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INTRODUCTION |
Extracellular proteolytic enzymes including serine proteinases and
metalloproteinases have been implicated in remodeling of the
extracellular matrix in lung injury and lung neoplasia (1-3). These
enzymes influence inflammatory cell traffic or cancer cell invasiveness
via the breakdown of basement membranes and extracellular matrix
(4-7). Plasmin, a serine protease, is involved in the dissolution of
extracellular matrix and basement membrane during tissue degradation.
This protease is generated via the action of plasminogen activators
such as urokinase (uPA)1 or
tissue plasminogen activator and can influence tissue remodeling either
directly or through activation of latent collagenases. Urokinase is
mainly involved in extravascular proteolysis in stromal remodeling in
acute and chronic lung injury (8-10) and in metastatic neoplasia (7).
During the last decade, evidence for involvement of the uPA system in
lung injury and repair or lung neoplasia (1) has steadily increased,
and it now seems clear that uPA-dependent plasminogen
activation is central to these processes. Increased expression of uPA
or uPAR has been inversely correlated with prognosis in lung cancer
(11, 12).
uPA-mediated plasminogen activation is tightly regulated by several
factors, including its high affinity receptor (uPAR), and two specific
and fast-acting inhibitors (PAI-1 and PAI-2) (13). Synthesis of
fibrinolytic components (uPA, uPAR, and plasminogen activator
inhibitor-1 and -2) is regulated by a variety of hormones, growth
factors, and cytokines either at the transcriptional or posttranscriptional level (13-18). The uPAR binds to both uPA and its
proenzyme pro-uPA to enhance cell surface plasminogen activation severalfold compared with that of fluid phase uPA (7, 13). Receptor-bound uPA can be inhibited by PAI-1 and PAI-2, and uPAR provides a mechanism for internalization of PAI-1-inactivated uPA. The
uPAR therefore plays an important role both in localizing and
modulating cell surface plasminogen activation. Human uPAR is heavily
glycosylated and is attached to the cell membrane by a
glycosylphosphatidylinositol (GPI) anchor. Both uPA and uPAR as well as
PAI-1 and -2 are expressed by lung epithelial cells (19-21),
indicating that autocrine regulation of this fibrinolytic system by the
epithelium could influence the course of either lung injury or lung cancer.
Expression of uPA and uPAR controls several cellular functions,
including epithelial cell adhesion, signaling, and mitogenesis, and
most of the biological activities of uPA are dependent on its
association with the uPAR (4, 13, 22). uPA is reported to generate
intracellular signals either by uPAR-dependent or uPAR-independent mechanisms (23). The expression of these components by
the lung epithelium is tightly regulated during normal physiological processes and is disordered in lung injury or lung cancer. It is
noteworthy that the signaling pathways activated by uPA/uPAR seem to be
the same pathways that induce their own expression (23). We therefore
postulated that interactive regulation between uPA and uPAR was
plausible, although not previously described. This possibility was
investigated using cultured Beas2B cells and primary bronchial
epithelial cells as a model system. In these studies, we describe a
novel regulatory pathway by which uPA induces uPAR expression by Beas2B
as well as primary bronchial epithelial cells.
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EXPERIMENTAL PROCEDURES |
Materials--
Culture media, penicillin, streptomycin, and
fetal calf serum were purchased from Life Technologies, Inc.; tissue
culture plastics were from Becton Dickinson Labware (Lincoln Park, NJ).
-Thrombin, herbimycin, genistein, bovine serum albumin (BSA), ovalbumin, Tris-base, aprotinin, dithiothreitol (DTT),
phenylmethylsulfonyl fluoride, silver nitrate, ammonium persulfate, and
phorbol myristate acetate (PMA) were from Sigma. Acrylamide,
bisacrylamide, and nitrocellulose were from Bio-Rad. Recombinant high
molecular weight (HMW) uPA was a generous gift from Drs. Jack Henkin
(Abbott) and Andrew Mazar (Angstrom Pharmaceuticals, San Diego, CA).
The low molecular weight (LMW), amino-terminal fragment (ATF),
anti-uPA, and anti-uPAR antibodies were obtained from American
Diagnostics (Greenwich, CT). A5 and B428 compounds were generous gifts
from Dr. Andew Mazar. XAR x-ray film was purchased from Eastman Kodak Co.
Cell Cultures--
Small airway epithelial cells (SAEC) were
obtained from Clonetics (San Diego, CA). Human bronchial epithelial
cells (Beas2B) or lung epithelial tumor cells, including H1395 and A549
human lung adenocarcinoma cells, H157 human lung squamous cells, H460 large cell lung carcinoma cells, and H146 human lung small cell lung
carcinoma cells were obtained from the ATCC. Human pleural mesothelial
(MeT5A) and spindle-shaped (M33K) or epithelioid (M9K) pleural
malignant mesothelioma cells were obtained from Dr. Brenda Gerwin,
National Institutes of Health. These cells, as well as primary cultures
of human pleural mesothelial cells obtained from pleural fluid
aspirates, were maintained in RPMI 1640 medium containing 10%
heat-inactivated fetal calf serum, 1% glutamine, and 1% antibiotics as described previously (24).
Total Cellular Membrane Extraction and Western
Blotting--
Cells grown to confluence were serum-starved overnight
with RPMI-glutamine media containing 0.5% BSA. The cells were treated with or without various agents for indicated times and were washed with
PBS. Receptor-bound uPA was removed by glycine HCl treatment as
described earlier (25). We used SDS-gel electrophoresis and Western
blotting to measure functional uPAR at the cell surface. Membrane
proteins isolated as described earlier (26) from Beas2B, SAEC, MeT5A,
selected lung tumor cell lines, and pleural mesothelioma cell lines
were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 1% BSA in wash buffer for
1 h at room temperature followed by overnight hybridization with
uPAR monoclonal antibody in the same buffer at 4 °C and washed, and
uPAR proteins were detected by enhanced chemiluminescence (ECL).
125I-uPA Binding--
Recombinant uPA (5 µg/ml)
was treated with 2 mCi of 125I-Na and 50 mg IODO-GEN for 5 min at 4 °C. The reaction was stopped by addition of excess KI, and
unincorporated label was removed by passage through a Sephadex-25
column as described earlier (27).
In a separate experiment, membrane proteins of Beas2B cells treated
with uPA for varying times (0-24 h) were separated on SDS-PAGE and
transferred to nitrocellulose membrane as described above. The
nitrocellulose membrane was subjected to ligand blotting assay, using
125I-uPA as described earlier (27).
We also measured binding of 125I-uPA by the method of Waltz
et al. (28) with modifications. Beas2B cells grown to
confluence in 24-well plates were treated with or without uPA for
24 h in serum-free media containing 0.5% BSA. The cells were
acid-treated and subjected to 125I-uPA binding as we
described earlier (27). The nonspecific binding was measured in the
presence of a 400-fold molar excess of cold uPA. Activation of
plasminogen by Beas2B cells was measured by a modification of the
esterolytic method as we described previously (27).
Plasmid Construction--
Plasmid uPAR/pBluescript was obtained
from the ATCC. The human uPAR mRNA template containing a complete
sequence of uPAR cDNA (nucleotides
16 to 1144) from uPAR
pBluescript was subcloned to HindIII and XbaI
sites of pRC/CMV (Invitrogen), and the sequences of the clones were
confirmed by sequencing. The uPAR insert was released by
HindIII or XbaI, purified on 1% agarose gels,
extracted with phenol/chloroform, and used as a cDNA probe for
Northern blotting.
Random Priming of uPAR cDNA--
The full-length template of
uPAR was released with HindIII or XbaI, purified
on 1% agarose gels, and labeled with [32P]dCTP using a
RediPrime labeling kit (Amersham Pharmacia Biotech). Passage through a
Sephadex G-25 column removed unincorporated radioactivity. The specific
activity of the product was 6 × 108 cpm/µg.
Nuclear Run-on Transcription Activation Assay--
Cells grown
to confluence in two T182 flasks were serum-starved overnight in
RPMI/BSA media. The cells were later treated with PBS, uPA, or TGF-
(2 ng/ml) for 12 h at 37 °C and then washed with ice-cold PBS.
Cells were resuspended in 0.4 ml of lysis buffer (10 mM
Hepes buffer, pH 7.9, 10 mM KCl, 0.1 mM EDTA,
0.1 mM EGTA, 1 mM DTT, 0.5 mM
phenylmethylsulfonyl fluoride, 2 µg/ml each leupeptin and aprotinin,
and 0.5 mg/ml benzamidine). The cells were homogenized and incubated on
ice for 1 h to release the nuclei. The nuclear pellet was
subjected to sucrose gradient centrifugation by careful layering on top
of 7.5 ml of sucrose buffer (1.3 M) followed by centrifugation at 10,000 × g for 15 min at 4 °C.
The nuclear pellet was resuspended in 200 µl of lysis buffer. The
nuclei (10-20 × 106) were subjected to the
transcription reaction in the presence of 250 µCi of
[32P]UTP at 30 °C for 30 min. 32P-Labeled
nuclear RNA was isolated using TRI-reagent, and unincorporated radioactivity was removed by repeated precipitation and washing by cold
ethanol. RNA pellets were dissolved in 40 µl of RNase-free water.
Recombinant plasmid DNA (10 µg/slot) was denatured by heating at
95 °C in NaOH/EDTA solution for 5 min and chilling on ice. Denatured
DNA solution was spotted on a nitrocellulose membrane using a slot blot
apparatus under vacuum. The membranes were vacuum-baked, prehybridized
overnight at 42 °C in a hybridization buffer (50 mM
Hepes, pH 7.0, 50% formamide, 4× SSC, 2× Denhardt's solution, 2 mM EDTA, 0.1% SDS, 0.225 mg/ml tRNA, 0.1 mg/ml
poly(A)+, and 0.2 mg/ml salmon sperm DNA).
32P-Labeled RNA (10 × 106 cpm/ml) was added to the
hybridization solution, and the membranes were incubated in a
hybridization oven for 48 h. The filters were washed with 2-0.1×
SSC with in-between RNase digestion, air-dried, and exposed to x-ray film.
Northern Blotting of uPAR mRNA--
A Northern blotting
assay was used to assess the level of uPAR mRNA. Beas2B cells grown
to confluence were serum-starved overnight in RPMI/BSA media and then
treated with uPA for varying times (0-24 h) in the same media. Total
RNA was isolated using TRI-reagent. RNA (20 µg) was isolated on
agarose/formaldehyde gels. After electrophoresis, the RNA was
transferred to Hybond N+ according to the instructions of
the manufacturer. Prehybridization and hybridization were done at
65 °C in NaCl (1 M)/SDS (1%) and 100 µg/ml salmon
sperm DNA. Hybridization was performed with a uPAR cDNA probe (1 ng/ml) labeled to ~6 × 108 cpm/µg of DNA
overnight. After hybridization, the filters were washed twice for 15 min at 65 °C with 2× SSC, 1% SDS; 1× SSC, 1% SDS; and 0.1% SSC,
1% SDS, respectively. The membranes were next exposed to x-ray film at
70 °C overnight. The intensity of the bands was measured by
densitometry and normalized against that of
-actin.
Overexpression of uPA, Transfection of Beas2B Cells with uPA
cDNA--
The uPA cDNA (29) was subcloned to eukaryotic
expression vector pRc/CMV2 (Invitrogen) containing the CMV promoter at
HindIII/NotI sites. The orientations and
sequences were confirmed by sequencing. Beas2B cells were transfected
with the prepared chimeric plasmid constructs by lipofection using
LipofectAMINE (Life Technologies, Inc.). Stable cell lines were made by
treating Beas2B cells with neomycin for 3 months. Cells carrying
plasmid DNA that survived after neomycin treatment were scraped off
from 6-well plates and grown in T75 flasks, and the presence of plasmid
DNA was confirmed by polymerase chain reaction using specific primers.
The overexpression of uPA by cDNA-transfected cells was confirmed
by Western blotting Beas2B cell lysates as well as conditioned media
using a uPA monoclonal antibody. The effect of endogenous uPA
overexpression on uPAR expression was measured by Western blotting for
uPAR as described above.
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RESULTS |
Expression of uPAR in Lung Epithelial Cells--
Since we
previously found that MeT5A mesothelial cells and MS-1 mesothelioma
cells both express uPAR in vitro (25, 27), we initially
wanted to determine if uPAR is differentially expressed in cultured
nonmalignant, bronchial epithelial (Beas2B) cells or small airway
epithelial (SAE) cells versus an array of malignant lung
carcinoma-derived cells. Pulmonary artery smooth muscle cells and human
pulmonary microvascular endothelial cells were also analyzed. Western
blotting assays were used to determine the level of uPAR expression at
the surface of primary cultures of lung small airway epithelial cells,
Beas2B cells, human lung carcinoma-derived cells (H1395, A549, H157,
H460, and H146), and from human pleural malignant mesothelioma cells
(M33K, M9K, and MS-1). Basal expression of uPAR in unstimulated cells
was greatest in H157, H460, and A549 cells among the lung cancers (Fig.
1).

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Fig. 1.
Expression of urokinase receptors in normal
and carcinoma-derived lung cells. Membrane proteins (100 µg)
isolated from cells of epithelial lineage (a) (lanes
1-7, SAEC, Beas2B, H157, H460, A549, H1395, and H146), lung
vascular-derived (b) (lanes 1 and 2,
pulmonary artery smooth muscle cells and human pulmonary microvascular
endothelial cells), and pleural (c) (lanes 1-5,
Met5A, Ren, MS-1, M9K, and M33K) were separated on 8% SDS-PAGE and
electroblotted to nitrocellulose membranes. The membranes were
subjected to Western blotting using a urokinase receptor monoclonal
antibody.
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Time-dependent Expression of uPAR by
uPA--
Signaling pathways activated by the uPA/uPAR system could be
the same pathways that induce their own expression (23). We therefore
explored the possibility that expression of uPA/uPAR in epithelial
cells leads to a nested signaling loop and/or activation of additional
mechanisms that could contribute to enhanced uPAR expression. We used
Beas2B epithelial cells as a lung epithelial cell model system to
initiate these studies. We treated these cells with uPA (the high
molecular weight, two-chain form) for varying times (0-24 h). uPAR
expression at the cell surface was assessed by Western blotting using
an anti-uPAR antibody. Results of these experiments (Fig.
2a) demonstrate that uPA
induces uPAR in Beas2B cells in a time-dependent manner and
that the induction starts between 3 and 6 h after addition of uPA.
Maximal induction of uPAR by uPA was achieved around 12 h, and the
elevated level is maintained for at least 24 h. uPA also induced
uPAR expression in primary bronchial epithelial cells but not in uPAR
over-producing H157 or A549 cells (Fig. 2b). We also
confirmed expression of increased functional uPAR in uPA-treated Beas2B
cells by ligand blotting assay, demonstrating receptor binding
consistent with the results of Western blotting experiments (data not
shown).

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Fig. 2.
Time-dependent uPAR expression by
uPA in lung epithelial (Beas2B) and SAEC cells. a,
confluent Beas2B cells were treated with or without two-chain uPA (1 µg/ml) for 0, 3, 6, 12, and 24 h (lanes 1-5) or PBS
24 h (lane 6) at 37 °C in basal medium containing
0.5% BSA, and membrane proteins were isolated. The total membrane
proteins were separated on 8% SDS-polyacrylamide gels and transferred
to nitrocellulose membranes. The membrane was immunoblotted with
anti-uPAR antibody. The data illustrated are representative of at least
three independent experiments, and mean density of the individual bands
is presented in the line graph. The p values are
0.05 for the 3-, 6-, and 24-h treatment periods versus the
0- or 24-h PBS treatment time points. Relatively large error
bars are attributable to the semiquantitative nature of the
densitometric analyses and the difference between signal/background
ratios in individual experiments. b, Western blot for
urokinase receptor of primary bronchial epithelial cells treated with
PBS (lane 1) or uPA (lane 2) or identically
treated H157 (lanes 3 and 4) or A549 cells
(lanes 5 and 6) for 24 h.
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To see if the high molecular weight uPA (HMW uPA) preparation we used
in this study contains lipopolysaccharides (LPS), we used the Limulus
Amebocyte lysate enzyme-linked immunosorbent assay method. We found
that this HMW uPA preparation contains very negligible amounts (about 1 pg/ml) of LPS. We next treated Beas2B cells with the same
concentration, 1 pg/ml as well as a 10-fold increment, 10 pg/ml LPS,
and measured uPAR expression by Western blotting as we described above.
We found that these concentrations of LPS failed to induce uPAR
expression, indicating that the induction of uPAR by uPA could not be
attributable to LPS contamination.
Induction of uPAR by Endogenous uPA--
We next prepared stable
uPA-overexpressing Beas2B cells and vector-treated controls by
transfecting these cells with the eukaryotic expression vector
PRc\CMV2 containing uPA cDNA or PRc\CMV2 cDNA using
lipofection. We analyzed the uPA expression of the stable cell lines by
Western blotting. As shown in Fig.
3a, Beas2B cells transfected
with uPA cDNA expressed a relatively large amount of uPA in both
the conditioned media and at the cell surface, in comparison to
vector-transfected or non-transfected controls. We then measured the
uPAR expression by these cells by Western blotting using a uPAR
monoclonal antibody, and we confirmed that Beas2B cells transfected
with uPA cDNA produced large amounts of uPAR at the cell surface.
Vector-transfected or non-transfected control did not demonstrate
increased uPAR expression, suggesting that induction of increased uPAR
expression was attributable to stimulation effected by increased
endogenous uPA (Fig. 3b).

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Fig. 3.
Overexpression of endogenous uPA induces uPAR
expression at the cell surface. a, Western blotting for
uPA expression in uPA cDNA-transfected Beas2B cells. Proteins from
cell lysates (CL) and conditioned media (CM) of
untreated Beas2B cells alone (lane 1) or Beas2B cells were
transfected with uPA cDNA in eukaryotic expression vector pRc/CMV
(lane 2) or Beas2B cells transfected with expression vector
pRc/CMV alone (lane 3). Proteins were separated on 8%
SDS-PAGE, transferred to a nitrocellulose membrane, and developed by
Western blotting using anti-uPA monoclonal antibody. b,
Western blotting for uPAR expression in uPA cDNA-transfected cells.
Membrane proteins from Beas2B cells treated as above were subjected to
Western blotting using anti-uPAR monoclonal antibodies.
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Binding of 125I-uPA--
We next wanted to
determine the specificity of uPA binding to the surface of
uPA-stimulated Beas2B cells. Accordingly, we treated PBS or
uPA-stimulated Beas2B cells with 0-61 nM
125I-uPA for 2 h at 4 °C with or without a 400-fold
molar excess of unlabeled uPA. The specific binding was calculated from
the difference between total and nonspecific counts (30). The data in
Fig. 4 show that exogenously added uPA
bound progressively with increasing concentration. Unstimulated Beas2B
cells demonstrated functional uPAR; however, uPA stimulation enhanced
125I-uPA binding at least 2-3-fold (Fig. 4a).
Binding of 125I-uPA was competed for by increasing
concentrations of unlabeled uPA, and ~60% was accounted for specific
binding (Fig. 4b). This observation is consistent with our
previous finding that 30-35% of 125I-uPA binding is
nonspecific in PMA-treated mesothelial cells (25, 27). Because the
binding experiments in this study were performed at 4 °C, it is
unlikely that the magnitude of the nonspecific binding is attributable
solely to internalization of uPA·uPAR·PAI-1 complexes. To determine
if uPA enhances plasminogen activation through induction of functional
uPAR, unstimulated or uPA-treated cells were preincubated with uPA for
2 h at 4 °C, washed, and incubated with plasminogen (6 µg)
for 20 min at 37 °C. The rate of plasminogen activation was obtained
by measuring the liberated plasmin. uPA induced plasmin generation in a
concentration-dependent manner (Fig. 4c).

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Fig. 4.
a, saturation binding of
125I-uPA to Beas2B cells treated with or without uPA.
Beas2B cells grown in multiwell plates were treated with PBS or uPA (1 µg/ml) for 24 h at 37 °C. The receptor-bound endogenous uPA
was removed by glycine HCl treatment, and the cells were later treated
with varying concentrations of 125I-uPA for 2 h at
4 °C. Cell-bound radioactivity was measured, and specific binding
was calculated based on nonspecific binding in the presence of 400-fold
molar excess of unlabeled uPA. b, competitive inhibition of
125I-uPA binding to uPA treated Beas2B cells by HMW uPA.
Beas2B cells treated with uPA were incubated with varying amounts of
unlabeled uPA for 2 h at 4 °C, followed by 2 h with
125I-uPA. Bound radioactivity was measured, and the percent
binding was calculated from cell-associated radioactivity in the
absence of unlabeled uPA. c, effect of uPA concentration on
uPA receptor-mediated plasminogen activation in Beas2B cells. Beas2B
cells treated with or without varying concentrations of uPA were
preincubated with 2.5 nM uPA for 2 h at 4 °C,
washed, and assayed for activation of added plasminogen (6 µg).
Background endogenous uPA activity of glycine HCl-treated cells has
been subtracted from the values shown.
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uPAR mRNA Expression by uPA in Beas2B Lung Epithelial
Cells--
By having determined that uPA mediates
time-dependent uPAR expression at the lung epithelial cell
surface, we wanted to confirm that the increased cell surface uPAR is
attributable to an increased level of uPAR mRNA. We next measured
the levels of uPAR mRNA in uPA-treated Beas2B epithelial cells by
Northern blotting using a uPAR cDNA probe and densitometric
scanning. As shown in Fig. 5, uPA induces
uPAR mRNA, and the induction is observed as early as 3 h after
the treatment. Maximum accumulation of uPAR mRNA is achieved
between 12 and 24 h after the treatment. These data confirm the
induction of uPAR expression by uPA as determined by Western blot. The
level of uPAR mRNA was quantitated by densitometric scanning and
normalized against
-actin loading controls. As shown in Fig. 5,
resting Beas2B cells express small amounts of uPAR mRNA. However,
uPAR mRNA levels increase about 20-fold by 24 h after uPA
treatment.

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Fig. 5.
Time-dependent induction of uPAR
mRNA by uPA. Beas2B cells were treated with two-chain uPA (1 µg/ml) for 0, 3, 6, 12, and 24 h (lanes 1-5). Total
RNA (20 µg/lane) was isolated using TRI-reagent and separated on an
agarose-formaldehyde gel and subjected to Northern blotting using
32P-labeled uPAR and -actin cDNAs. The figure shown
is representative of the results of at least three independent
experiments, and the bar graph illustrates the mean band
densities from these experiments. The p values for the uPA
treatments versus the 0 h treatment interval are
0.05. The relatively large error bars are attributable to
the semiquantitative nature of the densitometric analyses and the
difference between signal/background ratios in individual
experiments.
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Transcriptional Activation of uPA, Run-on Transcription
Experiments--
In order to determine whether uPA enhances uPAR gene
transcription, we treated Beas2B cells with uPA for 6 and 12 h and
isolated nuclei, and 32P-labeled RNA was hybridized with
uPAR cDNA immobilized on a nitrocellulose membrane. The results of
nuclear run-on transcription analyses demonstrated that uPA did not
induce transcriptional activation of the uPAR gene (n = 3 data not shown).
The Effect of uPA Concentration on uPAR Expression--
We next
treated Beas2B cells with varying amounts (0-3 µg/ml) of two-chain
uPA for 24 h and then measured cell surface uPAR expression by
Western blot assay. Fig. 6 shows that uPA
induced uPAR in a concentration-dependent manner. Induction
is apparent with as low as 10 ng/ml uPA. Maximal uPAR expression was
observed with 500 ng/ml uPA, beyond which there was a steady decline in uPAR expression. The steady decrease in uPAR expression at higher uPA
concentration could be due to degradation of uPAR by uPA (31). These
data suggest that the induction of uPAR by uPA is specific and
concentration-dependent.

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Fig. 6.
Effect of uPA concentration on uPAR
expression. Beas2B cells grown to confluence were treated with
varying amounts of uPA (0, 10, 50, 100, 250, 500, 750, 1000, 2000, and
3000 ng/ml lanes 1-10) for 24 h at 37 °C in basal
medium containing 0.5% BSA, and membrane proteins were isolated. The
total membrane proteins were separated on 8% SDS-polyacrylamide gel
and transferred to nitrocellulose membrane. The membrane was
immunoblotted with anti-uPAR antibody. The figure is representative of
the results of three separate experiments, and the bar graph
illustrates the mean band densities of these experiments. Relatively
large error bars are attributable to the semiquantitative
nature of the densitometric analyses and the difference between
signal/background ratios in individual experiments. The p
values for the uPA treatments versus the PBS control
p 0.05.
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Effects of Phosphatase and Phosphotyrosine Kinase Inhibitors on
uPA-mediated Induction of uPAR--
To determine whether uPA-mediated
uPAR expression involves cellular signaling, we pretreated Beas2B cells
with herbimycin A (2 µM) and genistein (6 µg/ml),
protein tyrosine kinase inhibitors, separately or in combination with
uPA. As shown in Fig. 7, neither herbimycin A nor genistein alone induced uPAR expression (Fig. 7,
lanes 3 and 5). However, when Beas2B cells were
treated with uPA, pretreatment with these inhibitors reversed
uPA-mediated uPAR expression (Fig. 7, lanes 4 and
6). Pretreatment of cells with vanadate (10 µM) (a tyrosine phosphatase inhibitor), on the other
hand, did not block uPA-induced uPAR expression but appeared to enhance
the uPA-mediated effect. These data suggest protein phosphorylation may
be involved in the process.

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Fig. 7.
Effect of tyrosine kinase and phosphatase
inhibitors on uPA-mediated uPAR expression. The cells grown to
confluence were treated with or without herbimycin A (2 µM), genistein (6 µg/ml), and sodium orthovanadate (10 µM) for 2 h followed by uPA (1 µg/ml) for 24 h at 37 °C in basal medium containing 0.5% BSA, and membrane
proteins were isolated. The total membrane proteins were separated on
an 8% SDS-polyacrylamide gel and then transferred to a nitrocellulose
membrane. The membrane was immunoblotted with anti-uPAR antibody. The
cells treated with PBS (lane 1), uPA (lane 2),
herbimycin A (Herb, lane 3), herbimycin A and uPA
(lane 4), genistein (Gene, lane 5),
genistein and uPA (lane 6), sodium orthovanadate (Na
Orth, lane 7), and sodium orthovanadate and uPA
(lane 8). The data illustrated are representative of the
findings of three separate experiments.
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The Role of uPAR in uPA-mediated uPAR Expression--
We next
sought to determine if uPA interacts with cell surface uPAR to induce
the receptor. To investigate this possibility, we pretreated Beas2B
cells with anti-uPAR antibody for 2 h and then treated with uPA
for 24 h. As shown in Fig.
8a, uPAR antibody (2 µg/ml)
alone failed to induce cell surface uPAR expression, and pretreatment
of Beas2B cells with this antibody failed to block uPA-induced uPAR
expression (Fig. 8a). To confirm that uPA-mediated uPAR
expression does not involve uPAR, we next treated Beas2B cells with A5
(1 µg/ml) compound (a receptor agonist that blocks association of uPA
with uPAR) (32) alone or in combination with uPA. As shown in Fig.
8a, A5 alone minimally induced uPAR expression but, in
combination with uPA, did not inhibit expression of uPAR at the cell
surface. By using a third independent approach, we removed uPAR from
cells by treating with PI-PLC (10 units/ml) and then tested to see if
uPA would stimulate uPAR expression. It is known that uPAR is a
GPI-linked protein, and PI-PLC completely removes GPI-linked proteins,
including uPAR from the cell surface. Under these conditions, we still
observed uPAR induction by uPA (Fig. 8a). In a separate
experiment, we found that PI-PLC completely cleaved uPAR from the cell
surface by Western blotting of membrane fractions (data not shown),
providing further evidence that the induction of uPAR by uPA is not
mediated by their association at the cell surface.

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Fig. 8.
a, role of uPAR in uPA-mediated uPAR
induction. Beas2B cells were grown to confluence and then treated with
or without anti-uPAR antibody (RAb, 2 µg/ml), A5 (1 µg/ml)
compound, and PI-PLC (10 units/ml) for 2 h followed by uPA (1 µg/ml) for 24 h at 37 °C in basal medium containing 0.5%
BSA. Membrane proteins were then isolated. The total membrane proteins
were separated on an 8% SDS-polyacrylamide gel and transferred to a
nitrocellulose membrane. The membrane was immunoblotted with an
anti-uPAR antibody. Data representative of three independent
experiments are shown. b, effect of uPA inhibitors on uPA
mediated uPAR expression. Beas2B cells grown to confluence were treated
with or without B-428 (0.02 mM), anti-uPA monoclonal
antibody (PAAb) (2 µg/ml), or plasminogen activator inhibitor
(PAI-1) (4 µg/ml) for 2 h followed by uPA (1 µg/ml)
for 24 h at 37 °C in basal medium containing 0.5% BSA, and
membrane proteins were isolated. The total membrane proteins were
separated on an 8% SDS-polyacrylamide gel and then transferred to a
nitrocellulose membrane. The membrane was immunoblotted with anti-uPAR
antibody. Data representative of three independent experiments are
illustrated. c, effect of different fragments of uPA on uPAR
expression. Beas2B cells grown to confluence were treated with or
without ATF (1 µg/ml) and LMW (1 µg/ml) of uPA for 2 h
followed by uPA (1 µg/ml) for 24 h at 37 °C in basal medium
containing 0.5% BSA. Membrane proteins were then isolated. The total
membrane proteins were separated on an 8% SDS-polyacrylamide gel and
then transferred to a nitrocellulose membrane. The membrane was
immunoblotted with an anti-uPAR antibody. The data are representative
of the findings of three separate experiments.
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The Effect of uPA Enzymatic Activity on Induction of uPAR--
By
having confirmed that uPA-mediated uPAR induction is not mediated
through interaction with uPAR, we next wanted to determine if enzymatic
activity of uPA is required for uPA-mediated uPAR induction. To address
this possibility, we tested the ability of an anti-uPA monoclonal
antibody to inhibit uPA expression. As shown in Fig. 8b, an
anti-uPA antibody (2 µg/ml) did not induce uPAR expression, whereas
inactivation of uPA catalytic activity by this antibody reversed the
effect. Similarly PAI-1 (4 µg/ml) (Fig. 8b) inhibited
uPA-mediated uPAR induction, indicating that uPA activity is required.
uPA antibody or PAI-1 are large molecules and could therefore be
inhibiting uPA-mediated functions by interfering with its interaction
at distal sites rather than by inactivating enzymatic activity. We
therefore pretreated uPA with B-428 (0.02 mM) (an active
site inhibitor of uPA) (33) and found that inactivation of uPA activity
by B-428 inhibited uPA-mediated uPAR expression (Fig. 8b).
Likewise, chloromethyl ketone-inactivated uPA (27) failed to induce
uPAR expression (n = 2 data not shown).
The Effect of ATF or LMW uPA on Induction of uPAR--
We next
treated Beas2B cells with the amino-terminal fragment of uPA (ATF) or
the active low molecular weight (LMW) fragment of uPA to confirm that
the uPA effect is not mediated by receptor occupancy. As shown in Fig.
8c, ATF (1 µg/ml) alone did not induce uPAR nor did
pretreatment alter uPA-mediated uPAR expression. The uPA LMW fragment
(1 µg/ml), conversely, induced uPAR at the cell surface.
The Effect of Proteases and Protease Inhibitors on uPA-mediated
uPAR Expression--
Since we found that uPAR is not involved in
uPA-mediated uPAR expression, we inferred that proteases might have
been induced by uPA to stimulate uPAR expression. To address this
possibility, we treated Beas2B cells with uPA in the presence of
aprotinin, a broad spectrum protease inhibitor that inactivates
trypsin-like activity. As shown in Fig.
9, neither aprotinin (1 µg/ml) alone nor in combination with uPA inhibited uPAR induction. We also investigated the possibility that uPA induces uPAR expression through
plasmin, the end product of uPA-mediated plasminogen activation. Neither plasmin (1 µg/ml) nor plasmin and uPA altered uPAR expression (Fig. 9). We also treated Beas2B cells with
-thrombin (1 µg/ml) another serine protease alone or in combination with uPA. Thrombin alone did not induce uPAR expression; however, it reversed the uPA
effect when the cells were treated with both proteases in combination
(Fig. 9).

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Fig. 9.
Effect of proteases and protease inhibitors
on uPA-mediated uPAR induction. Beas2B cells were grown to
confluence and then treated with or without aprotinin (1 µg/ml),
plasmin (1 µg/ml), or -thrombin (1 µg/ml) alone for 2 h
followed by uPA (1 µg/ml) for 24 h at 37 °C in basal medium
containing 0.5% BSA. Membrane proteins were then isolated. The total
membrane proteins were separated on 8% SDS-polyacrylamide gels and
then transferred to nitrocellulose membranes. The membranes were
immunoblotted with anti-uPAR antibody. The cells were treated with PBS
(lane 1), uPA (lane 2), aprotinin (lane
3), aprotinin and uPA (lane 4), plasmin (lane
5), plasmin and uPA (lane 6), thrombin (lane
7), and thrombin and uPA (lane 8). The figure shown is
representative of three separate experiments, and the bar
graph illustrates the mean band densities from these
experiments.
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DISCUSSION |
Local generation of plasmin by uPA is a central mechanism by which
cells degrade extracellular matrices to relocate from one anatomical
location to another. uPA/plasmin-mediated proteolysis is critical for
cellular migration and tissue remodeling following either lung injury
or the propagation and metastasis of lung neoplasms (4, 7). uPAR is
essential for uPA-dependent pericellular proteolysis and is
localized at the leading edge of migrating cells (13). The interaction
between uPA and uPAR at the cancer cell surface appears to influence
neoplastic growth and metastasis by mediating effects on tissue
remodeling, tumor cell invasion, cellular adhesion, and proliferation
(1, 33, 34). Tumor cell invasion is also facilitated by saturation of
uPAR with either exogenously supplemented uPA or overexpressed
endogenous uPA (34, 35). In addition, the binding of uPA to uPAR
mediates cell proliferation in several cell types including
nonmalignant and malignant epithelial cells and mesothelioma cells (25,
26).
Epithelial carcinomas originating in the lung and other tissues
including breast, ovary, prostate, and kidney all express increased
amounts of uPA and its receptor (34-40). This observation suggested
the possibility that interaction between these molecules might account
for the relative overexpression of the receptor. Since uPAR expression
is implicated in the pathogenesis of lung cancer as well as lung injury
and fibrosis (2, 5, 8, 10, 21), we sought to elucidate how uPAR is
regulated in lung epithelial cells derived from neoplasms or
nonmalignant epithelium. We chose Beas2B cells as an in
vitro lung epithelial cell model system to determine if uPAR
expression is regulated by uPA, and we found that this was the case.
Regulation of uPAR expression in different cell populations involves
both transcriptional and posttranscriptional mechanisms. In previous
studies, we found that a posttranscriptional pathway influences levels
of uPAR mRNA in lung cancer and malignant mesothelioma cells (26)
as well as rabbit mesothelial cells and fibroblasts (41). Cytokines
that occur in both the tumor microenvironment and in lung inflammation
and repair increase uPAR expression at the cell surface through this
mechanism. Similar findings were previously reported in PMA and
TGF-
-treated U937 cells (14). In this study, we found that
uPA-mediated expression of uPAR did not involve transcriptional
activation of the uPAR gene, implicating regulation at alternative levels.
It has also been reported previously that the specific binding capacity
for uPA and cell surface uPAR expression is increased by PMA, epidermal
growth factor, LPS, TGF-
, and tumor necrosis factor-
in various
cell lines (28, 41). To extend our understanding of the regulation of
uPAR by epithelial cells, we analyzed the effect of uPA on uPAR and
uPAR mRNA expression. uPA strongly and rapidly induces cell surface
uPAR expression, an effect that can be traced back to a rapid
antecedent increase in the cellular level of uPAR mRNA. PMA,
TGF-
, and tumor necrosis factor-
increase the rate of
transcription and stability of uPAR mRNA, whereas epidermal growth
factor increases transcription alone (14). We found that
cytokine-induced uPAR was posttranscriptionally regulated in these
cells by a mechanism known to influence uPAR mRNA stability at this
level (16, 26). The increased uPAR mRNA stability in lung tumor and
pleural mesothelioma cells correlates with the increased uPAR mRNA
and cell surface uPAR. It may be that uPA-mediated induction of uPAR
involves posttranscriptional pathways, a possibility that will require
future, detailed investigation.
We showed previously that the binding of uPA to its receptor was
mitogenic for MS-1 and MeT5A cells and that proliferation of these
cells could be blocked by antisense oligonucleotides directed against
uPAR (25). Our present study demonstrates that uPA-mediated uPAR
expression is concentration-dependent. This pathway
represents a potentially versatile regulatory system in which uPA in
the tumor stroma or lung microenvironment could favor amplification of
its receptor. This molecular mechanism may be crucial to our
understanding of the participation of the uPA/uPAR system in
pathological conditions such as cancer or organizing alveolitis. uPA
expression under such circumstances could lead to excessive
pericellular proteolysis and a change in the behavior of epithelial
cells, predicated upon increased expression of uPAR.
The mechanism by which uPA induces uPAR appears to be
proteolysis-dependent and uPAR-independent. The regulatory
mechanism further involves activation of tyrosine kinases. The
sustained effect of uPA on both uPAR protein and mRNA levels
suggests that the specific message sent by tyrosine kinase likely
prolongs the half-life of uPAR mRNA. uPA also induces synthesis of
growth factors or cytokines which, in turn, may induce a late increase
in the level of uPAR mRNA. These possible mechanisms also warrant
further investigation.
Studies performed in other cell types have demonstrated the involvement
of uPAR in uPA-mediated signaling. Along this line, activation of
focal adhesion kinase and mitogen-activated protein kinases in
cultured endothelial cells has been reported. Similarly, activation of
a 38-kDa tyrosine-phosphorylated uPAR-associated protein has been
identified in U937 cells (42). In both cases, signaling by uPA was
abolished when the cells were treated with PI-PLC, and other studies
also support the association of uPAR with PKC and cytokeratin (43).
However, in our study, pretreatment of Beas2B cells with PI-PLC for
2 h followed by uPA treatment for 24 h did not inhibit uPAR
expression. The explanation could be that prolonged exposure to uPA (24 h) reverses the effect of removal of PI-PLC. uPAR also appears to
associate with
2 integrins and members of the Src family
of kinases (44, 45). In vascular smooth muscle cells, uPAR was
associated with JAK1, Tyk2, and Src kinases (46), whereas in kidney
epithelial tumor cells, uPAR associates with gp130 and JAK1 (47).
The mechanism by which uPA induces uPAR in Beas2B cells is at present
unknown. Clearly the mechanism is not plasmin-dependent because exposure of cells to aprotinin or treatment with plasmin did
not, respectively, alter uPAR expression. One possibility is that uPA
may proteolytically activate a transmembrane protein to initiate
signaling. It has been reported that uPA interacts with a novel, high
affinity binding protein in platelet membranes (48). Similarly, in
melanoma cells uPA appears to bind to an unidentified membrane protein
to initiate signal transduction. Whatever the receptor for uPA-mediated
uPAR induction proves to be, the magnitude of induction is greater than
that effected by PMA or TGF-
, which are among the most potent
inducers of cellular uPAR expression (27).
In summary, we confirmed that uPA stimulates cell surface expression of
uPAR by cultured Beas2B and primary lung epithelial cells. This newly
recognized pathway represents another mechanism by which
uPAR-dependent responses of the lung epithelium may be regulated in the context of lung injury and repair or in the
transformation, growth, and spread of lung neoplasms.