugino, D., Okushima, M., Kobayashi, H., and Terao, T. (1998) Biotechnol. Appl. Biochem. 27, 145-152[Medline] [Order article via Infotrieve] |
19. |
Takigawa, M.,
Okawa, T.,
Pan, H.,
Aoki, C.,
Takahashi, K.,
Zue, J.,
Suzuki, F.,
and Kinoshita, A.
(1997)
Endocrinology
138,
4390-4400 |
20. |
Kobayashi, H.,
Sun, G. W.,
Hirashima, Y.,
and Terao, T.
(1999)
Endocrinology
140,
3835-3842 |
21. | Kobayashi, H., Hirashima, Y., Sun, G. W., Fujie, M., Shibata, S., Tamotsu, S., Kato, K., Morishita, H., and Terao, T. (1998) Pflug. Arch. Eur. J. Physiol. 436, 16-25[CrossRef][Medline] [Order article via Infotrieve] |