Transforming Growth Factor-{beta}1 Produced by Ovarian Cancer Cell Line HRA Stimulates Attachment and Invasion through an Up-regulation of Plasminogen Activator Inhibitor Type-1 in Human Peritoneal Mesothelial Cells*

Yasuyuki Hirashima, Hiroshi Kobayashi {ddagger}, Mika Suzuki, Yoshiko Tanaka, Naohiro Kanayama and Toshihiko Terao

From the Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handayama 1-20-1, Hamamatsu, Shizuoka 431-3192, Japan

Received for publication, December 2, 2002 , and in revised form, April 7, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) and its specific inhibitor (plasminogen activator inhibitor type-1 (PAI-1)) could be involved in the pathogenesis of peritoneal dissemination. We showed previously that expression of uPA and PAI-1 in the human ovarian cancer cell line HRA can be down-regulated by exogenous bikunin (bik), a Kunitz-type protease inhibitor, via suppression of transforming growth factor-{beta}1 (TGF-{beta}1) up-regulation and that overexpression of the bik gene can specifically suppress the in vivo growth and peritoneal dissemination of HRA cells in an animal model. We hypothesize that the plasminogen activator system in mesothelial cells can be modulated by HRA cells. To test this hypothesis, we used complementary techniques in mesothelial cells to determine whether uPA and PAI-1 expression are altered by exposure to culture media conditioned by HRA cells. Here we show the following: 1) that expression of PAI-1, but not uPA, was markedly induced by culture media conditioned by wild-type HRA cells but not by bik transfected clones; 2) that by antibody neutralization the effect appeared to be mediated by HRA cell-derived TGF-{beta}1; 3) that exogenous TGF-{beta}1 specifically enhanced PAI-1 up-regulation at the mRNA and protein level in mesothelial cells in a time- and concentration-dependent manner, mainly through MAPK-dependent activation mechanism; and 4) that mesothelial cell-derived PAI-1 may promote tumor invasion possibly by enhancing cell-cell interaction. This represents a novel pathway by which tumor cells can regulate the plasminogen activator system-dependent cellular responses in mesothelial cells that may contribute to formation of peritoneal dissemination of ovarian cancer.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bikunin (bik),1 a Kunitz-type protease inhibitor, also known as urinary trypsin inhibitor, is proposed as a main participant in the inhibition of tumor cell invasion and metastasis, possibly through the direct inhibition of cell-associated plasmin activity and suppression of urokinase-type plasminogen activator (uPA) mRNA expression (13). The dissemination of ovarian cancer cells within the abdominal cavity involves interaction of tumor cells with the peritoneal mesothelium. In the previous study (4), we transfected the human ovarian carcinoma cell line HRA, highly invasive cells, with an expression vector harboring a cDNA encoding for human bik to investigate the effect of bik overexpression and changes in tumor cell phenotype and invasiveness in the stably transfected clones. We found that transfection of HRA with the bik gene constitutively suppresses ERK1/2 phosphorylation/activation and uPA mRNA and protein expression as well as significantly reduced invasion relative to the parental cells (4). Animals inoculated with bik+ clones intraperitoneally induced reduced peritoneal dissemination and long term survival (4). We conclude that transfection of HRA cells with the bik cDNA constitutively suppresses ERK1/2 activation, which results in inhibition of uPA mRNA and protein expression and subsequently reduces dissemination of bik+ clones. In conditions of peritoneal dissemination, there is extensive remodeling of the peritoneum, involving proteolysis of the extracellular matrix (ECM), cell proliferation, attachment, migration, and angiogenesis (58). The metastatic process in epithelial ovarian cancer is thought to involve surface shedding and subsequent dissemination of ovarian cancer cells, facilitated by localized proteolysis at the interface between ovarian cancer cells and peritoneal surfaces.

One of the factors regulating the metastatic process is considered to be TGF-{beta}, which is a multifunctional cytokine that elicits numerous cellular effects pertinent to the metastatic process (8). TGF-{beta} is an effective inducer of matrix deposition/turnover, cell locomotion, and PAI-1 expression (9). Rodriguez et al. (8) suggest that TGF-{beta} may enhance the invasiveness of ovarian cancers. Receptor complexes of the TGF-{beta} superfamily consist of a ligand-binding type II receptor serine-threonine kinase that, following ligand binding, binds to and transphosphorylates the signal transducing type I serine-threonine kinase (10). These receptors activate, through a phosphorylation event, members of a family of downstream signaling intermediates, the Smads (11). Although the Smad pathway is widely represented in most of the cell types and tissues studied, additional pathways may be activated following treatment with TGF-{beta} in specific contexts (12). For example, activation of Ras, ERK1/2, and c-Jun N-terminal kinase (JNK) by TGF-{beta} signaling has been reported in nonmalignant and malignant cell lines (11), whereas activation of protein kinase A contributes to TGF-{beta}-signaling responses in murine cells. In addition, TGF-{beta}-activated kinase-1, a member of the MAPK kinase kinase family and activator of JNK and p38 MAPK pathways, is rapidly activated by TGF-{beta} in certain cell systems (11, 12). PAI-1 may be regulated by different cytokines and growth factors among which TGF-{beta} plays a pivotal role. Indeed, TGF-{beta} stimulates PAI-1 secretion in various cell lines and in vivo (1315).

In considering these data, we asked whether the PA system expressed by mesothelial cells could be involved in the pathogenesis of peritoneal dissemination by ovarian cancer. Although the PA system has been identified in the mesothelial cells, it is not known whether ovarian cancer exposure might alter PA system expression. In this study we determined whether culture media conditioned by human ovarian cancer cell line HRA induced uPA and PAI-1 expression in cultured human peritoneal mesothelial cells and dermal fibroblast cells as a control, either by incubation with culture medium conditioned by the bik+ clones or the controls (wild-type HRA cells and the luciferase transfected (luc+) clones). By using antibody neutralization techniques, we tested whether the effects were mediated by selected cytokines. Our results demonstrate that culture medium conditioned by wild-type cells, rather than bik+ clones, increases PAI-1 expression of cultured mesothelial cells at least via a TGF-{beta}1-mediated MAPK pathway and that TGF-{beta}1 enhances tumor cell adhesion and subsequent invasion via overexpression of PAI-1. We also demonstrate the involvement of PAI-1 in an increased cell-cell interaction between ovarian cancer cells and mesothelial cells. These findings suggest that altered expression of PAI-1 by the mesothelium could influence the processes of peritoneal dissemination in ovarian cancer cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Ultrapure natural human TGF-{beta}1 was from Genzyme (Cambridge, MA) and R & D Systems (Minneapolis, MN). The antibodies against uPA (Ab 3689, recognizes uPA B-chain; Ab 3471, reacts with uPA A-chain; interferes with binding of uPA to its receptor), PAI-1 (Ab 379, recognizes all forms of PAI-1 and its complexes and neutralizes PAI-1 inhibition), and uPAR (3932, equally reactive with occupied and unoccupied uPAR; and Ab 3936, interferes with receptor binding) were gifts from Dr. R. Hart (American Diagnostics, Greenwich, CT). High molecular weight recombinant uPA, synthetic chromogenic substrate of uPA (Spectrozyme UK), chromogenic plasmin substrate (Spectrozyme PL), Glu-type plasminogen, and purified plasmin were also obtained from American Diagnostics. Rabbit polyclonal antibodies against the total and phosphorylated ERK1/2 were from Zymed Laboratories Inc. (San Francisco, CA) and Calbiochem, respectively. Polyclonal antibodies that recognize the total and phosphorylated (activated) forms of JNK and p38 were from Santa Cruz Biotechnology (Santa Cruz, CA). Neutralizing antibody against TGF-{beta}1 was purchased from R & D Systems and Sigma. Neutralizing mouse monoclonal IgG antibodies to TNF-{alpha} or IL-1{beta} were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Horseradish peroxidase-conjugated goat anti-rabbit Ig were obtained from Dako (Copenhagen, Denmark). MAPK kinase inhibitor PD98059, p38 MAPK inhibitor SB202190, and protein kinase A inhibitor H-89 were from New England Biolabs (Beverly, MA). Culture media, penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Invitrogen. Tissue culture plastics were purchased from Costar/Corning (Cambridge, MA) and Falcon (BD Biosciences). Bovine serum albumin (BSA), Tris base, dithiothreitol, phenylmethylsulfonyl fluoride, and ammonium persulfate were commercially obtained from Sigma. Acrylamide, bisacrylamide, and PVDF membranes were from Bio-Rad. Matrigel was purchased from Collaborative Research (Bedford, MA). X-ray film was purchased from Eastman Kodak Co. The enhanced chemiluminescence (ECL) was purchased from Amersham Biosciences.

Cell Culture—Human ovarian cancer cell line HRA was grown and cultured as described previously (16). The HRA cells were maintained in RPMI 1640 medium supplemented with 10% FBS in a cell culture incubator (constantly set at 37 °C with 5% CO2).

Human peritoneal mesothelial cells were isolated during elective surgery from patients operated on for a nonperitonitis cause and without disseminated cancer. Isolation of human peritoneal mesothelial cells and primary cultures were performed as already reported (17, 18). Human peritoneal mesothelial cells were routinely maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2. Cells were subcultured weekly by incubation at 37 °C for 2 min with 0.0125% trypsin in 0.02% EDTA, followed by addition of complete medium, washing, and resuspension in fresh medium. Fresh medium was given three times a week, twice at 2-day intervals and once after a weekend interval. Confluent cultures of mesothelial cells were routinely split 1:4 and grown in gelatin-coated culture dishes or flasks in the presence of antibiotics. All of the experiments described below were performed in DMEM containing 10% FBS and 1 mM sodium pyruvate with mesothelial cells between passage 2 and 3 and dermal fibroblasts between passages 2 and 5, respectively. Confluent cultured peritoneal mesothelial cells were incubated in serum-free medium for 24 h before testing the effects of compounds. For some experiments, ~5 x 105 cells/well were plated out in 35-mm dishes and transferred to serum-free DMEM for 24 h and then exposed to the different agents at the indicated concentrations and for the times indicated. For the inhibition studies, optimized concentrations of SB202190, PD98059, and H-89 were added 1 h prior to TGF-{beta} treatment. Incubation of cells with doses of tested compounds for up to 48 h did not have a significant effect on cell viability. At confluence, growth medium was removed and replaced with fresh medium (control), with culture media containing concentrated culture media supernatants from ovarian cancer cells, or TGF-{beta}1 at concentrations indicated in the figures for different times at 37 °C in 5% CO2.

Human dermal fibroblasts were isolated from full thickness human skin (19). The isolated fibroblasts were cultured in DMEM containing 10% FBS with antibiotics. Before exposure to TGF-{beta}1, the culture medium was replaced with serum-free DMEM.

Preparation of Conditioned Medium and Cell Lysate—For the preparation of conditioned medium, subconfluent cultures of ovarian cancer cell clones were washed three times with PBS and incubated with serum-free DMEM for 24 h. The conditioned medium was collected, centrifuged to remove cell debris, filtered, lyophilized, and stored at –80 °C until needed.

The cell monolayers treated with or without various agents for the indicated times were washed with PBS. 1 x 106 cells were lysed in 750 µl of lysis buffer containing 20 mM Tris-HCl (pH 7.5), 12.5 mM 2-glycerophosphate, 150 mM NaCl, 1.5 mM MgCl2, 2 mM EGTA, 10 mM NaF, 0.5% Triton X-100, 2 mM dithiothreitol, 1 mM sodium vanadate, and 1 mM phenylmethylsulfonyl fluoride at 4 °C for 15 min and scraped with a rubber policeman. Cell extracts were then centrifuged at 3,000 x g to remove cell debris. All samples were stored at –20 °C until use. In parallel, cells treated in the same condition in different dishes were harvested and counted using a hemocytometer.

Detection of Cell Surface-associated Plasminogen Activators by Colorimetric Assay—Cells were seeded at 10,000 cells/well in 96-well plates in the maintenance medium and allowed to reach ~90% confluence. The cell monolayers treated with or without various agents for the indicated times were washed with PBS. The cells were washed once with PBS and analyzed in a PBS-based reaction buffer (100 µl/well). The reactions were initiated by the addition of the chromogenic amidolytic substrate specific for either uPA (Spectrozyme UK) to a final concentration of 0.2 mM in the presence or absence of amiloride (1 mM) or neutralizing antibodies to uPA (10 µg/ml). Cell surface-associated plasmin activity was determined spectrophotometrically using a Spectrozyme PL. No amidolytic substrate was added in the blank reactions. The photometric absorbance of the reaction mixtures at 405 nm was monitored at 37 °C over the next 30 min.

Lactate Dehydrogenase and Total Protein Assays—TGF-{beta}1 cytotoxicity was approximated by release of lactate dehydrogenase (LDH) and compared between treatment groups and control. LDH was measured using the ultraviolet method kit (Sigma). In addition, the trypan blue cell staining procedure was performed. Total protein determinations were made using a spectrophotometric technique using bicinchoninic acid (Pierce). Determinations of LDH and proteins were made in triplicate.

Quantitation of uPA, PAI-1, and TGF-{beta}1 Levels, Enzyme-linked Immunosorbent Assays—The amount of uPA was measured in the cellconditioned medium and in the cell lysate using the Imubind uPA ELISA kit from American Diagnostics Inc. PAI-1 levels were determined using the Imubind PAI-1 ELISA kit from the same vendor. Lower detection levels were set at 10 pg/ml for the uPA kit and below 1 ng/ml for the PAI-1 kit. The uPA assay measured all forms of uPA and its complexes. PAI-1 ELISA detected the active and latent forms of PAI-1, as well as PAI-1 in complex with PAs. The active and total (active plus latent) TGF-{beta}1 were assayed with a human TGF-{beta}1 ELISA kit (BIOSOURCE, Tokyo, Japan) according to the manufacturer's instructions. To measure total TGF-{beta}1, samples were acidified with acetic acid and then neutralized as described previously (20).

Flow Cytometric Analysis—Culture cells were harvested and washed with washing buffer (PBS supplemented with 2% bovine serum albumin and 0.1% NaN3 (pH 7.4)). Because uPAR is trypsin-sensitive, cells were treated with hyaluronidase and used for further experiments. A single cell suspension (106/ml) was incubated with affinity-purified antibodies or isotype control antibodies on ice for 1 h. Cells were washed three times with washing buffer, and 2 µl of the primary antibody (Ab 3471 plus Ab 3689 (uPA), Ab 379 (PAI-1), and 3932 (uPAR)) and 3 µl of fluorescein isothiocyanate-conjugated second antibody (Dako) were added for 1 h on ice, respectively. The cells were washed twice with fluorescence-activated cell sorter buffer and then subjected to flow cytometry using the FACScan flow cytometer (BD Biosciences), and data (mean fluorescence intensity) were quantified. At least 10,000 cells were analyzed per sample in all experiments. All experiments were performed at least twice.

Western Immunoblot Analysis—Equal protein amounts of each sample (conditioned media and cell lysates) were resolved on a 12% acrylamide, 0.5% bisacrylamide SDS gel under nonreducing and reducing conditions and transferred to PVDF membranes. The membrane was blocked with 2% BSA in wash buffer for 1 h at room temperature followed by overnight hybridization with a 1:1000 dilution of each antibody in the same buffer at 4 °C and washed and then with a goat anti-rabbit/mouse IgG conjugated to horseradish peroxidase. Finally, the membranes were washed and developed with a chemiluminescence detection reagent (ECL system, Amersham Biosciences). Antibodies used are as follows: anti-uPA (Ab 3471 plus Ab 3689), anti-PAI-1 (Ab 379), anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK, anti-p38 MAPK, anti-phospho-p38 MAPK, anti-total Smad2, and anti-phospho-Smad2. As loading controls, Western blots were also performed using antibodies against total ERK1/2, JNK, p38, and Smad2.

Isolation of Cytoplasmic RNA and Northern Blot Analysis—The HRA cells were plated at a density of 106 cells/100-mm dish. When cells grown in monolayer reached an early phase of confluency, total cellular RNA samples were extracted by Trizol (from Invitrogen) according to the manufacturer's protocol. For Northern blot analysis, cytoplasmic RNA (20 µg) was electrophoresed onto a formaldehyde, 1.0% agarose gel and blotted onto a nylon filter. After transfer, RNA was cross-linked onto the membranes by UV irradiation. The nylon filter was hybridized with a 32P-labeled cDNA probe in 50% formamide, 5x saline/sodium phosphate/EDTA, 0.1% SDS, 5x Denhardt's solution, and 100 µg/ml salmon sperm DNA at 42 °C for 20 h. Quantitative analysis was performed with a Bio-Rad model 620Video Densitometer with a one-dimensional analyst software package for Macintosh.

The uPA cDNA probe was prepared as described previously (21). pSP64-hPAI-1, a 1.4-kbp EcoRI-BglII fragment (position 54–1480 (22)) isolated from pPAI1-C1, a plasmid containing a 2.2-kbp human PAI-1 cDNA insert (23), was subcloned between the BamHI and EcoRI sites of pSP64 (24).

Adhesion Assay—To quantify tumor cell adhesion to the peritoneal mesothelial cells and fibroblast cells, a standardized cell adhesion assay was developed according to methods described previously (25, 26). Mesothelial and fibroblast monolayers were established in a 96-well plate precoated with gelatin. In order to determine the effect of TGF-{beta}1 on tumor cell adhesion, mesothelial and fibroblast monolayers were preincubated with increasing doses of TGF-{beta}1 for 12 h in the presence or absence of specific antibodies. Non-preincubated monolayers served as controls.

Tumor cells were labeled with calcein-AM solution as described previously (26). The labeled tumor cells were washed to remove free dye. Two hundred µl of DMEM, 0.5% BSA containing 3 x 104 calcein-labeled tumor cells were added. After plates were incubated at 37 °C for 60 min, the medium of each well was removed and washed twice with DMEM, 0.5% BSA. The remaining fluorescence per well was measured on a plate reader (PerkinElmer Life Sciences), using 485 excitation and 530 emission filters. The amount of adherent cells was determined by calibrating the measured fluorescence of the experimental wells in relation to the standard.

Invasion Assay of Cancer Cells—An invasion assay was performed according to the method reported by Mizutani et al. (7). An invasion assay upper chamber with an 8-µm porosity cell-permeable polycarbonate filter covered with Matrigel was placed on a cell culture plate. Peritoneal mesothelial cells or dermal fibroblast cells were cultured fully on Matrigel, pretreated with or without 10 ng/ml TGF-{beta}1 for 12 h in the presence or absence of neutralizing antibodies, and washed, and 2 x 105 HRA cells were then added to the invasion assay upper chamber. As a chemoattractant, NIH3T3 fibroblast conditioned medium was used. After cultivation for 36 h, the chambers were immersed in 100% methanol for 1 min for fixation, and all cells were then stained by hematoxylin. The cells remaining on the top surface of the membrane were completely removed with a cotton swab, and the membrane was removed from the chamber and mounted on a glass slide. These preparations were examined under a microscope at x100 magnification. The number of infiltrating HRA cells were counted in five regions selected at random, and the extent of invading HRA cells was determined by the mean count. Duplicate filters were used, and the experiments were repeated three times.

Statistics—Data are expressed as mean ± S.D. of at least three independent triplicate experiments. Statistical analysis was performed by one-way analysis of variance followed by the Student's t test. p < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We transfected the HRA cells with an expression vector harboring a cDNA encoding for human bikunin (4). The bikunin expression vector pCMV-bikunin-IRES-bsr (bik+) and the control vector pCMV-luciferase-IRES-bsr encoding luciferase (luc+) were transfected into HRA cells by the standard calcium phosphate precipitation method. We conclude that bik+ clones significantly reduced invasion and that animals inoculated with bik+ clones induced reduced peritoneal dissemination and long term survival (4). Therefore, we examined whether ovarian cancer cells produce factors that can stimulate PA system expression in mesothelial cells.

Expression of uPA and PAI-1 in Human Peritoneal Mesothelial Cells and Dermal Fibroblast Cells—The culture media conditioned by mesothelial and fibroblast cells were initially tested by ELISA for uPA and PAI-1. Basal secretion of PAI-1 was greater in mesothelial cells and to a lesser extent in fibroblast cells. Unlike PAI-1, uPA was secreted to a lesser degree by these cells (Fig. 1, A and B). Western blot assays were used to determine the level of PA system expression in the cell lysate and conditioned medium. Expression of PAI-1 was greater in mesothelial cell lysate and to a lesser extent in fibroblast cell lysate (Fig. 1C). uPA expression is low in these cell lysates, as suggested by the inability to detect protein in cell lysates by Western blot. The faint band of uPA·PAI-1 complex was detected in these conditioned media. These cells failed to detect free uPA protein in the medium (Fig. 1D).



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FIG. 1.
Expression of uPA and PAI-1 in human peritoneal mesothelial cells and dermal fibroblast cells. A and B, serum-free conditioned media were obtained by incubating mesothelial cells and fibroblast cells in 2-cm2 dishes at 37 °C for 24 h with 0.5 ml of incubation medium. uPA and PAI-1 levels in the cell-conditioned medium were determined using the Imubind uPA and PAI-1 ELISA kits. Values are means ± S.D. of three experiments as ng/ml. C and D, proteins (100 µg) isolated from cells (CL) and conditioned medium (CM, 50 µl) were separated on 12% SDS-PAGE and electroblotted to nitrocellulose membranes. The membranes were subjected to Western blotting using monoclonal antibodies to PAI-1 (C) or uPA (D). The Western blot data are typical of duplicate experiments. M, mesothelial cells; F, fibroblast cells.

 

Time-dependent Expression of uPA and PAI-1 in Mesothelial Cells in Response to Culture Medium Conditioned by Ovarian Cancer Cells—We tested the ability of each medium from ovarian cancer cells (wild-type HRA cells, luc+ clone, and bik+ clone) to influence expression of PAI-1 protein in the mesothelial cells and fibroblast cells for varying times (0–48 h). PAI-1 expression in the cell lysates was assessed by Western blotting. Results of the experiments using mesothelial cells (Fig. 2A) demonstrate that culture medium conditioned by wild-type cells and luc+ clone significantly induces mesothelial cell PAI-1 expression in a time-dependent manner and that the induction starts 6–12 h after addition of both media. Maximal induction of PAI-1 was achieved around 12–24 h, and the elevated level is not maintained for at least 48 h. Fig. 2B shows that PAI-1 protein levels increase about 3.5-, 2.8-, and 1.5-fold by 12 h after exposure of mesothelial cells to media from wild-type cells, luc+ clone, and bik+ clone, respectively. The conditioned media supernatants from wild-type and luc+ clone significantly increased PAI-1 expression in mesothelial cells compared with those of bik+ clone. In fibroblasts exposed to these media, PAI-1 levels increase, but there was no significant increase in PAI-1 expression over that in cells treated with fresh media alone (data not shown).



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FIG. 2.
Time-dependent PAI-1 protein expression by each medium from ovarian cancer cells in mesothelial cells. Confluent mesothelial cells were exposed to media from wild-type cells, luc+ clone, or bik+ clone (200 µl (10x concentrated each medium) plus 1800 µl fresh basal medium containing 0.5% BSA) or fresh medium alone for 0, 6, 12, 24, and 48 h (1st to 5th lanes) at 37 °C, and cell lysate proteins were isolated. The total proteins (100 µg) were separated on 12% SDS-polyacrylamide gels and transferred to PVDF membranes. A, the membrane was immunoblotted with anti-PAI-1 antibody. B, densitometric analysis of the Western blot shown in A. The results are expressed as ratio relative to untreated control (0-h incubation). The data illustrated are representative of at least three independent experiments, and the mean density ± S.D. of the individual bands is presented in the bar graph. The letters a and b, or a' and b', or a'', b'', and c'' indicate statistical differences (p < 0.05).

 

We tested the ability of each culture medium conditioned by ovarian cancer cells to influence expression of uPA protein in the mesothelial and fibroblast cell extracts. ELISA data using mesothelial cells and fibroblast cells revealed that there was no significant increase in uPA expression by 48 h after exposure of both cells to media from wild-type cells, luc+ clone, and bik+ clone, respectively (data not shown).

uPA and PAI-1 mRNA Expression by Mesothelial Cells and Fibroblast Cells Exposed to Culture Medium Conditioned by Each Cell Line, Northern Blot Analysis—By having determined that culture media from wild-type cells stimulates a time-dependent PAI-1 protein expression in the mesothelial cells, we next assessed the ability of each medium to influence PAI-1 message expression by mesothelial and fibroblast cells. Culture media from bik clones (wild-type cells and luc+ clone) significantly induces PAI-1 mRNA, and the induction is observed 6 h after the treatment (Fig. 3). Maximum accumulation of PAI-1 mRNA is achieved 6–12 h after the treatment. PAI-1 mRNA levels increase about 4.0-, 3.8-, and 1.3-fold by 12 h after exposure of mesothelial cells to media from wild-type cells, luc+ clone, and bik+ clone, respectively. We did not detect any effect of the tumor cell-conditioned media on GAPDH mRNA content. Thus, the increased level of PAI-1 mRNA is attributable to increased secreted and cell-associated PAI-1 proteins.



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FIG. 3.
Time-dependent induction of uPA and PAI-1 mRNA by each medium from ovarian cancer cells in mesothelial cells. Left panel, mesothelial cells were exposed to each medium from wild-type cells, luc+ clone, and bik+ clone for 0, 3, 6, 12, and 24 h (1st to 5th lanes). Total RNA (20 µg/lane) was isolated using Trizol reagent and separated on an agarose-formaldehyde gel and subjected to Northern blotting using 32P-labeled uPA, PAI-1, and GAPDH cDNAs. Right panel, densitometric analysis of the Northern blot shown in the left panel. The level of PAI-1 mRNA was quantitated by densitometric scanning and normalized against GAPDH loading controls. The results are expressed as ratio relative to untreated control (0-h incubation). The figure shown is representative of the results of two independent experiments, and the bar graph illustrates the mean band densities from two experiments with a similar result.

 

Unlike the results from ELISA, uPA mRNA levels increase about 3.5-, 4.3-, and 1.8-fold by 6 h after exposure of mesothelial cells to culture media from wild-type cells, luc+ clone, and bik+ clone, respectively (Fig. 3). In contrast, fibroblast cells exposed to each medium demonstrated no significant increased expression of uPA and PAI-1 mRNA (data not shown).

TGF-{beta}1 Produced by Wild-type HRA Cells Specifically Stimulates PAI-1 Expression in Mesothelial Cells—To investigate whether wild-type HRA cells released several types of cytokines participating in an up-regulation of PAI-1 production, the conditioned medium of HRA cells was tested by ELISA for IL-1{beta}, TNF-{alpha}, and TGF-{beta}1. Our recent ELISA data (4) revealed that TGF-{beta}1 is significantly produced by HRA cells (TGF-{beta}1, 396 ± 29.1; TNF-{alpha}, 63.1 ± 10.5; and IL-1{beta}, <20 pg/ml/106 cells/24 h). Twenty four-hour, serum-free conditioned medium from HRA cells contained both latent and activated TGF-{beta}1 (latent TGF-{beta}1, 128 ± 25.1, and active TGF-{beta}1, 180 ± 32.5 pg/ml/106 cells/24 h). We analyzed the TGF-{beta}1 expression of the stable cell lines by ELISA. bik+ clone expressed a relatively small amount of TGF-{beta}1 in the conditioned media (105 ± 13.6 pg/ml/106 cells/24 h), in comparison to luc+ clone (330 ± 78.5 pg/ml/106 cells/24 h) or nontransfected controls (396 ± 29.1 pg/ml/106 cells/24 h).

In follow-up experiments, we tested, by a specific ELISA for PAI-1, the ability of neutralizing antibodies to TNF-{alpha}, IL-1{beta},or TGF-{beta}1 to block the up-regulation of PAI-1 expression by the culture medium conditioned by wild-type cells. We found that only the antibody to TGF-{beta}1 (10 µg/ml) attenuated concentrations of PAI-1 protein (a 12-h exposure) in the conditioned medium of mesothelial cells (Fig. 4A).



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FIG. 4.
Anti-TGF-{beta}1 specifically inhibits PAI-1 production by mesothelial cells exposed to media from wild-type cells. A, human mesothelial cells were exposed to medium from wild-type HRA cells for 12 h at 37 °C in the presence of varying concentrations of anti-TGF-{beta}1 (0, 1, 5, and 10 µg/ml (B)), anti-TNF-{alpha} (10 µg/ml), anti-IL-1{beta} (10 µg/ml), or nonimmune IgG (10 µg/ml). PAI-1 levels in the cell-conditioned medium were determined using the specific ELISA kit. Data (means ± S.D.) illustrated are representative of the findings in three separate experiments. The letters a and b indicate statistical differences (p < 0.05).

 

Culture media conditioned by wild-type HRA cells were incubated with different concentrations of anti-TGF-{beta}1 antibody (Fig. 4B) or other neutralizing antibodies (data not shown). ELISA revealed a significant decrease in PAI-1 secretion in mesothelial cells incubated with culture media mixed with 5–10 µg/ml of the neutralizing anti-TGF-{beta}1 antibody. In contrast, antibodies to other cytokines (TNF-{alpha} and IL-1{beta}) or nonimmune IgG had no significant effect on secretion of PAI-1 (data not shown). No morphologically identifiable signs of cytotoxicity were observed in cells incubated with media containing these antibodies.

uPA and PAI-1 mRNA Levels in TGF-{beta}1-treated Mesothelial and Fibroblast Cells—When confluent cultures of mesothelial cells were incubated for 12 h in the presence of increasing concentrations of TGF-{beta}1, PAI-1 mRNA levels were increased in a dose-dependent manner, with a maximal 5–6-fold increase at 2–10 ng/ml (Fig. 5A). A kinetic analysis revealed that in the presence of 10 ng/ml TGF-{beta}1, PAI-1 induction in mesothelial cells began between 3 and 6 h of incubation and was maximal (5.2-fold increase) after 12 h (Fig. 5B). TGF-{beta}1 also increased uPA mRNA expression in mesothelial cells in a dose-dependent manner (Fig. 5A). The kinetics of uPA mRNA induction was very similar to those seen for PAI-1 mRNA, although uPA mRNA induction was ephemeral, being maximal after 3 h and not returning to base-line levels even after 24 h (Fig. 5B).



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FIG. 5.
TGF-{beta}1 stimulates uPA and PAI-1 mRNAs in mesothelial cells. A, dose dependence. Mesothelial cells were incubated for 12 h in the presence of the indicated concentrations of TGF-{beta}1. B, time dependence. The cells were incubated for the indicated times in the presence of TGF-{beta}1 (10 ng/ml). Replicate filters containing total cellular RNA (20 µg/lane) were hybridized with 32P-labeled human uPA and PAI-1 cDNA probes. GAPDH staining demonstrates uniformity of loading and RNA integrity. Right panel, densitometric analysis of the Northern blot shown in left panel. The results are expressed as ratio relative to the 1st lane. Data illustrated are representative of the findings in two separate experiments with a similar result.

 

When confluent cultures of fibroblast cells were incubated in the presence of increasing concentrations of TGF-{beta}1 or for different times, uPA and PAI-1 mRNA levels were not significantly increased (data not shown). Thus, TGF-{beta}1 showed a stronger impact on uPA and PAI-1 mRNA expression in mesothelial cells.

uPA and PAI-1 Protein Levels in TGF-{beta}1-treated Mesothelial and Fibroblast Cells—We measured the levels of uPA and PAI-1 in both cells exposed to TGF-{beta}1 by ELISA (for uPA) and Western blotting (for PAI-1; Fig. 6). As expected, TGF-{beta}1 induces PAI-1 protein production in the mesothelial cell lysates in a dose- and time-dependent manner. PAI-1 levels in cell lysates were increased, with a maximal 4.0-fold increase at 2 ng/ml (Fig. 6A). PAI-1 protein induction in mesothelial cells was maximal (4.3-fold increase) after 12 h (Fig. 6B). However, TGF-{beta}1 failed to induce PAI-1 protein production in the fibroblast cell lysates (data not shown). In a parallel experiment, uPA ELISA data revealed that TGF-{beta}1 showed no induction of uPA protein in these cells (data not shown). It is unlikely that the increased expression of uPA mRNA is attributable to an increased level of uPA protein. To determine the sensitivity and specificity of the ELISA used is sufficient to reliably determine uPA under the conditions used, we measured, in a parallel experiment, the uPA levels in cell lysate and conditioned medium of human ovarian cancer HRA cells. This ELISA showed that 10 ng/ml TGF-{beta}1 markedly enhanced uPA expression in HRA cells (see Ref. 27), demonstrating that the signal observed is in fact due to uPA antigen, rather than a nonspecific background.



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FIG. 6.
Regulation by TGF-{beta}1 of uPA and PAI-1 protein production in mesothelial cells. A, dose dependence. Mesothelial cells were incubated for 12 h in the presence of the indicated concentrations of TGF-{beta}1. B, time dependence. The cells were incubated for the indicated time in the presence of TGF-{beta} (10 ng/ml). Cell lysate proteins (100 µg/lane) were hybridized with monoclonal antibody to PAI-1. Lower panel, densitometric analysis of the Western blot shown in upper panel. The results are expressed as ratio relative to the 1st lane. Data illustrated are representative of the findings in two separate experiments with a similar result.

 

Flow Cytometric Analysis of Expression of Membrane-bound uPA, PAI-1, and uPAR Proteins on HRA Cells and Unstimulated and TGF-{beta}1-stimulated Mesothelial Cells—Following in vitro culture of mesothelial cells with TGF-{beta}1, there were 1.2- (37.5 versus 30.1 (fluorescence mean channel)), 4.3- (446.8 versus 104.0), and 1.4-fold (29.5 versus 20.4) increases in cell surface expression of uPA, PAI-1, and uPAR, compared with unstimulated mesothelial cells (Fig. 7). Furthermore, HRA cells were strongly positive for uPA (210.4) and uPAR (319.3), as well as to a lesser degree for PAI-1 (58.7). These results demonstrated that mesothelial cells produced a small amount of uPA, but they abundantly expressed plasma membrane-bound uPA, uPAR, and PAI-1.



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FIG. 7.
Expression of membrane-bound uPA, PAI-1, and uPAR proteins on HRA cells and unstimulated and TGF-{beta}1-stimulated mesothelial cells. FACS analysis was performed as described under "Materials and Methods." A typical FACS diagram was shown. HRA cells as well as unstimulated and TGF-{beta}1-stimulated cells (10 ng/ml, 12 h) were stained with antibodies to uPA (A), PAI-1 (B), or uPAR (C), respectively. These cells were not fixed with ethanol in order to detect plasma membrane-bound protein expression. A representative result of two independent experiments is shown. The data (x axis; fluorescence mean intensity) was quantified by flow cytometric scanning. Peak 1, nonimmune IgG (NI-IgG); peak 2, unstimulated mesothelial cells; peak 3, TGF-{beta}1-stimulated mesothelial cells; and peak 4, wild-type HRA cells.

 

Phosphorylation of ERK1/2, JNK, p38 MAPK, and Smad2 in Mesothelial Cells Exposed to TGF-{beta}1—We examined the effects of TGF-{beta}1 on the activation of ERK1/2, JNK, p38 MAPK, and Smad2 in semi-confluent mesothelial cells. Here we show that TGF-{beta}1 induced rapid, transient phosphorylation of ERK1/2, p38 MAPK, and Smad2. The increased phosphorylation of ERK1/2 (Fig. 8A), p38 MAPK (Fig. 8C), and Smad2 (Fig. 8D) appeared as early as 15 min, reached the peak at 30 min, and was sustained until 1 h after TGF-{beta}1 treatment (10 ng/ml). TGF-{beta}1 increased phosphorylation of ERK1/2 and p38 MAPK in the cells ~3–4 times that of the basal level. TGF-{beta}1 also dramatically phosphorylated Smad2. In contrast, JNK was not phosphorylated by TGF-{beta}1 (Fig. 8B). Total ERK1/2, p38 MAPK, Smad2, and JNK proteins were abundantly expressed in these cells.



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FIG. 8.
Phosphorylation and activation of ERK1/2, JNK, p38 MAPK, and Smad2 in mesothelial cells exposed to TGF-{beta}1. Mesothelial cells were treated with TGF-{beta}1 (10 ng/ml). At the indicated times, cells were collected, and equal amounts of total cell lysates (100 µg/lane) were analyzed by SDS-PAGE and immunoblotted. Phosphorylations of either ERK1/2 (A), JNK (B), p38 MAPK (C), or Smad2 (D) were analyzed. Upper and lower panels indicate patterns of phospho- and total protein in mesothelial cells, detected by their specific antibodies, respectively. E and F, mesothelial cells were pretreated with SB202190 or PD98059 for 1 h and subsequently treated with TGF-{beta}1 (10 ng/ml) for 30 min at 37 °C as indicated. The data are typical of duplicate experiments with a similar result.

 

Furthermore, we examined whether phosphorylation of ERK1/2 occurs independently of p38 MAPK activation in mesothelial cells. Inhibition of p38 MAPK activation using the inhibitor SB202190 did not affect ERK1/2 phosphorylation (Fig. 8E). Also, inhibition of ERK1/2 activation using the inhibitor PD98059 did not affect p38 MAPK phosphorylation (Fig. 8F). Thus, the TGF-{beta}1-inducible p38 MAPK and ERK pathways appear to function independently of each other in mesothelial cells.

ERK1/2 and p38 MAPK Pathways Are Independently Required for TGF-{beta}1-induced PAI-1 Gene Expression—Treatment of mesothelial cells with PD98059 almost completely abrogated the up-regulation of TGF-{beta}1-induced PAI-1 mRNA expression (Fig. 9A). Furthermore, treatment of the cells with SB202190 partially reversed the up-regulation of TGF-{beta}1-induced PAI-1 gene expression. On the other hand, H-89 had no inhibitory effects, further demonstrating the specificity of these findings.



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FIG. 9.
Inhibition of the MAPK and p38 MAPK pathways reverses the TGF-{beta}1 stimulatory effects of PAI-1 mRNA expression in mesothelial cells. Mesothelial cells were treated for 12 h with or without TGF-{beta}1 (10 ng/ml) in the presence or absence of SB202190 (2 µM), PD98059 (10 µM), or H-89 (10 µM). A, PAI-1 mRNA and GAPDH mRNA were assessed using Northern blot analysis. B, PAI-1 protein in cell lysates was assessed using Western blot analysis. Right panel, densitometric analysis of the Northern/Western blot shown in left panel. The results are expressed as ratio relative to untreated control (1st lane). The data (means ± S.D.) are typical of triplicate experiments. The letters a–d indicate statistical differences (p < 0.05).

 

We next assessed the ability of each inhibitor to influence PAI-1 protein expression by mesothelial cells. PD98059 and SB202190, but not H-89, abrogated TGF-{beta}1-induced strong expression of PAI-1 protein in the cells (Fig. 9B). Our data established that the MAPK pathway is a main cascade and is activated independently of the p38 MAPK pathway.

Cell Surface-associated Plasminogen Activators by Colorimetric Assay—To determine directly the significance of TGF-{beta}1-dependent activation of MAPK or p38 MAPK in the induction of the up-regulation of functional PAI-1 protein expression, we evaluated the effects of PD98059 and SB202190 on cell-associated uPA activity in TGF-{beta}1-stimulated mesothelial cells. TGF-{beta}1 reduced cell-associated uPA amidolytic activity in a dose-dependent manner (data not shown), and concomitant treatment with PD98059 or SB202190 reversed such a reduction (Fig. 10).



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FIG. 10.
Detection of cell surface-associated plasminogen activators by colorimetric assay. The mesothelial cell monolayers treated with various agents for 12 h were washed with PBS. The color reactions were initiated by the addition of the chromogenic amidolytic substrate specific for either uPA (Spectrozyme UK). Amiloride (1 mM)or neutralizing antibodies to uPA (10 µg/ml; not shown here) almost completely block the color reaction. The photometric absorbance of the reaction mixtures at 405 nm was monitored at 37 °C over the next 30 min. The data (means ± S.D.) are typical of triplicate experiments. The letters a–d indicate statistical differences (p < 0.05).

 

Tumor Cell Adhesion to and Invasion through Mesothelial and Fibroblast Monolayers—Maximum adhesion of wild-type HRA cells to a non-preincubated mesothelial monolayer was seen after 3 h, whereas maximum adhesion of tumor cells to fibroblast monolayer was seen after 2 h (data not shown). The attached cells seemed to be weakly adhered, because vigorous pipetting could easily detach the cells from the monolayers. Nonstimulated cell adhesion was ~23 (for mesothelial cells) and 31% (for fibroblast cells) of the total amount of tumor cells added. When assayed for their ability to adhere to these monolayers, there was no detectable effect on the cellular adhesion in the bik+ clone compared with the parental and luc+ cells (Fig. 11, A and B). We next tested the ability of each subline to attach to mesothelial and fibroblast monolayers pretreated with TGF-{beta}1. Preincubation of the mesothelial monolayer with TGF-{beta}1 (10 ng/ml, 12 h) resulted in enhanced tumor cell adhesion (about 20% increase) (Fig. 11A). In contrast, tumor cell adhesion to fibroblast monolayers was not stimulated in response to TGF-{beta}1 (Fig. 11B). Also, there was no effect on the cellular adhesion in the bik+ clone compared with the parental and luc+ cells, irrespective of whether monolayers were preincubated with or without TGF-{beta}1.



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FIG. 11.
A and B, tumor cell adhesion before and after preincubation of mesothelial and fibroblast monolayers with TGF-{beta}1. Tumor cells adhered to mesothelial and fibroblast monolayers about 3 and 2 h after the seeding, respectively. Tumor cell adhesion to mesothelial (A) and fibroblast (B) monolayer was measured after preincubation with (open square) or without 10 ng/ml TGF-{beta}1 (filled square). The data are typical of triplicate experiments. C and D, effect of mesothelial and fibroblast monolayers pretreated with or without TGF-{beta}1 on ovarian cancer cell invasion. Invasion assay showed that TGF-{beta}1-stimulated mesothelial (C) or fibroblast (D) monolayers cultured on Matrigel caused a significant increase in the invasion of HRA cells compared with the assay using TGF-{beta}1-untreated monolayers, in all cell clones. The data (means ± S.D.) are typical of triplicate experiments. The letters a and b or a' and b' indicate statistical differences (p < 0.05).

 

When the monolayers were not pretreated with TGF-{beta}1, the number of infiltrating cells was significantly larger in the fibroblast cell-layered chambers than in the mesothelial cell-layered chamber (40.3 ± 4.9 versus 16.9 ± 1.7; Fig. 11, C and D). As shown in Fig. 11C, each of the three ovarian cancer clones enhanced their infiltration potency by placing peritoneal mesothelial monolayer pretreated with TGF-{beta}1 on the Matrigel. In the wild-type HRA cells, TGF-{beta}1-dependent effect was saturable and dose-dependent with a maximum 1.8-fold increase in tumor cell invasion compared with the unstimulated control group. The number of invasive HRA cells in response to 0, 1, and 10 ng/ml of TGF-{beta}1 was 16.9 ± 1.7, 21.3 ± 2.6, and 30.2 ± 2.5, respectively. bik+ clones significantly reduced the invasive capacity through mesothelial monolayers than do wild-type HRA cells and luc+ clones, irrespective of whether mesothelial cells were pretreated with or without TGF-{beta}1. In contrast, none of these clones enhanced their infiltration potency by placing fibroblast monolayers pretreated with TGF-{beta}1 on the Matrigel (Fig. 11D). bik+ clones significantly reduced the invasive capacity through fibroblast monolayers compared with wild-type HRA cells and luc+ clone.

In order to elucidate directly the influence of the PA system in unstimulated and TGF-{beta}1-stimulated mesothelial cells on HRA cell adhesion (Fig. 12A) and invasion (Fig. 12B), confluent mesothelial cell cultures were pretreated with or without TGF-{beta}1 (10 ng/ml) for 12 h. Neutralizing anti-uPA (Ab 3471 plus Ab 3689), anti-PAI-1 (Ab 379), and anti-uPAR (Ab 3936) antibodies were used for stimulatory or inhibitory effect in the HRA cell adhesion and invasion. Treatment of mesothelial cells with anti-PAI-1 antibody decreased the HRA cell adhesion as compared with the unstimulated and TGF-{beta}1-stimulated control. The effect of TGF-{beta}1 on tumor cell invasion was also inhibited with anti-PAI-1 antibody. However, HRA cell adhesion was enhanced by addition of anti-functional uPA antibody to the cells. On the other hand, anti-uPA antibody-treated cultures decreased HRA cell invasion as compared with that from control cultures. HRA cell invasion was markedly inhibited by anti-functional antibody to the uPAR, whereas cell adhesion was enhanced by this antibody. Nonimmune IgG does not affect HRA cell adhesion or invasion.



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FIG. 12.
Neutralizing antibodies to uPA, PAI-1, or uPAR modulate HRA cell adhesion to and invasion through unstimulated or TGF-{beta}1-stimulated mesothelial cells. HRA cells were added to mesothelial cell layers pretreated with or without TGF-{beta}1 (10 ng/ml, 12 h) in the presence of several different antibodies for cell adhesion (A) and cell invasion (B), respectively. {alpha}-uPA, Ab 3471 plus Ab 3689 (50 µg/ml); {alpha}-PAI-1, Ab 379 (50 µg/ml); {alpha}-uPAR, Ab 3936 (50 µg/ml); and NI-IgG, nonimmune mouse IgG (50 µg/ml). The data (means ± S.D.) are typical of triplicate experiments. The letters a–d indicate statistical differences (p < 0.05).

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have demonstrated previously (27) the following. 1) HRA produced secreted and cell-associated uPA and PAI-1, and the plasma membrane of the HRA cells showed enzymatically active uPA even in the presence of high levels of PAI-1. 2) HRA cells specifically released TGF-{beta}1. 3) HRA cells leading to invasion through Matrigel are induced through TGF-{beta}1-dependent up-regulation of uPA expression participating in regulation of cell invasion. 4) Elimination of endogenous TGF-{beta}1 could induce change in uPA and PAI-1 expression, which could in turn modify the invasive behavior of the cells. These results indicate that the TGF-{beta}1 stimulatory response involved the PA system, which in turn stimulates cell invasiveness. Our recent data also demonstrated that exogenously added bikunin decreases levels of TGF-{beta}1 expression in vitro (27), and bikunin gene transfection reduces the peritoneal dissemination of these cells in an in vivo mouse model (4). In the present study we attempt to explain the latter effect in terms of altered mesothelial cell PAI-1 expression, using cell adhesion and invasion assays as in vitro surrogates. As models we used HRA cells to produce substantial amounts of TGF-{beta}1 and uPA constitutively, and they are an aggressive and invasive line (4).

We investigated the mechanism(s) by which TGF-{beta}1, which is produced by ovarian cancer cells, mediates the adhesion and invasive behavior of tumor cells through an overproduction of PAI-1 from peritoneal mesothelial cells. Here we show that peritoneal mesothelial cells markedly up-regulate expression of PAI-1 mRNA and protein when mesothelial cells were affected by TGF-{beta}1 secreted by HRA cells, and we show that this exposure resulted in enhanced tumor cell adhesion and subsequently facilitated invasion. We also show that HRA cell adhesion to mesothelial monolayers is enhanced by neutralization of uPA activity and uPAR function but suppressed by anti-functional antibodies to PAI-1, whereas HRA cell invasion through mesothelial monolayers is suppressed by neutralization of uPA, uPAR, and PAI-1. It is thus likely that uPA and uPAR may suppress HRA cell adhesion but promote invasion. Furthermore, neutralization of PAI-1 appears to abolish completely the increase in adhesion and invasion mediated by TGF-{beta}1. These results allow us to speculate that PAI-1 derived from peritoneal mesothelial cells is deeply involved not only in the adhesion of ovarian cancer cells but also in local invasion and peritoneal dissemination. This would show definitively that induction of PAI-1 mRNA and protein expression in mesothelial cells is modest in response to culture medium conditioned by the bik+ clone, because bik transfection down-regulates TGF-{beta}1 production. Although these observations may be relatively novel in relation to ovarian cancer cell interaction with mesothelial cells, the effect of TGF-{beta}1 on mesothelial cell PAI-1 expression is well documented (1315), and the adhesion of other cancer cells to mesothelial cells has also been shown previously to be increased by TGF-{beta} (26). Therefore, the novelty in the present study lies in the relationship between alterations in mesothelial cell PAI-1 expression and the interaction of these cells with HRA cells.

The binding of uPA to its receptor has been shown to induce intracellular signaling involving proto-oncogene expression (30), mitogenic effects (31), cell adhesion (32), or migration (33), depending on cell type. All these effects are independent of the proteolytic activity of uPA but require its receptor-binding site (34). Because the number of uPAR was limited in TGF-{beta}1-treated mesothelial cells, it is possible that a binding of free uPA or the complex occurs to uPAR on adjacent cell types such as HRA cells. In our cell-cell interaction system, we found that uPA may inhibit cell adhesion but enhance cancer cell invasion through mesothelial cell layers. These findings may be supported by the fact that enhanced uPA expression leads to activation of cell motility but reduced cell-cell adhesion (35). The anti-uPA antibody 3689 used in the experiment shown in Fig. 12 neutralizes catalytic activity, so it can only exert its effect via plasminogen activation and not via uPAR signaling. Although the anti-uPAR antibody also has an effect (which is in fact larger), uPAR is also needed to maximize the efficiency of plasminogen activation. Taken together, the uPA binding to uPAR on the HRA cell surface and/or receptor-bound enzymatically active uPA may be necessary for initiating cell invasion. uPA, free or complexed, released from mesothelial cells and HRA cell themselves induced by TGF-{beta}1 could function as an autocrine/paracrine signal to modulate HRA cell adhesion to and invasion through mesothelial cell layers.

uPA is constitutively expressed by HRA themselves, and TGF-{beta}1 could stimulate up-regulation of uPA and PAI-1 in HRA cells (27). Furthermore, exposure of mesothelial cells to ng/ml concentrations of TGF-{beta}1 results in an increase in uPA and PAI-1 mRNA levels. Although the magnitude of the TGF-{beta}1-induced increase in uPA and PAI-1 mRNA levels was similar in mesothelial cells, TGF-{beta}1 does not enhance uPA protein expression in association with increases in uPA mRNA. These findings suggest that different post-transcriptional mechanisms, including uPA mRNA translation efficiency and changes in half-life, may account for the differences in uPA protein expression or possibly as a result of rapid uptake and degradation by the uPAR (28), because uPAR is thought to be expressed on mesothelial cells (see Fig. 7) (29). These data allow us to hypothesize that the TGF-{beta}1-mediated decrease in cell-bound PA activity in mesothelial cells may be mediated by an increase in functional PAI-1 that binds to and inhibits uPA, which is not up-regulated at the protein level.

Numerous studies showed that expression of high levels of tumor-associated PAI-1 indicates a poor prognosis for many cancer patients (3638). Consistent with this fact, high levels of PAI-1 were found in aggressive tumor tissues. Thus, combined determination of the invasion factors uPA and PAI-1 supports risk-adapted individualized therapeutic strategies in patients with cancer. It is likely that excessive uPA activity is associated with reduced attachment of tumor cells to ECM and that PAI-1 may promote tumor invasion and metastasis by enhancing cell adhesion and suppressing detachment from the ECM. The critical importance of an appropriate balance between proteases and protease inhibitors in the invasion process is suggested by the following two sets of observations (39): 1) that ECM invasion is inhibited in the presence of an excess of protease inhibitors; 2) that when unchecked by protease inhibitors, excessive proteolysis is incompatible with subsequent firm invasion. Thus, our data could help to understand the dual function of PAI-1: to control the enzymatic activity of uPA and to promote cell adhesion as well as cell invasion (40). The TGF-{beta}1-mediated induction of PAI-1 expression in mesothelial cells is in agreement with the hypothesis that finely tuned proteolytic activity is required for cancer cell invasion. Our present findings of PAI-1-mediated cell adhesion and invasion are consistent with previous results showing that antibodies against PAI-1 resulted in the inhibition of cultured cancer cell invasion (41). Therefore, we hypothesize that uPA is thought to be responsible for detaching tumor cells from the basement membrane or ECM and thus promote their migration to cover exposed connective tissue, and although PAI-1 is believed to control or inhibit proteolytic activity and cell migration, this inhibitor is necessary for optimal tumor invasion (36). This hypothesis may be also strengthened by the fact that PAI-1 is proangiogenic at physiological concentrations (39). Furthermore, it has been reported that mesothelial cells express both the {alpha}v{beta}3 vitronectin receptor (42) and the uPAR (28), which both may be involved in adhesion to vitronectin and in mesothelial cell migration. Therefore, PAI-1 is able to promote migration of cells probably by lowering the strength of uPAR adhesion to vitronectin (13, 43). Although PAI-1 has been linked with cell adhesion, in particular with adhesion to vitronectin, in the experiment described here the adhesion of HRA cells to mesothelial monolayers is observed, and cell-cell interaction would therefore seem to be more important. An example is the observation that TGF-{beta}1-mediated changes in adhesion molecule expression have been reported previously, and in particular an up-regulation of CD44 expression in mesothelial cells has been shown to increase adhesion of colon cancer cells (44).

Finally, we investigated the TGF-{beta}1-mediated signal transduction cascades of the PAI-1 gene to identify the molecular target sites of such regulation. Smad family and the ERK1/2 pathways have been shown to participate in TGF-{beta} signaling in human peritoneal mesothelial cells (45). Our experiments demonstrate that, at the molecular level, this induction is mediated largely by a signal via the MAPK. TGF-{beta}1 also operate partially through the p38 MAPK signaling pathway. Therefore, these studies establish a signal transduction cascade or crosstalk emanating from TGF-{beta}1 to PAI-1. Our study represents the first report that TGF-{beta}1 stimulates PAI-1 up-regulation at the mRNA and protein level, at least in part, through MAPK-dependent activation mechanisms in human peritoneal mesothelial cells. However, at this time, we could not exclude the possibility that the PAI-1 up-regulation effect of TGF-{beta}1 is associated with activation of Smads. Thus, elucidation of signal transduction cascades in response to TGF-{beta}1 may support individual therapeutic strategies in ovarian cancer patients with peritoneal dissemination.

In conclusion, our findings represent the first demonstration that mesothelium plays an active role in inducing the adhesion and invasion of ovarian cancer cells. These data suggest a novel mechanism by which ovarian cancer cells could influence mesothelial cell functions relevant to peritoneal dissemination, although uPA overexpression by HRA cells may be transiently inhibited by PAI-1 produced from stimulated mesothelial cells, mesothelial cell-derived PAI-1 may promote tumor invasion possibly by enhancing cell adhesion and suppressing detachment from the mesothelial cells. Taken together, bikunin may be involved in the inhibition of development of ovarian cancer in peritoneal dissemination through suppression of cancer cell-derived TGF-{beta}1 production.


    FOOTNOTES
 
* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan (to H. K.) and by a grant from the Yamanouchi Foundation for Research on Metabolic Disorders. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Fax: 81-53-435-2308; E-mail: hirokoba{at}hama-med.ac.jp.

1 The abbreviations used are: bik, bikunin; BSA, bovine serum albumin; JNK, c-Jun N-terminal kinase; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; IL-1{beta}, interleukin-1{beta}; LDH, lactate dehydrogenase; luc, luciferase; MAPK, mitogen-activated protein kinase; PA, plasminogen activator; PAI-1, plasminogen activator inhibitor type-1; PBS, phosphate-buffered saline; TAK1, TGF-{beta}-activated kinase-1; TGF-{beta}, transforming growth factor-{beta}; TNF-{alpha}, tumor necrosis factor-{alpha}; uPA, urokinase-type plasminogen activator; PVDF, polyvinylidene difluoride; IL, interleukin; Ab, antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FACS, fluorescence-activated cell sorter. Back


    ACKNOWLEDGMENTS
 
We thank Drs. M. Fujie, K. Shibata, T. Noguchi, and A. Suzuki (Equipment Center and Photo Center; Hamamatsu University School of Medicine) for helping with the biochemical analysis. We are also grateful to Drs. H. Morishita, Y. Kato, and H. Sato (BioResearch Institute, Mochida Pharmaceutical Co., Gotenba, Shizuoka, Japan), Drs. Y. Tanaka and T. Kondo (Chugai Pharmaceutical Co. Ltd., Tokyo, Japan), and Drs. S. Miyauchi and M. Ikeda (Seikagaku Kogyo Co. Ltd., Tokyo, Japan) for their continuous and generous support of our work.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. 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]
  2. Kobayashi, H., Suzuki, M., Tanaka, Y., Hirashima, Y., and Terao, T. (2001) J. Biol. Chem. 276, 2015–2022[Abstract/Free Full Text]
  3. Hirashima, Y., Kobayashi, H., Suzuki, M., Tanaka, Y., Kanayama, N., Fujie, M., Nishida, T., Takigawa, M., and Terao, T. (2001) J. Biol. Chem. 276, 13650–13656[Abstract/Free Full Text]
  4. Suzuki, M., Kobayashi, H., Tanaka, Y., Hirashima, Y., Kanayama, N., Takei, Y., Saga, Y., Suzuki, M., Itoh, H., and Terao, T. (2003) Int. J. Cancer 104, 289–302[CrossRef][Medline] [Order article via Infotrieve]
  5. Hashizume, K., Uchiyama, A., Sasaki, N., Noshiro, H., Morisaki, T., Tanaka, M., and Katano, M. (2002) Anticancer Res. 22, 1781–1786[Medline] [Order article via Infotrieve]
  6. Fishman, D. A., Kearns, A., Chilukuri, K., Bafetti, L. M., O'Toole, E. A., Georgacopoulos, J., Ravosa, M. J., and Stack, M. S. (1998) Invasion Metastasis 18, 15–26[CrossRef][Medline] [Order article via Infotrieve]
  7. Mizutani, K., Kofuji, K., and Shirouzu, K. (2000) Surg. Today 30, 614–621[CrossRef][Medline] [Order article via Infotrieve]
  8. Rodriguez, G. C., Haisley, C., Hurteau, J., Moser, T. L., Whitaker, R., Bast, R. C., Jr., and Stack, M. S. (2001) Gynecol. Oncol. 80, 245–253[CrossRef][Medline] [Order article via Infotrieve]
  9. Kutz, S. M., Hordines, J., McKeown-Longo, P. J., and Higgins, P. J. (2001) J. Cell Sci. 114, 3905–3914[Abstract/Free Full Text]
  10. Massague, J., and Weis-Garcia, F. (1996) Cancer Surv. 27, 41–64[Medline] [Order article via Infotrieve]
  11. Frey, R. S., and Mulder, K. M. (1997) Cancer Res. 57, 628–633[Abstract]
  12. Watanabe, H., de Caestecker, M. P., and Yamada, Y. (2001) J. Biol. Chem. 276, 14466–14473[Abstract/Free Full Text]
  13. Rougier, J. P., Guia, S., Hagege, J., Nguyen, G., and Ronco, P. M. (1998) Kidney Int. 54, 87–98[CrossRef][Medline] [Order article via Infotrieve]
  14. Tietze, L., Elbrecht, A., Schauerte, C., Klosterhalfen, B., Amo-Takyi, B., Gehlen, J., Winkeltau, G., Mittermayer, C., and Handt, S. (1998) Thromb. Haemostasis 79, 362–370[Medline] [Order article via Infotrieve]
  15. Falk, P., Ma, C., Chegini, N., and Holmdahl, L. (2000) Scand. J. Clin. Lab. Invest. 60, 439–447[CrossRef][Medline] [Order article via Infotrieve]
  16. Kikuchi, Y., Kizawa, I., Oomori, K., Miyauchi, M., Kita, T., Sugita, M., Tenjin, Y., and Kato, K. (1987) Cancer Res. 47, 592–596[Abstract]
  17. Rougier, J. P., Moullier, P. H., Piedagnel, R., and Ronco, P. M. (1997) Kidney Int. 51, 337–347[Medline] [Order article via Infotrieve]
  18. Stylianou, E., and Jenner, L. A. (1990) Kidney Int. 37, 1563–1570[Medline] [Order article via Infotrieve]
  19. Garner, W. L., Karmiol, S., Rodriguez, J. L., Smith, D. J., Jr., and Phan, S. H. (1993) J. Invest. Dermatol. 101, 875–879[Abstract]
  20. Zhang, H., Morisaki, T., Matsunaga, H., Sato, N., Uchiyama, A., Hashizume, K., Nagumo, F., Tadano, J., and Katano M. (2000) Clin. Exp. Metastasis 18, 343–352[Medline] [Order article via Infotrieve]
  21. 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]
  22. Pannekoek, H., Veerman, H., Lambers, H., Diergaarde, P., Verweij, C. L., van Zonneveld, A. J., and van Mourik, J. A. (1986) EMBO J. 5, 2539–2544[Abstract]
  23. Andreasen, P. A., Riccio, A., Welinder, K. G., Douglas, R., Sartorio, R., Nielsen, L. S., Oppenheimer, C., Blasi, F., and Dano, K. (1986) FEBS Lett. 209, 213–218[CrossRef][Medline] [Order article via Infotrieve]
  24. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Nucleic Acids Res. 12, 7035–7056[Abstract]
  25. Catterall, J. B., and Gardner, M. J. (1994) Cancer Lett. 87, 199–203[Medline] [Order article via Infotrieve]
  26. van Rossen, M. E., Hofland, L. J., van den Tol, M. P., van Koetsveld, P. M., Jeekel, J., Marquet, R. L., and van Eijck, C. H. (2001) J. Pathol. 193, 530–537[CrossRef][Medline] [Order article via Infotrieve]
  27. Kobayashi, H., Suzuki, M., Tanaka, Y., Kanayama, N., and Terao, T. (2003) J. Biol. Chem. 278, 7790–7799[Abstract/Free Full Text]
  28. Sitter, T., Toet, K., Fricke, H., Schiffl, H., Held, E., and Kooistra, T. (1996) Am. J. Physiol. 271, R1256–R1263[Medline] [Order article via Infotrieve]
  29. Shetty, S., Kumar, A., Johnson, A. R., and Idell, S. (1995) Antisense Res. Dev. 5, 307–314[Medline] [Order article via Infotrieve]
  30. Dumler, I., Petri, T., and Schleuning, W. D. (1994) FEBS Lett. 343, 103–106[CrossRef][Medline] [Order article via Infotrieve]
  31. Rabbani, S. A., Desjardins, J., and Bell, A. W. (1990) Biochem. Biophys. Res. Commun. 173, 1058–1064[Medline] [Order article via Infotrieve]
  32. Waltz, D. A., Sailor, L. Z., and Chapman, H. A. (1993) J. Clin. Invest. 91, 1541–1552[Medline] [Order article via Infotrieve]
  33. Gyetko, M. R., Todd, R. F., Wilkinson, C. C., and Sitrin, R. G. (1994) J. Clin. Invest. 93, 1380–1387[Medline] [Order article via Infotrieve]
  34. Sandberg, T., Casslén, B., Gustavsson, B., and Benraad, T. J. (1998) Biol. Reprod. 59, 759–767[Abstract/Free Full Text]
  35. Kondoh, N., Yamada, T., Kihara-Negishi, F., Yamamoto, M., and Oikawa, T. (1998) Br. J. Cancer 78, 718–723[Medline] [Order article via Infotrieve]
  36. Duffy, M. J. (2002) Clin. Chem. 48, 1194–1197[Abstract/Free Full Text]
  37. Harbeck, N., Alt, U., Berger, U., Kates, R., Kruger, A., Thomssen, C., Janicke, F., Graeff, H., and Schmitt, M. (2000) Int. J. Biol. Markers 15, 79–83[Medline] [Order article via Infotrieve]
  38. Harbeck, N., Schmitt, M., Kates, R. E., Kiechle, M., Zemzoum, I., Janicke, F., and Thomssen, C. (2002) Clin. Breast Cancer 3, 196–200[Medline] [Order article via Infotrieve]
  39. Pepper, M. S., and Montesano, R. (1990) Cell Differ. Dev. 32, 319–327[CrossRef][Medline] [Order article via Infotrieve]
  40. Planus, E., Barlovatz-Meimon, G., Rogers, R. A., Bonavaud, S., Ingber, D. E., and Wang, N. (1997) J. Cell Sci. 110, 1091–1098[Abstract/Free Full Text]
  41. Liu, G., Shuman, M. A., and Cohen, R. L. (1994) Int. J. Cancer 60, 501–506
  42. Devy, L., Blacher, S., Grignet-Debrus, C., Bajou, K., Masson, V., Gerard, R. D., Gils, A., Carmeliet, G., Carmeliet, P., Declerck, P. J., Noel, A., and Foidart, J. M. (2002) FASEB J. 16, 147–154[Abstract/Free Full Text]
  43. Waltz, D. A., Natkin, L. R., Fujita, R. M., Wei, Y., and Chapman, H. A. (1997) J. Clin. Invest. 100, 58–67[Abstract/Free Full Text]
  44. Nakashio, T., Narita, T., Akiyama, S., Kasai, Y., Kondo, K., Ito, K., Takagi, H., and Kannagi, R. (1997) Int. J. Cancer 70, 612–618[CrossRef][Medline] [Order article via Infotrieve]
  45. Hung, K. Y., Chen, C. T., Huang, J. W., Lee, P. H., Tsai, T. J., and Hsieh, B. S. (2001) Kidney Int. 60, 1249–1257[CrossRef][Medline] [Order article via Infotrieve]