Characterization of Binding Properties of Urinary Trypsin Inhibitor to Cell-associated Binding Sites on Human Chondrosarcoma Cell Line HCS-2/8*

Yasuyuki HirashimaDagger , Hiroshi KobayashiDagger §, Mika SuzukiDagger , Yoshiko TanakaDagger , Naohiro KanayamaDagger , Michio Fujie, Takashi Nishida||, Masaharu Takigawa||, and Toshihiko TeraoDagger

From the Dagger  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.




    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Urinary trypsin inhibitor (UTI)1 is produced as a light chain of inter-alpha -inhibitor (Ialpha 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.

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)-alpha -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-alpha 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.

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.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 -20 °C until use.

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 gamma -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.

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 gamma -counter. The assays were performed with duplicate samples. The nonspecific binding was subtracted from all raw data to give the specific bound counts.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



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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).

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.



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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.

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.



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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 gamma -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.

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).



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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 (open circle ) or 1 nM 125I-NG-UTI () with increasing amounts of unlabeled UTI. The cell-bound ligand was quantified in a gamma -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).



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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 gamma -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.

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.



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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 gamma -counter. The mean ± S.D. of three experiments is shown. For concentrations of competitors, see the Fig. 2 legend.

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.



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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 (open circle ) 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 (black-square) or 100 µg/ml soluble C4S (). Cells were also incubated with PMA (1.0 µM) supplemented with NG-UTI (500 nM; triangle ) 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.

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha -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.

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.


    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; Ialpha I, inter-alpha -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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