Urokinase-type plasminogen activator induces tyrosine
phosphorylation of a 78-kDa protein in H-157 cells
G. Jayarama
Bhat,
Jagadambika J.
Gunaje, and
Steven
Idell
Department of Specialty Care Services, The University of Texas
Health Center at Tyler, Tyler, Texas 75708
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ABSTRACT |
Studies from our laboratory have shown that
exposure of human lung epithelial cells to urokinase plasminogen
activator (uPA) induces their proliferation. This effect of uPA is
likely to occur via activation of signal transduction pathways. To
elucidate uPA-induced signal transduction mechanisms, we exposed H-157
cells to uPA and determined the induced tyrosine phosphorylation
profile of proteins. We demonstrate that, in these cells, uPA
prominently induced tyrosine phosphorylation of a 78-kDa protein. This
effect was observed as early as 30 min and was sustained for at least 24 h. Treatment of cells with agents that abrogate uPA
receptor (uPAR) function, including neutralizing anti-uPAR antibody,
phosphatidylinositol-specific phospholipase C, or a selective
antagonist that blocks the association of uPA with uPAR (Å5
compound), all failed to prevent uPA-induced tyrosine phosphorylation.
B-428, an active site inhibitor of uPA activity, prevented the uPA
effect. Treatment of cells with hepatocyte growth factor, vascular
endothelial growth factor, or transforming growth factor-
, all of
which are known to be activated by a uPA-dependent pathway, did not
stimulate tyrosine phosphorylation of the 78-kDa protein. uPA induced
an increase in
[3H]thymidine
incorporation into DNA, and cell numbers were unaffected in the
presence of Å5. These results demonstrate that, in H-157 cells, uPA induces tyrosine phosphorylation of a 78-kDa protein via a
proteolysis-dependent but uPAR-independent mechanism. This novel
signaling pathway represents a putative mechanism by which uPA could
influence epithelial cell proliferation.
cell signaling; urokinase plasminogen activator
receptor; tyrosine phosphorylation
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INTRODUCTION |
THE UROKINASE PLASMINOGEN activator (uPA) is a serine
protease that plays an important role in physiological and pathological processes in which cell adhesion, migration, or tissue remodeling is
required (4, 8). It converts plasminogen to plasmin and degrades fibrin
and other protein components of the extracellular matrix (e.g.,
fibronectin). Through proteolysis, uPA also activates pro-hepatocyte
growth factor (HGF; see Ref. 14) and vascular endothelial growth factor
(VEGF; see Ref. 15), and, via generation of plasmin, it activates
transforming growth factor-
(TGF-
; see Ref. 18).
Beyond its role as a facilitator of extracellular proteolysis and
activator of growth factors, uPA is reported to generate intracellular
signals via both a high-affinity receptor (uPA receptor or
uPAR)-dependent (1, 2, 5-7, 13, 16, 22) or -independent (12)
mechanism. The high-affinity uPAR has been cloned (17); however, recent
evidence suggests that uPA may also interact with other membrane
proteins to initiate signaling (9, 12). uPAR is a heavily glycosylated
protein linked to the plasma membrane by a glycosylphosphatidylinositol
(GPI) moiety. uPAR lacks both cytoplasmic and transmembrane domains,
but binding of uPA to uPAR is known to activate several signaling
intermediates. These include tyrosine phosphorylation of a 38-kDa
protein in U-937 cells (6), activation of protein kinase C-
in WISH
cells (2), diacylglycerol formation in epidermal cells (5), activation
of Janus kinase-signal transducers and activators of transcription
(JAK-STAT) signaling in the epithelial cell line TCL-598
(13) and vascular smooth muscle cells (7), Src kinases in
THP-1 and monocytic cells (1, 16), and p125 focal adhesion kinase (FAK)
and p42/44 mitogen-activated protein (MAP) kinase in endothelial cells
(22). These signaling intermediates, either individually or
collectively, may contribute to uPA-mediated cellular responses,
including cellular proliferation.
We have previously demonstrated that H-157 (a human lung epithelial
cell line) and M9K (a human mesothelioma cell line) cells express high
levels of uPAR and that exposure of these cells to uPA induces their
proliferation (19, 20). The potent growth effects of uPA prompted us to
examine the signaling pathway initiated by uPA in these cells. In this
study, we demonstrate that uPA prominently induces tyrosine
phosphorylation of a 78-kDa protein via a uPAR-independent signaling
pathway. This requires uPA proteolytic activity and is not mediated by
secondary activation of growth factors such as VEGF, HGF, and TGF-
.
This novel pathway may contribute to uPA-induced cellular responses in
epithelial and mesothelioma cells, including cell migration, cellular
proliferation, or adhesion during tissue remodeling or metastasis.
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MATERIALS AND METHODS |
Materials. Tissue culture flasks, FBS,
and cell culture media were purchased from Life Technologies
(Gaithersburg, MD). Nitrocellulose membranes were purchased from
Amersham (Arlington Heights, IL); [3H]thymidine was
purchased from NEN (Boston, MA). Anti-phosphotyrosine antibody was
purchased from Upstate Biotechnology (Lake Placid, NY), and goat
anti-mouse IgG coupled to horseradish peroxidase was purchased from
Bio-Rad (Melville, NY). The two-chain high-molecular-mass uPA was a
generous gift from Dr. Jack Henkin, Abbott Laboratories (Abbott Park,
IL). The low-molecular-mass uPA, amino-terminal fragment (ATF), and
neutralizing anti-uPAR antibody were obtained from American Diagnostics
(Greenwich, CT). HGF, VEGF, and TGF-
were purchased from R&D Systems
(Minneapolis, MN); Å5 compound was a gift from Dr. A. Mazar
(Ångstrom Pharmaceuticals, San Diego, CA); all other
chemicals were purchased from Sigma (St. Louis, MO).
Cell culture. H-157, a cell line
derived from human squamous cell lung carcinoma, cells were obtained
from American Type Culture Collection (Manassas, VA); M9K mesothelioma
cells were provided by Dr. Brenda Gerwin (National Cancer Institute,
Bethesda, MD). Both cell types were grown in RPMI medium containing
10% FBS for 12-24 h and were serum starved for 12 h in RPMI
medium before the addition of uPA or other agents.
Total cell extraction and Western
blots. Cells were treated with various agents for
indicated times and were washed in PBS. Cells were scraped and lysed in
buffer (10 mM Tris-Cl, pH 7.4, 100 mM NaCl, 1% Triton X-100, 15%
glycerol, 1 mM sodium orthovanadate, and 10 µg/ml aprotinin and
leupeptin). Twenty-five micrograms of protein were run on an 8%
polyacrylamide gel, transferred to nitrocellulose, and probed with
anti-phosphotyrosine antibodies. Alternatively, 200 µg of the samples
were immunoprecipitated with respective antibodies, and complexes were
run on an 8% polyacrylamide gel and immunoblotted with
anti-phosphotyrosine antibodies. Tyrosine-phosphorylated proteins were
detected by enhanced chemiluminescence.
Subcellular fractionation. Cells were
scraped and washed with PBS two times and swelled in hypotonic buffer
(10 mM Tris-Cl, pH 7.5, 1 mM
MgCl2, 10 mM KCl, 1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 3 µg/ml
aprotinin). The cells were then homogenized with a Dounce
homogenizer and centrifuged at 600 g
for 10 min at 4°C. The resultant supernatant was centrifuged at
100,000 g for 1 h. The pellets were
referred to as membrane fraction, and the supernatant was referred to
as the cytosolic fraction. The plasma membrane was isolated as
previously described (11). For this, after thorough washing with PBS,
the cells were homogenized in 0.3 M sucrose at 4°C in a Dounce
homogenizer. After sedimentation at 1,500 g for 15 min, the homogenized
membranes were resuspended in 1.56 M sucrose, overlaid with 0.3 M
sucrose, and centrifuged at 105,000 g
for 2 h. The plasma membrane fraction was collected from the
interface and was stored at
80°C until used.
[3H]thymidine incorporation
assay.
H-157 cells were plated on 24-well plates and were grown to 80%
confluence (19). The cells were then serum starved for 24 h and treated
with various agents and uPA for 36 h. Cells were then pulse labeled
with 1 µCi/ml
[3H]thymidine. The
cells were then fixed with 1 ml of 15% TCA. This was followed by
10-min washes with 10% TCA, first at 4°C and then at room
temperature. Cells were then dissolved in 1 N NaOH and placed in
Ready-safe scintillation fluid, and radioactivity was counted using a
scintillation counter. To determine cell numbers, the cells were
detached and counted using a hemacytometer.
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RESULTS |
uPA induces tyrosine phosphorylation of a 78-kDa
protein in H-157 and M9K cells. Signal transduction
mechanisms via membrane receptors commonly involve protein tyrosine
phosphorylation. To elucidate uPA-induced signal transduction
mechanisms in H-157 cells, we treated cells with uPA (the
high-molecular-mass, two-chain form) for various lengths of time (from
15 min to 24 h), and tyrosine-phosphorylated proteins were visualized
in Western blots using anti-phosphotyrosine antibody. Figure
1A
demonstrates that, in these cells, uPA prominently induces tyrosine
phosphorylation of a 78-kDa protein. This was detected as early as 15 min and was sustained for at least 24 h. In a few blots (2 out of 7),
we observed weak tyrosine phosphorylation of two additional proteins of
molecular masses 75 and 58 kDa (Fig. 1A). The reason for the weaker
induction of these proteins by uPA is not clear at this stage; however,
it may be related to their expression levels at the time of cellular
isolation. It is also possible that differences in the levels of
secretion of endogenous uPA by these cells (19) at the time of
isolation may cause a variation in the ability of exogenously added uPA to induce signaling. Because tyrosine phosphorylation of the 78-kDa protein was prominently detected in all experiments, we sought to
characterize the activation of this protein by uPA in the present study. We also observed that uPA induced tyrosine phosphorylation of a
78-kDa protein in the mesothelial cell line M9K (Fig.
1B). Using H-157 cells for more
detailed analyses, we next characterized uPA-induced tyrosine
phosphorylation of the 78-kDa protein. Figure 1C demonstrates that the effect of uPA
is concentration dependent and is observed at a concentration as low as
0.1 nM. We also sought to determine if uPA-mediated tyrosine
phosphorylation could be blocked by herbimycin A, a tyrosine kinase
inhibitor. Figure 1D demonstrates that
herbimycin A inhibits uPA-mediated tyrosine phosphorylation. Similar
results were obtained in M9K cells (data not shown). These results
demonstrate that, in H-157 and M9K cells, uPA, via activation of a
signal transduction pathway, induces tyrosine phosphorylation of a
78-kDa protein.

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Fig. 1.
Urokinase plasminogen activator (uPA)-induced tyrosine phosphorylation
of proteins in H-157 (A) and M9K
(B) cells. Serum-starved cells were
left untreated or were treated with uPA (10 nM) for different periods
of time, and total cell extracts were prepared and immunoblotted with
anti-phosphotyrosine antibody. The position of the 78-kDa protein
tyrosine phosphorylated by uPA and positions of tyrosine-phosphorylated
75- and 58-kDa proteins are shown by arrows.
C: concentration-dependent effect of
uPA on tyrosine phosphorylation of the 78-kDa protein. Serum-starved
cells were treated with different concentrations of uPA for 3 h. Total
cell lysates were then prepared and immunoblotted with
anti-phosphotyrosine antibody. D:
effect of herbimycin A on uPA-induced tyrosine phosphorylation.
Serum-starved cells were left untreated (lane
1) or were treated with uPA (10 nM;
lane 2), herbimycin A (1 µM;
lane 3), or first with herbimycin A
(1 µM) for 30 min and then with uPA (10 nM; lane
4). After 3 h of treatment with uPA, total cell
lysates were prepared and immunoblotted with anti-phosphotyrosine
antibody. Blots are representative of the findings of 3 independent
experiments.
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The tyrosine-phosphorylated 78-kDa protein associates
with intracellular membrane fractions in uPA-treated
cells. To localize the 78-kDa tyrosine-phosphorylated
protein in uPA-stimulated H-157 cells, we isolated particulate
(membrane), cytoplasmic, and nuclear fractions. Proteins from these
fractions were analyzed in Western blots by probing with
anti-phosphotyrosine antibody. Figure
2A demonstrates that the 78-kDa
protein is present in the particulate fraction in uPA-stimulated cells
(lane 2). It is not detectable in
cytoplasmic (Fig. 2A, lane 4) or
nuclear (Fig. 2A, lane 6) fractions.
Further analysis showed that the uPA-induced protein is not present in
plasma membrane fractions (Fig. 2B).

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Fig. 2.
Localization of the 78-kDa protein in the particulate fraction.
A: serum-starved H-157 cells were left
untreated or were treated with uPA (10 nM) for 4 h. After treatment
with uPA, cytoplasmic, particulate, nuclear, and plasma membrane
fractions were prepared and solubilized, and proteins were
immunoblotted with anti-phosphotyrosine antibody.
B: as a positive control for plasma
membrane preparation, we used cells treated with epidermal growth
factor (EGF; 10 ng/ml) for 10 min. Experiments are representative of 4 separate experiments. EGFR, EGF receptor.
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Enzymatic activity is required for uPA-mediated
tyrosine phosphorylation. Depending upon the cell type
analyzed, uPA-mediated signaling effects are reported to occur by both
proteolysis-dependent (10) or -independent (16, 22) mechanisms. For
example, it was shown in THP-1 cells that the addition of the ATF of
uPA was sufficient to activate
p56/p59hck kinases and promote
their association with uPAR (16). Interestingly, the 33-kDa
lower-molecular-mass form of uPA was ineffective. The uPA ATF does not
contain enzyme activity; however, it binds to uPAR with high affinity
(4). The low-molecular-mass form of uPA does not bind uPAR; however, it
possesses enzyme activity. To establish if enzyme activity is required
for uPA-induced tyrosine phosphorylation in H-157 cells, we tested the
ability of ATF and low-molecular-mass uPA to mimic the effects of
high-molecular-mass two-chain uPA. Figure 3
demonstrates that ATF failed to induce tyrosine phosphorylation of the
78-kDa protein (lane 2), whereas low-molecular-mass uPA induced this phosphorylation reaction
(lane 5). Preexposure of cells to
ATF (which binds uPAR) did not prevent the ability of uPA to induce
tyrosine phosphorylation (Fig. 3, lane
4). Figure 4 demonstrates
that B-428, an active site inhibitor of uPA activity (23), completely
abolished the uPA effect. These results demonstrate that uPA enzymatic
activity is required for tyrosine phosphorylation of the 78-kDa protein
in H-157 cells.

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Fig. 3.
Effect of amino-terminal fragment (ATF) and small-molecular-mass uPA on
induction of tyrosine phosphorylation. Serum-starved H-157 cells were
left untreated (lane 1) or were
treated with ATF (10 nM; lane 2),
uPA (10 nM; lane 3), ATF (10 nM) + uPA (10 nM; lane 4), or
low-molecular-mass uPA (10 nM; lane
5). ATF was added 30 min before the addition of uPA
for 4 h. Cell lysates were prepared, and proteins were immunoblotted
with anti-phosphotyrosine antibody. Blots are representative of 3 separate experiments.
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Fig. 4.
Effect of B-428 on uPA-induced tyrosine phosphorylation. Serum-starved
H-157 cells were left untreated (lane
1) or were treated with uPA (10 nM;
lane 2), B-428 (10 µM;
lane 3), or B-428 (10 µM) + uPA
(10 nM; lane 4). After 4 h of
treatment, cell lysates were prepared, and proteins were immunoblotted
with anti-phosphotyrosine antibody. Blots are representative of the
findings of 3 separate experiments.
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uPA-induced tyrosine phosphorylation occurs via a
uPAR-independent mechanism. The ability of uPA to
stimulate tyrosine phosphorylation in cells preexposed to ATF (Fig. 3)
suggests that the response may occur via a uPAR-independent mechanism.
We used three independent experimental approaches to establish the
involvement of uPAR in uPA-mediated tyrosine phosphorylation in H-157
cells. First, we exposed the cells to neutralizing anti-uPAR antibody
and then sought to determine if uPA would induce tyrosine
phosphorylation of the 78-kDa protein under these conditions. Figure
5A
demonstrates that the neutralizing antibody did not block the uPA
effect (lanes 2 and
4), suggesting that a
uPAR-independent signaling pathway may be involved in the induction of
tyrosine phosphorylation of the 78-kDa protein. Densitometric analysis
of Fig. 5A showed that treatment with
anti-uPAR antibody slightly increased (15%) uPA-mediated tyrosine
phosphorylation of the 78-kDa protein. The reason for this increased
uPA-induced response in the presence of anti-uPAR antibody is not
clear. However, one possibility is that binding of uPAR antibody to
uPAR may increase the total availability of uPA for uPAR-independent
signaling. In the second approach, we used the recently developed
Å5 compound (a uPAR antagonist) to determine if it would
block uPA-induced tyrosine phosphorylation at different time points (30 min to 3 h). Å5 is an 11-mer cyclic peptide that inhibits the
binding of human uPA to uPAR with an IC50 of 11 nM (Dr. A. Mazar,
personal communication). Exposure of cells to Å5 failed to
block the uPA effect at all of the time points examined (Fig.
5B). This also suggests that
localization of uPA activity by uPAR is not required for generating the
signals. In a third approach, we stripped uPAR from cells by treating
with phosphatidylinositol-phospholipase C (PI-PLC) and then tested to
see if uPA would stimulate tyrosine phosphorylation of the 78-kDa
protein. PI-PLC cleaves GPI-linked proteins from the cell surface,
including uPAR. We observed that, in PI-PLC- treated cells, uPA still
induced tyrosine phosphorylation (Fig.
5C, lane 4). In these cells, PI-PLC completely removed uPAR
from the cell surface (Fig. 5D,
positive control), providing further evidence that the effect is not
mediated by the association of uPA with uPAR at the cell surface.

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Fig. 5.
Effect of anti-uPA receptor (uPAR) antibody
(A), Å5 compound
(B), and
phosphatidylinositol-phospholipase C (PI-PLC;
C) on uPA-induced tyrosine
phosphorylation. In A, H-157 cells
were left untreated (lane 1) or were
treated with uPA (lane 2), anti-uPAR
antibody (lane 3), or first with
anti-uPAR antibody (1 µg/ml) and then with uPA (lane
4). Anti-uPAR antibody was added 30 min before the
addition of uPA for 4 h. Total cell lysates were prepared, and proteins
were immunoblotted with anti-phosphotyrosine (anti-P-tyr) antibody.
B: H-157 cells were left untreated
(lane 1) or were treated with uPA
for different time points (lanes
2-5) or first with Å5 for 30 min and
then with uPA over the same time intervals (lanes
7-10). Lane 6 represents protein from cells treated with Å5 for 3 h and 30 min (control). Total cell lysates were prepared and immunoblotted with
anti-phosphotyrosine antibody. C:
cells were left untreated (lane 1)
or were treated with PI-PLC (PLC; 10 U/ml; lane
2), uPA (10 nM; lane
3), or first with PLC for 1 h and then with uPA
(lane 4). After 4 h of treatment
with uPA, proteins from the cell lysates were immunoblotted with
anti-phosphotyrosine antibody. D:
samples from C were immunoblotted with
anti-uPAR (human) antibody. uPAR band appears heterogeneous due to
heavy glycosylation (4). Blots are representative of the findings of 3 separate experiments.
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uPA-induced signaling is not mediated by HGF, VEGF, or
TGF-
. uPA has been reported to convert
the inactive pro-HGF form to the mature, active form of HGF in MRC-5
fibroblasts (14). In CHO-K1 cells, uPA was demonstrated to convert the
50-kDa nonmitogenic form of VEGF to the mature 38-kDa mitogenic form by
proteolytic action (15). In another study, it was shown that plasmin
generated by uPA activity in the extracellular matrix can convert
latent TGF-
to the active species in cocultures of endothelial and
smooth muscle cells (18). Based upon these observations, we next
considered the possibility that uPA may induce phosphorylation of the
78-kDa protein via a secondary activation of HGF, VEGF, or TGF-
in
the extracellular matrix. To determine if this was the case, we tested the ability of these growth factors to stimulate tyrosine
phosphorylation of the 78-kDa protein in H-157 cells. Figure
6 demonstrates that only uPA, but not HGF,
VEGF, or TGF-
, induces tyrosine phosphorylation in these cells. We
also investigated the possibility that uPA mediates its effect through
the generation of plasmin. To examine this possibility, we performed
experiments to see if uPA would induce tyrosine phosphorylation of the
78-kDa protein in the presence of the protease inhibitor aprotinin.
Figure 7A
demonstrates that aprotinin did not block the ability of uPA to induce
tyrosine phosphorylation. Figure 7B
demonstrates that exposure of cells to plasmin did not induce tyrosine
phosphorylation of the 78-kDa protein (lane
3). Also, treatment of cells with another protease, namely
-thrombin, failed to mimic the uPA effect
(lane 4). These observations
demonstrate that tyrosine phosphorylation of the 78-kDa protein is
specifically mediated by uPA and that plasmin is not involved in
uPA-mediated signaling effects in H-157 cells.

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Fig. 6.
Effect of transforming growth factor (TGF)- , vascular endothelial
growth factor (VEGF), and hepatocyte growth factor (HGF) on protein
tyrosine phosphorylation. In A, cells
were left untreated (lane 1) or were
treated with uPA (lane 2) for 4 h or
TGF- (10 ng/ml; lane 3), VEGF (20 ng/ml) (lane 4), or HGF (20 ng/ml;
lane 5) for 20 min. Cell lysates
were prepared and immunoblotted with anti-phosphotyrosine antibody.
Blots are representative of the findings of 3 separate experiments.
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Fig. 7.
uPA-mediated induction of tyrosine phosphorylation is not mediated via
generation of plasmin. A: H-157 cells
were left untreated (lane 1) or were
treated with uPA (10 nM) for 1 h (lane
2), aprotinin (20 µg/ml) for 1 h
(lane 3), or aprotinin and uPA (10 ng/ml) for 1 h (lane 4). Cell
lysates were prepared and immunoblotted with anti-phosphotyrosine
antibody. B: serum-starved cells were
left untreated or were treated with uPA (lane
2), plasmin (lane
3), or -thrombin (lane
4). Total cell lysates were prepared and
immunoblotted with anti-phosphotyrosine antibody. Blots are
representative of the findings of 3 separate experiments.
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Å5 compound does not block the uPA-mediated increase in
[3H]thymidine incorporation and
cell number.
To determine if a uPAR-independent signaling pathway in H-157 cells
potentially contributes to the uPA-induced proliferative effects, we
measured [3H]thymidine
incorporation in DNA in the presence of uPA or Å5 plus uPA.
Table 1 demonstrates that uPA induces an
~1.6-fold stimulation of
[3H]thymidine
incorporation in H-157 cells. Å5 did not block the uPA-induced increase in
[3H]thymidine
incorporation. In addition, Å5 did not block the ability of
uPA to increase the cell number (~2-fold), suggesting that
uPA-induced cellular proliferation does not require binding of uPA to
uPAR.
A possible role for the 78-kDa protein in uPA-induced
increase in cell proliferation. To establish a
correlation between uPA-induced tyrosine phosphorylation of the 78-kDa
protein and cell proliferation, we investigated if inhibition of
tyrosine phosphorylation with herbimycin A would prevent the
uPA-mediated increase in
[3H]thymidine
incorporation and cell number. Table 2
demonstrates that herbimycin A inhibits the uPA-induced increase in DNA
synthesis and cell number. Pretreatment of the cells with vanadate (a
tyrosine phosphatase inhibitor) had no effect on the uPA-induced
response. Pretreatment of cells with B-428 (inhibitor of uPA)
completely blocked uPA-induced DNA synthesis and cell number. This
suggests that uPA enzyme activity is required for uPA-induced cellular responses. Also, treatment with plasmin did not induce DNA synthesis and cell number. These results in aggregate suggest that the 78-kDa protein may be involved in uPA-mediated effects on DNA synthesis and
proliferation in H-157 cells.
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Table 2.
Effect of B-428, herbimycin A, and vanadate on uPA-induced uptake of
[3H]thymidine and cell proliferation
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DISCUSSION |
In this study, we have identified a proteolysis-dependent but
uPAR-independent signaling pathway mediated by uPA in H-157 and M9K
cells. Activation of this pathway involves tyrosine kinases and results
in tyrosine phosphorylation of a 78-kDa protein. This protein
associates with the intracellular membrane fraction in uPA-stimulated
cells. Its absence in the nucleus suggests that it is not a
transcription factor. Although the identity of this protein is not
known at this stage, it may represent a kinase or a structural protein
phosphorylated by a kinase during uPA-induced cellular signaling. This
uPAR-independent signaling pathway represents a putative mechanism by
which uPA could influence cellular responses in epithelial and
mesothelioma cell types.
Studies performed in other cell types have demonstrated the involvement
of uPAR in uPA-mediated signaling. For example, activation of FAK and
MAP kinases in cultured endothelial cells is clearly a uPAR-dependent
process (22). Similarly, activation of a 38-kDa protein in U-937 cells
occurs via uPAR (6). In both of these cases, signaling by uPA was
abolished when cells were treated with PI-PLC. Other studies also
implicate uPAR in signal transduction processes. For example, one
report demonstrated the association of uPAR with protein kinase C-
and cytokeratin 8 and 18 in uPA-treated WISH cells (2). uPAR also was
shown to associate with
2-integrins and members of the
Src kinase family in uPA-treated human monocytes / THP-1
cells (1, 16). In aortic vascular smooth muscle cells, uPAR was shown
to associate with JAK1, Tyk2, and Src tyrosine kinases (7). In the
kidney epithelial tumor cell line TCL-598, an association of uPAR with
the signal transducer protein gp130 and JAK1 has been demonstrated
(13). In these cells, the assembly of uPAR with tyrosine/serine kinases
and membrane-spanning
2-integrins in one receptor
complex indicates functional cooperation between these molecules.
However, it is to be noted that the uPA-induced signaling effects that
have been reported are not observed in all cell lines examined and, in
fact, are quite diverse (4). The reason for such differential
activation of signaling intermediates in different cell types is not
clear. However, this phenomenon may be related to the expression of
different levels of signaling proteins in each cell type. Our current
demonstration of a novel pathway of uPAR-independent signaling in H-157
and M9K cells adds further diversity to the previously described
mechanisms by which uPA-mediated signaling can be accomplished.
The mechanism by which uPA induces tyrosine phosphorylation of the
78-kDa protein in H-157 and M9K cells is not known at this time.
Clearly, it is not due to the secondary activation of HGF, VEGF, and
TGF-
. The response is probably not mediated via generation of
plasmin because exposure of cells to aprotinin did not block tyrosine
phosphorylation of the 78-kDa protein. Also, treatment of cells with
plasmin failed to induce phosphorylation of the 78-kDa protein. One
possibility is that uPA, through proteolysis, may activate a
transmembrane protein to initiate this signaling pathway in H-157
cells. In this context, it is important to note that uPA was shown to
interact with a novel, high-affinity binding protein in platelet
membranes (9). Another recent study indicates that the mitogenic
effects of uPA on IF6 and M14 melanoma cells are independent of
high-affinity binding of uPA to the uPAR (12). In that study, it was
shown that blocking the proteolytic activity decreased the mitogenic
effect by 30%; however, blocking the interactions of uPA with uPAR,
using a specific monoclonal antibody, did not alter the mitogenic
effect induced by uPA. Thus, in melanoma cells, uPA appears to bind to
an unidentified membrane-associated protein to mediate signal
transduction. Although a similar scenario may be operative in H-157
cells, the delineation of the exact mechanisms by which uPA initiates
signal transduction in these cells requires further study.
In immunoprecipitation experiments, antibodies against protein kinase
C-
, protein kinase C-
, protein tyrosine phosphatase (PTP)-1C,
PTP-1D, pp60src substrate, and
phosphatidylinositol 3-kinase (p85) all failed to recognize the 78-kDa
protein (unpublished results). Additional studies are required to
identify this protein and to determine how it affects uPA-induced
cellular responses in H-157 and M9K cells. However, we have developed
evidence to suggest that uPAR-independent signaling has the potential
to contribute to uPA-induced cellular responses. In support of this
idea, we observed that uPA addition to H-157 cells induced an
~1.6-fold increase in
[3H]thymidine
incorporation and a comparable 2-fold increase in cell number.
Preexposure of cells to the uPAR antagonist Å5 did not
prevent this uPA-induced response, suggesting that uPA-induced cellular
proliferation does not require binding of uPA to uPAR. Inhibition of
tyrosine phosphorylation of the 78-kDa protein with herbimycin A
decreased the uPA-induced DNA synthesis and cell number. This
observation establishes a link between activation of the 78-kDa protein
and cell proliferation in uPA-treated cells. Direct confirmation of the
role of this 78-kDa protein in uPA-induced cellular proliferation will
require its future identification.
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ACKNOWLEDGEMENTS |
We thank Dr. Andrew Mazar, Ångstrom Pharmaceuticals, San
Diego, CA, for providing Å5 compound; Dr. Jack Henkin, Abbott
Laboratories, Abbott Park, IL, for providing the two-chain
high-molecular-mass urokinase plasminogen activator; and Dr. Brenda
Gerwin, National Cancer Institute, Bethesda, MD, for providing M9K cells.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-45018 (to S. Idell) and by American Heart Association (National Center) Grant no. 96006630 (to G. J. Bhat).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. J. Bhat,
Dept. of Specialty Care Services, The Univ. of Texas Health Center at
Tyler, 11937 US Hwy. 271, Tyler, TX 75708 (E-mail:
jbgunaje{at}uthct.edu).
Received 24 December 1998; accepted in final form 30 March 1999.
 |
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