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.
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ABSTRACT |
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INTRODUCTION |
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One of the factors regulating the metastatic process is considered to be
TGF-, which is a multifunctional cytokine that elicits numerous cellular
effects pertinent to the metastatic process
(8). TGF-
is an effective
inducer of matrix deposition/turnover, cell locomotion, and PAI-1 expression
(9). Rodriguez et al.
(8) suggest that TGF-
may
enhance the invasiveness of ovarian cancers. Receptor complexes of the
TGF-
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-
in
specific contexts (12). For
example, activation of Ras, ERK1/2, and c-Jun N-terminal kinase (JNK) by
TGF-
signaling has been reported in nonmalignant and malignant cell
lines (11), whereas activation
of protein kinase A contributes to TGF-
-signaling responses in murine
cells. In addition, TGF-
-activated kinase-1, a member of the MAPK kinase
kinase family and activator of JNK and p38 MAPK pathways, is rapidly activated
by TGF-
in certain cell systems
(11,
12). PAI-1 may be regulated by
different cytokines and growth factors among which TGF-
plays a pivotal
role. Indeed, TGF-
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-1-mediated MAPK pathway and that TGF-
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.
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MATERIALS AND METHODS |
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Cell CultureHuman 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-
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-
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-1, the culture medium was replaced with serum-free DMEM.
Preparation of Conditioned Medium and Cell LysateFor 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 AssayCells 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 AssaysTGF-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-1 Levels,
Enzyme-linked Immunosorbent AssaysThe 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-
1 were assayed with a human
TGF-
1 ELISA kit (BIOSOURCE, Tokyo, Japan) according to the
manufacturer's instructions. To measure total TGF-
1, samples were
acidified with acetic acid and then neutralized as described previously
(20).
Flow Cytometric AnalysisCulture 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 AnalysisEqual 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 AnalysisThe 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 541480 (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 AssayTo 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-1 on tumor cell
adhesion, mesothelial and fibroblast monolayers were preincubated with
increasing doses of TGF-
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 CellsAn 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-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.
StatisticsData 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.
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RESULTS |
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Expression of uPA and PAI-1 in Human Peritoneal Mesothelial Cells and Dermal Fibroblast CellsThe 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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Time-dependent Expression of uPA and PAI-1 in Mesothelial Cells in Response to Culture Medium Conditioned by Ovarian Cancer CellsWe 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 (048 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 612 h after addition of both media. Maximal induction of PAI-1 was achieved around 1224 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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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 AnalysisBy 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 612 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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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-1 Produced by Wild-type HRA Cells Specifically
Stimulates PAI-1 Expression in Mesothelial CellsTo 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
, TNF-
, and TGF-
1. Our recent
ELISA data (4) revealed that
TGF-
1 is significantly produced by HRA cells (TGF-
1, 396 ±
29.1; TNF-
, 63.1 ± 10.5; and IL-1
, <20
pg/ml/106 cells/24 h). Twenty four-hour, serum-free conditioned
medium from HRA cells contained both latent and activated TGF-
1 (latent
TGF-
1, 128 ± 25.1, and active TGF-
1, 180 ± 32.5
pg/ml/106 cells/24 h). We analyzed the TGF-
1 expression of
the stable cell lines by ELISA. bik+ clone expressed a
relatively small amount of TGF-
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-, IL-1
,or TGF-
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-
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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Culture media conditioned by wild-type HRA cells were incubated with
different concentrations of anti-TGF-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 510 µg/ml of the neutralizing anti-TGF-
1 antibody.
In contrast, antibodies to other cytokines (TNF-
and IL-1
) 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-1-treated Mesothelial
and Fibroblast CellsWhen confluent cultures of mesothelial cells
were incubated for 12 h in the presence of increasing concentrations of
TGF-
1, PAI-1 mRNA levels were increased in a dose-dependent
manner, with a maximal 56-fold increase at 210 ng/ml
(Fig. 5A). A kinetic
analysis revealed that in the presence of 10 ng/ml TGF-
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-
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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When confluent cultures of fibroblast cells were incubated in the presence
of increasing concentrations of TGF-1 or for different times,
uPA and PAI-1 mRNA levels were not significantly increased
(data not shown). Thus, TGF-
1 showed a stronger impact on uPA
and PAI-1 mRNA expression in mesothelial cells.
uPA and PAI-1 Protein Levels in TGF-1-treated
Mesothelial and Fibroblast CellsWe measured the levels of uPA and
PAI-1 in both cells exposed to TGF-
1 by ELISA (for uPA) and Western
blotting (for PAI-1; Fig. 6).
As expected, TGF-
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-
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-
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-
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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Flow Cytometric Analysis of Expression of Membrane-bound uPA, PAI-1,
and uPAR Proteins on HRA Cells and Unstimulated and
TGF-1-stimulated Mesothelial CellsFollowing in
vitro culture of mesothelial cells with TGF-
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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Phosphorylation of ERK1/2, JNK, p38 MAPK, and Smad2 in Mesothelial
Cells Exposed to TGF-1We examined the effects of
TGF-
1 on the activation of ERK1/2, JNK, p38 MAPK, and Smad2 in
semi-confluent mesothelial cells. Here we show that TGF-
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-
1 treatment (10 ng/ml).
TGF-
1 increased phosphorylation of ERK1/2 and p38 MAPK in the cells
34 times that of the basal level. TGF-
1 also dramatically
phosphorylated Smad2. In contrast, JNK was not phosphorylated by TGF-
1
(Fig. 8B). Total
ERK1/2, p38 MAPK, Smad2, and JNK proteins were abundantly expressed in these
cells.
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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-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-1-induced PAI-1 Gene ExpressionTreatment of
mesothelial cells with PD98059 almost completely abrogated the up-regulation
of TGF-
1-induced PAI-1 mRNA expression
(Fig. 9A).
Furthermore, treatment of the cells with SB202190 partially reversed the
up-regulation of TGF-
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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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-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
AssayTo determine directly the significance of
TGF-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-
1-stimulated mesothelial cells. TGF-
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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Tumor Cell Adhesion to and Invasion through Mesothelial and Fibroblast
MonolayersMaximum 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-
1.
Preincubation of the mesothelial monolayer with TGF-
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-
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-
1.
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When the monolayers were not pretreated with TGF-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-
1 on the Matrigel.
In the wild-type HRA cells, TGF-
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-
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-
1. In contrast, none of these clones enhanced their infiltration
potency by placing fibroblast monolayers pretreated with TGF-
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-1-stimulated mesothelial cells on HRA cell adhesion
(Fig. 12A) and
invasion (Fig. 12B),
confluent mesothelial cell cultures were pretreated with or without
TGF-
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-
1-stimulated control.
The effect of TGF-
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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DISCUSSION |
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We investigated the mechanism(s) by which TGF-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-
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-
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-
1 production. Although these
observations may be relatively novel in relation to ovarian cancer cell
interaction with mesothelial cells, the effect of TGF-
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-
(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-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-
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-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-
1 results in an
increase in uPA and PAI-1 mRNA levels. Although the
magnitude of the TGF-
1-induced increase in uPA and
PAI-1 mRNA levels was similar in mesothelial cells, TGF-
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-
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-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
v
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-
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-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-
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-
1 also operate partially through the p38
MAPK signaling pathway. Therefore, these studies establish a signal
transduction cascade or crosstalk emanating from TGF-
1 to PAI-1. Our
study represents the first report that TGF-
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-
1 is associated with activation of Smads.
Thus, elucidation of signal transduction cascades in response to TGF-
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-1 production.
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FOOTNOTES |
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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,
interleukin-1
; 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-
-activated kinase-1; TGF-
, transforming growth factor-
;
TNF-
, tumor necrosis factor-
; uPA, urokinase-type plasminogen
activator; PVDF, polyvinylidene difluoride; IL, interleukin; Ab, antibody;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FACS, fluorescence-activated
cell sorter.
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ACKNOWLEDGMENTS |
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REFERENCES |
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