From the Department of Specialty Care Services, the
University of Texas Health Center, Tyler, Texas 75708 and the
¶ Department of Pathology and Laboratory Medicine, the University
of Pennsylvania, Philadelphia, Pennsylvania 19104
Received for publication, July 24, 2002, and in revised form, March 11, 2003
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ABSTRACT |
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The plasminogen/plasmin system,
urokinase-type plasminogen activator (uPA), its receptor (uPAR), and
its inhibitor (PAI-1), influence extracellular proteolysis and cell
migration in lung injury or neoplasia. In this study, we sought to
determine whether tcuPA (two chain uPA) alters expression of its major
inhibitor PAI-1 in lung epithelial cells. The expression of PAI-1 was
evaluated at the protein and mRNA level by Western blot,
immunoprecipitation, and Northern blot analyses. We found that tcuPA
treatment enhanced PAI-1 protein and mRNA expression in Beas2B lung
epithelial cells in a time- and concentration-dependent
manner. The tcuPA-mediated induction of PAI-1 involves
post-transcriptional control involving stabilization of PAI-1 mRNA.
Inactivation of the catalytic activity of tcuPA had little effect on
PAI-1 induction and the activity of the isolated amino-terminal
fragment was comparable with full-length single- or two-chain uPA. In
contrast, deletion of either the uPA receptor binding growth factor
domain or kringle domain (^kringle) from full-length
single chain uPA markedly attenuated the induction of PAI-1. Induction
of PAI-1 by exposure of lung epithelial cells to uPA is a newly
recognized pathway by which PAI-1 could regulate local fibrinolysis and
urokinase-dependent cellular responses in the setting of
lung inflammation or neoplasia.
Proteolytic enzymes, including urokinase
(uPA)1 and
metalloproteinases, have been implicated in the pathogenesis of lung
inflammation and the growth of lung tumors. These proteases facilitate
remodeling of the transitional stroma via the breakdown of basement
membranes and extracellular matrix proteins, including fibrin (1-3).
Plasminogen activation can be mediated by urokinase-type (uPA) and
tissue-type plasminogen activators. The former is mainly involved in
extravascular proteolysis and is implicated in stromal remodeling and
neoplasia. Plasminogen activator inhibitor type-1 (PAI-1), a member of
the serpin family of serine protease inhibitors, binds and irreversibly inactivates both of these plasminogen activators (4), thereby regulating expression of plasminogen activator activity.
PAI-1 also modulates cell adhesion to extracellular matrix both by
preventing cell detachment as a consequence of excess plasmin formation
(5), but also through its interaction with vitronectin (1, 6, 7). PAI-1
binds to vitronectin exposed at sites of vascular interruption
(8). Binding of PAI-1 to vitronectin stabilizes its activity (9) and
alters its proteolytic specificity (10, 11). In turn, PAI-1 exposes but
transiently occludes the integrin binding site in vitronectin (12) and
inhibits uPA-induced uPAR-mediated adhesion (13). Binding of uPA to
PAI-1 lowers its affinity for vitronectin, restoring integrin binding,
while promoting the affinity of uPA for the low density
lipoprotein-related protein (14), which clears inactive complexes and
recycles unoccupied uPAR to the cell surface (15, 16). Thus, orderly
cell migration along the provisional matrix requires a coordinated
interaction between the expression and localization of uPA, PAI-1, and
their (sub)cellular binding sites. Theoretically, both untoward or
premature proteolysis, or excessive or ineffective development of
adhesion forces, could retard cell migration along a provisional matrix.
It is then not surprising that pathologic overexpression of PAI-1 has
been linked to a wide range of inflammatory and neoplastic lung
diseases (4, 17). PAI-1 is secreted by epithelial cells of many normal
tissues, including the lung
(18).2 A defect of
uPA-related fibrinolytic activity, in large part related to
overexpression of PAI-1, has been associated with lung dysfunction in
acute respiratory distress syndrome and interstitial lung
diseases (20, 21). There is also extensive and growing evidence for
involvement of PAI-1 in cell migration, tumor invasion, and metastasis,
where its mechanism of action is more complex (17). Growth of certain
tumors is attenuated by PAI-1 (5, 22). On the other hand, PAI-1 is
required for tumor-induced angiogenesis in other experimental models
and high levels of PAI-1, as well as uPA, and uPAR in lung tumor tissue
correlate with poor prognosis (23, 24). The mechanism underlying these
seeming opposing roles for PAI-1 is unexplained, but points to the
importance of factors that regulate the timing and level of PAI-1
expression as it relates to its dual anti-proteolytic and anti-adhesive activities.
Expression of PAI-1 is modulated by diverse stimuli including hormones,
growth factors, endotoxin, glucocorticoids, and cytokines, acting at
either the transcriptional or post-transcriptional levels (25-30).
However, to our knowledge, the possibility that uPA itself regulates
the expression of its inhibitor has not been studied. Overexpression of
uPA, uPAR, or PAI-1 by tumor cells (31-34) as well as autoregulatory
feedback in normal epithelial cells (35, 36) combined with the finding
that uPA stimulates proliferation of varied cell types (37-42) all
suggested to us the hypothesis that uPA might regulate PAI-1 expression
in lung epithelial cells.
Also, the mechanism by which uPA mediates transcellular signaling
remains unclear. uPA binds with high affinity through its growth factor
domain to its cellular receptor (uPAR). However, the fact that uPAR is
a glycolipid-anchored protein suggests that uPA may signal through
other portions of the molecule directly or through conformational
changes in uPAR via integrin ligands, integrins, or other transmembrane
adapters (13, 31). We, and others, have identified a signal-transducing
region in the kringle of uPA that stimulates smooth muscle cell
contraction and migration (43, 44), but the effect of this domain on
epithelial cells or on protein synthesis has not been explored.
Moreover, uPA but not uPAR is required for smooth muscle cell migration
and neointimal growth in vivo (45, 46). In this paper, we
describe a new paradigm through which PAI-1 expression by lung
epithelial cells is regulated by uPA. This pathway could influence
alveolar PAI-1 expression and thereby modulate uPA-mediated responses
of lung epithelial cells in lung injury or neoplasia.
Materials--
Culture media, penicillin, streptomycin, and
fetal calf serum were purchased from Invitrogen; tissue culture
plastics were from BD Bioscience. Cell Cultures--
Human bronchial epithelial cells (Beas2B)
were obtained from the ATCC. These cells were maintained in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 1%
glutamine, and 1% antibiotics as previously described (42). Primary
cultures of human small airway epithelial cells were obtained from
Clonetics (San Diego, CA) and cultured in the same media, as previously
described (48).
Total Protein Extraction and Western Blotting--
Cells were
grown to confluence and were serum starved overnight in RPMI 1640 media. The cells were then treated with or without recombinant human
two-chain uPA or other agents for selected times in serum-free media
supplemented with 0.5% BSA. Following these treatments, the cells were
suspended in lysis buffer (10 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Triton X-100, 15% glycerol, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
and 3-10 µg of aprotinin per 100 ml). The cell lysates were prepared
using three cycles of freezing and thawing. Proteins from Beas2B cell
lysates (50 µg) 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 anti-PAI-1 monoclonal antibody in the same buffer at
4 °C, washed, and PAI-1-immunoreactive proteins were detected by
enhanced chemiluminescence. The membranes were stripped with
Overexpression of uPA-Transfection of Beas2B Cells with uPA
cDNA--
uPA cDNA (49) was subcloned into the eukaryotic
expression vector pRc/CMV2 (Invitrogen) containing the CMV promoter at
HindIII/NotI sites. The orientations and
sequences were confirmed by nucleotide sequencing. Beas2B cells were
transfected with the prepared chimeric plasmid constructs using
LipofectAMINE (Invitrogen). Stable cell lines were created by
culturing Beas2B cells in neomycin-containing media for 3 months. Cells
carrying plasmid DNA that survived neomycin treatment were scrapped
from 6-well plates and grown in T75 flasks, and the presence of plasmid
DNA was confirmed by PCR using specific primers. The overexpression of
uPA by cDNA-transfected cells was confirmed by Western blotting of
Beas2B cell lysates as well as conditioned media using a uPA monoclonal
antibody. The effect of endogenous uPA overexpression on PAI-1
induction was then measured by Western blot and immunoprecipitation, as
described above (35).
Plasmid Construction--
Plasmid PAI-1/pGEM was obtained by
polymerase chain reaction amplification of a human lung cDNA
library. The cDNA corresponding to the coding region (0.5 kb) was
subcloned to pGEMR-T vector (Promega) and the sequence of
the clones was confirmed by nucleotide sequencing. The PAI-1 insert was
released by NcoI and PstI, purified on 1%
agarose gels, extracted with phenol/chloroform, and used as a cDNA
probe for Northern blotting.
Random Priming of PAI-1 cDNA--
The cDNA template of
PAI-1 was released with NcoI/PstI, purified on
1% agarose gels, and labeled with [32P]dCTP using a
Rediprime labeling kit (Promega). Passage through a Sephadex G-25
column removed unincorporated radioactivity. The specific activity of
the product was 6-7 × 108 cpm/µg.
Nuclear Run-on Transcription Activation Assay--
Confluent
cells grown in two T182 flasks were serum-starved overnight in RPMI-BSA
media. The cells were later treated with PBS or recombinant human
two-chain uPA (1 µg/ml) for 12 h at 37 °C and analyzed using
the transcription activation assay as described earlier (35).
PAI-1 mRNA Assessment by Northern Blotting--
A Northern
blotting assay was used to assess the steady-state level of PAI-1
mRNA. Confluent Beas2B cells were serum-starved overnight in
RPMI-BSA media, and treated with two-chain human recombinant uPA for
varying times (0-24 h) in the same media. Total RNA was isolated using
TRI reagent, RNA (20 µg) was separated 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 PAI-1 cDNA probes (1 ng/ml) labeled to ~6-7 × 108 cpm/µg of DNA overnight. After hybridization, the
filters were washed twice for 15 min at 65 °C, with, respectively,
2 × SSC, 1% SDS; 1 × SSC, 1% SDS; and 0.1% SSC, 1% SDS.
The membranes were exposed to x-ray film at
In separate experiments, Beas2B cells were treated with PBS or uPA for
24 h and then with cycloheximide to inhibit ongoing translation.
The stability of the PAI-1 protein expressed under these conditions was
then measured. PAI-1 protein concentrations were determined at varying
time periods (0-24 h) by a combination of metabolic labeling and
immunoprecipitation as described above.
Time-dependent Induction of PAI-1 by TcuPA--
We
previously found that lung carcinoma-derived cells differentially
express PAI-1 in vitro (18). Based on this observation, we
first sought to determine whether tcuPA induces PAI-1 expression in
Beas2B cells, a non-malignant lung epithelial cell line. We treated the
cells with the high molecular weight, two-chain form of uPA (tcuPA) for
varying lengths (0-24 h) of time. Total proteins from cell lysates
were used for Western blotting using an anti-PAI-1 antibody. The data
in Fig. 1a demonstrate that
tcuPA induces PAI-1 expression in Beas2B cells in a
time-dependent manner. The induction is detectable by
3 h after the addition of tcuPA and maintained for up to 24 h
(Fig. 1b). Identical tcuPA treatment also induced PAI-1
expression in primary small airway epithelial cells (Fig. 1c).
To see if contaminating lipopolysaccharides (LPS) in the high molecular
weight uPA (tcuPA) preparation we used might be responsible for the
observed activity, we first measured the LPS content by the Limulus
Amebocyte lysate enzyme-linked immunosorbent assay method. We found
that this tcuPA preparation contains negligible amounts (~1 pg/ml) of
LPS. To determine whether the LPS content of the preparation could
account for the induction of PAI-1, we next treated Beas2B cells with
this (1 pg/ml) as well as a 10-fold higher concentrations (10 pg/ml) of
LPS for up to 12 h. We then measured PAI-1 expression by Western
blotting as described above. We found that these concentrations of LPS
failed to induce PAI-1 expression (data not shown) in this cell type,
indicating that the induction of PAI-1 by the tcuPA preparation we used
could not be attributed to LPS contamination.
Induction of PAI-1 by Endogenous uPA--
We also prepared
uPA-overproducing 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 PAI-1 expression of the
stable cell lines by Western blotting. As shown in Fig.
2a, Beas2B cells transfected
with uPA cDNA expressed relatively large amounts of PAI-1 in
comparison to vector-transfected or non-transfected control cells. We
also found a comparable increase in PAI-1 protein expression by uPA
cDNA-transfected cells compared with vector cDNA or
non-transfected control cells when the cells were metabolically labeled
with [35S]methionine and the proteins immunoprecipitated
using an anti-PAI-1 monoclonal antibody (Fig. 2b).
Induction of PAI-1 mRNA Expression by TcuPA in Lung Epithelial
Cells--
To evaluate the possibility that part of this increase is
because of internalization of uPA-PAI-1 complexes by Beas2B cells, we
examined the effect of tcuPA on expression of PAI-1 mRNA. We measured the steady-state levels of PAI-1 mRNA in tcuPA-treated Beas2B cells by Northern blotting using a PAI-1 cDNA probe. As shown in Fig. 3, tcuPA induces PAI-1
mRNA in Beas2B cells, with the induction observed as early as
3 h after treatment. Maximum accumulation of PAI-1 mRNA is
achieved within 3 h. Whereas tcuPA induces both 3.2- and 2.4-kb
components of PAI-1 mRNA, induction of the 3.2-kb moiety was found
to be greater. These data confirm that tcuPA increases PAI-1 mRNA
expression and affects increased protein expression by Beas2B cells, as
determined by Western blotting. The level of PAI-1 mRNA was
quantified by densitometric scanning and normalized against Evaluation of Transcriptional Activation of PAI-1 by
TcuPA--
Nuclear run-on experiments indicated that addition of tcuPA
to Beas2B cells over 12 h did not induce PAI-1 mRNA (Fig.
4a).
Evaluation of the Effect of TcuPA on PAI-1 mRNA
Stability--
Because tcuPA did not enhance the rate of PAI-1
transcription, we next sought to determine whether tcuPA influenced the
stability of PAI-1 mRNA. To address this possibility, we treated
Beas2B cells with PBS or tcuPA for 12 h and then inhibited ongoing
transcription with
5,6-dichloro-1- Effect of TcuPA Concentration on PAI-1 Protein Expression--
We
next treated Beas2B cells with varying amounts (0-1 µg/ml, 0-18
nM) of tcuPA for 24 h and then measured PAI-1
expression by Western blotting. The data shown in Fig.
5 indicate that tcuPA induced PAI-1
expression by Beas2B cells in a concentration-dependent manner. The effect is apparent at concentrations as low as 10 ng/ml
(0.18 nM). Maximum PAI-1 expression was observed at
concentrations of tcuPA between 250 and 1000 ng/ml. These data
demonstrate that the induction of PAI-1 in Beas2B cells by tcuPA is a
high affinity, concentration-dependent process with a
Kd comparable with that reported for binding to
uPAR.
Effects of Phosphatase and Phosphotyrosine Kinase Inhibitors on
tcuPA-mediated PAI-1 Induction--
To determine whether
tcuPA-mediated PAI-1 expression involves one or more of the pathways
implicated in tcuPA signaling (31, 32, 35, 36) are the same ones as
responsible for the observed increase in mRNA stability, we
pretreated Beas2B cells with herbimycin A and geneticin separately or
in combination with tcuPA. As shown in Fig.
6a, herbimycin A and geneticin
alone do not induce PAI-1 expression nor reverse tcuPA-mediated PAI-1
expression by Beas2B cells. Pretreatment of cells with tcuPA and sodium
orthovanadate (a tyrosine phosphatase inhibitor), on the other hand,
inhibited PAI-1 expression. We next used Western blot analysis of the
cytosolic extracts of PBS or tcuPA-treated cells in the presence or
absence of sodium orthovanadate to determine whether the induction of PAI-1 by tcuPA involved the expression of phosphotyrosine phosphatase (PTP) 1C. The data illustrated in Fig. 6b demonstrate that
tcuPA induces PTP1C expression. However, pretreatment with sodium
orthovanadate inhibited PTP1C expression in both PBS- and tcuPA-treated
cells.
Role of the Catalytic Domain of TcuPA on PAI-1
Expression--
Experiments were then performed to determine whether
the induction of PAI-1 by tcuPA requires retention of its catalytic
activity. Induction of PAI-1 by tcuPA in Beas2B cells was partially
inhibited by a urokinase-specific small molecule inhibitor, B428, by a
monoclonal antibody directed at its catalytic site, and by the
irreversible active site titrant, chloromethyl ketone (Fig.
7). Role of the Non-catalytic Domains of tcuPA on PAI-1
Expression--
These data strongly suggest that the capacity of tcuPA
to induce PAI-1 resides in the non-proteolytic ATF. In accord with that
hypothesis, the induction of PAI-1 by ATF was similar to that induced
by scuPA or tcuPA (Fig. 8a).
In contrast, the isolated low molecular weight catalytic domain had
little activity, consistent with the amount of PAI-1 induced when the
catalytic activity of tcuPA was inhibited (Figs. 7 and 8a).
suPAR totally blocked the activity of tcuPA (Fig. 8, b and
c), excluding signaling through the uPA-uPAR complex
(50, 52) and eliminating the possibility of endotoxin or another
mechanism of PAI-1 induction.
The ATF is composed of the uPAR-binding GFD and a kringle. To determine
whether either or both domains are required for the inductive effect,
we studied scuPA variants lacking one or the other domain. Deletion of
the uPAR-binding GFD almost totally abolished the inductive effect, as
did deletion of the kringle (Fig. 8, b and c).
These data suggest that uPAR or, less likely, another cellular
receptor, must bind to the uPA GFD for induction of PAI-1 to occur, but
that the kringle is needed either because it contains the signaling
element or is required to induce the active conformation of the
GFD·uPAR complex. In addition, treatment of Beas2B cells with growth
factor domain (^GFD) or kringle (^kringle) uPA deletion
mutants inhibited PTP1C expression. These observations suggest that
tcuPA-mediated induction of PAI-1 at least in part involves
phosphotyrosine phosphatases, and that the induction of PTP1C by uPA
involves both the GFD and kringle regions (Fig. 8d).
uPA-dependent proteolysis is critical for cellular
migration and tissue remodeling in inflammation, tumor growth, and the development of metastasis (53, 54). The tcuPA-uPAR interaction can
promote cellular movement and regulate cellular attachment to
surrounding ground substance, processes that may contribute to
remodeling of the lung in the acute respiratory distress syndrome or in
the interstitial lung diseases (21, 55). The interaction between uPA
and uPAR at the cancer cell surface also appears to be a critical event
in the pathogenesis of neoplastic growth and metastasis, mediating
tissue remodeling, tumor cell invasion, adhesion, and proliferation (1,
39). Binding of uPA to uPAR mediates cell proliferation in several cell
types including nonmalignant lung epithelial cells, lung
carcinoma-derived cells, and mesothelioma (41, 42). Tumor cell invasion
is also facilitated by occupancy of uPAR with host- or tumor-derived
uPA (56, 57). Oligomerization of uPAR may facilitate
vitronectin-mediated cell adhesion and migration (58). Pathways that
regulate the uPA-uPAR-PAI-1 system are, therefore, germane to the
pathogenesis of lung injury, its repair and neoplasia.
Plasminogen activation is regulated in part by two specific,
fast-acting plasminogen inhibitors, PAI-1 and PAI-2. These inhibitors belong to the serpin family and are products of different genes. These
inhibitors bind and inactivate both receptor-bound and fluid-phase uPA.
PAI-1 is the major PAI in plasma and in most tissues, and a wide
diversity of hormones, cytokines, and growth factors regulate its
cellular expression. In the lung, PAI-1 is expressed at the surface of
nonmalignant lung epithelial cells, which also elaborate uPA and
thereby regulates the delicate PA-PAI balance that determines expression of fibrinolytic activity in the alveolar compartment (55,
59).
The balance between proteolytic enzymes and their inhibitors is also
critical in the regulation of tissue remodeling and normal angiogenesis
(60). Cell migration along provisional matrix involves sequential and
topographically directed adhesion/disadhesion. The traction forces
involved must fall within a critical range. The fact that both
excessive and inadequate cell contacts preclude coordinate
assembly/disassembly of focal contacts helps to explain the otherwise
seemingly paradoxical requirement for PAI-1 in cell migration and its
negative correlation with prognosis in a variety of human tumors. The
possibility that this balance is achieved through an autoregulatory
process, in this case initiated by uPA, has received little attention.
Therefore, we sought to determine whether uPA contributed to the
regulation of PAI-1 expression by lung epithelial cells.
In this study, we confirm that this is the case and demonstrate that
uPA induces expression of PAI-1 in cultured Beas2B as well as primary
small airway epithelial cells. This pathway provides a versatile
regulatory system through which the uPA concentration of the ambient
microenvironment could regulate PA activity and pericellular
proteolysis by up-regulating expression of PAI-1. This molecular
mechanism may be a crucial determinant of cellular invasiveness of lung
carcinomas, in which excessive uPA-dependent pericellular
proteolysis increases cellular invasiveness (17), an effect that can be
regulated by local expression of PAI-1.
Regulation of PAI-1 expression involves both transcriptional and
post-transcriptional mechanisms. In previous studies, we found that a
post-transcriptional pathway influences levels of PAI-1 mRNA in
lung cancer-derived cell lines and nonmalignant lung epithelial cells
(18). Similar findings were previously reported in phorbol myristate
acetate, insulin, insulin-like growth factor, and cyclic nucleotide
analogue-treated cells (61-64). Cytokines expressed in the setting of
acute lung injury or in the tumor microenvironment increased PAI-1
expression (65). The identification of a newly identified
post-transcriptional mechanism by which the lung epithelium regulates
PAI-1 suggested the possibility that other novel pathways could
likewise influence expression of this major PA inhibitor. Based upon
our previous observations, we therefore hypothesized that the induction
of PAI-1 in lung epithelial cells by uPA could involve
post-transcriptional regulation. We now confirm that PAI-1 mRNA is
stabilized by tcuPA treatment. We have previously shown that
tcuPA-mediated induction of its own expression as well as that of uPAR,
and that the processes also involve post-transcriptional regulation
(35, 36). However, tcuPA treatment did not stabilize the induced PAI-1
protein itself. Studies are in progress to study the inter-relationship
between these pathways and to identify the responsible regulatory
factors. Differences in cell receptors, transcription factors, or other components of the signaling pathway may participate in the
characteristic untoward migration of tumor cells compared with the
coordinated regulation of normal repair processes including
angiogenesis and wound healing.
The mechanism by which tcuPA induces PAI-1 in Beas2B cells appears to
be largely independent of its proteolytic activity. Inhibitors of the
catalytic site or proteolytic inactivation by thrombin caused little
loss of PAI-1-inducing activity. Moreover, essentially identical
amounts of PAI-1 were induced by ATF and full-length single- and
two-chain forms of uPA. In contrast, little or no PAI-1 was induced
when either the growth factor domain or the kringle domain were deleted
from the full-length molecule. Binding of GFD to uPAR may be required
to approximate a signaling element in the kringle with a yet to be
identified transmembrane adapter. Signaling mediated through the uPA
kringle domain has previously been reported in vascular smooth muscle
cells (43, 44). The alternative explanation, i.e.
that the kringle is required to generate a productive interaction of
the GFD·uPAR complex with an adapter, such as an integrin, cannot be
excluded. However, the fact that suPAR inhibited PAI-1 induction,
although suPAR·uPA complexes bind to vitronectin (50), makes this
latter explanation somewhat less likely. The regulatory mechanism may
also involve the participation of other factors. We previously reported
that tumor necrosis factor- The process also involves cellular signaling mainly through activation
of tyrosine phosphatases. The inhibitory effect of tyrosine phosphatase
inhibitors on PAI-1 expression indicates that tyrosine phosphorylation
is involved in the signaling process and that the signaling mechanism
could involve PTP1C. This possibility is supported by the induction of
PTP1C by the uPA GFD and kringle, regions that are otherwise implicated
in expression of PAI-1. uPA may also induce synthesis of growth factors
or cytokines, which in turn may induce increases in the level of PAI-1
mRNA. The elucidation of the mechanisms responsible for the
prolonged effect of uPA on the PAI-1 mRNA level remain to be
determined. The induction of PAI-1 by tcuPA and thrombin was additive
whereas plasmin did not influence the process (data not shown),
indicating that the induction mechanism was not subject to inhibitory
cross-talk involving these proteases.
Studies performed with 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
(68). Similarly, activation of a 38-kDa tyrosine-phosphorylated
uPAR-associated protein has been identified in U937 cells (32). In both
cases, signaling by uPA was abolished when the cells were treated with phosphatidylinositol-phospholipase C and other studies also
supported the association of uPAR with protein kinase C and cytokeratin (66). In vascular smooth muscle cells, uPAR has been associated with
JAK1, Tyk2, and Src kinases (33), whereas in kidney epithelial tumor
cells uPAR associates with gp130 and JAK1 (19). We now show that uPA
mediates induction of PAI-1 and that the signaling mechanism involves
phosphotyrosine phosphatases, including PTP1C.
In summary, we demonstrate that uPA stimulates expression of PAI-1 by
lung epithelial cells in culture. If operative in vivo, this
pathway could contribute to the relative local overexpression of PAI-1
and the paucity of alveolar fibrinolytic capacity associated with
inflammatory lung disease (55). On the other hand, failure to
counterbalance the exuberant production of uPA characteristic of tumor
cells by the timely production of sufficient PAI-1 by host cells or the
tumor cells themselves may contribute to neoplastic proliferation and
formation of metastasis. Identification of the intracellular mediators
of this process may provide a new handle on the regulation of impaired
or untoward cell migration. This newly identified pathway is, to our
knowledge, the first description of the ability of uPA to regulate the
expression of its major inhibitor, PAI-1, in any cell type. The
physiological and pathological implications of uPA-mediated PAI-1
expression will require further study.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Thrombin, herbimycin A,
genestein, bovine serum albumin (BSA), ovalbumin, Tris base, aprotinin,
dithiothreitol, phenylmethylsulfonyl fluoride, silver nitrate, ammonium
persulfate, and phorbol myristate acetate were from Sigma. Acrylamide,
bisacrylamide, and nitrocellulose were from Bio-Rad. Recombinant high
molecular weight two-chain uPA was a generous gift from Drs. Jack
Henkin and Andrew Mazar from Abbott Laboratories (Abbott Park, IL).
Anti-PAI-1 and anti-uPA antibodies were obtained from American
Diagnostics (Greenwich, CT). Antiphosphotyrosine phosphatase 1C
antibody was from Upstate Pharmaceuticals (Lake Placid, NY). The uPA
antagonist B428 was the generous gift of Dr. Andrew Mazar (Angstrom
Pharmaceuticals, San Diego, CA). XAR x-ray film was purchased from
Eastman Kodak. uPA deletion mutants were cloned and the recombinant
proteins were expressed in S2 cells and purified, including the
amino-terminal fragment (ATF) (amino acids 1-135), low molecular
weight uPA fragments (amino acids 136-411), and the deletion mutants
GFD-scuPA (amino acids 4-43) and kringle-scuPA (amino acids 47-135),
as previously described (47).
-mercaptoethanol and subjected to Western blotting with
-actin
monoclonal antibody. Alternatively, we measured uPA-mediated PAI-1
expression by metabolic labeling using [35S]methionine in
combination with immunoprecipitation as we described earlier (41). In
separate experiments, we also measured phosphotyrosine phosphatase 1C
expression by PBS or uPA-treated cells in the presence or absence of
sodium orthovanadate by Western blotting using an antiphotyrosine
phosphatase 1C antibody.
70 °C overnight. The
intensity of the bands was measured by densitometry and normalized
against that of
-actin. uPA mRNA stability was assessed by
transcription chase experiments. In these experiments, cells stimulated
with or without uPA were then treated with
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole to inhibit
ongoing transcription, after which total RNA was isolated at specific
time points. PAI-1 mRNA was measured by Northern blot as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Time-dependent PAI-1 expression
by tcuPA in Beas2B lung epithelial cells. a, confluent
cells were treated with or without recombinant human two-chain uPA (1 µg/ml) for 0-24 h at 37 °C in basal medium containing 0.5% BSA.
The total proteins from the cell lysates were separated on 8%
SDS-polyacrylamide gels and transferred to nitrocellulose membranes.
The membrane was immunoblotted with anti-PAI-1 monoclonal antibody.
b, the data illustrated are integrated from at least four
independent experiments, and mean density of the individual bands is
presented in the line graph. c, Western blot for
PAI-1 protein of primary small airway epithelial cells treated with PBS
or tcuPA for 24 h. The corresponding blots were stripped and
reprobed with -actin monoclonal antibodies for assessment of equal
loading.
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Fig. 2.
Overexpression of endogenous uPA induces
PAI-1 expression. a, Western blotting for PAI-1
expression in uPA cDNA-transfected Beas2B cells. Proteins from cell
lysates of untreated Beas2B cells (lane 1), Beas2B cells
transfected with expression vector pRc/CMV alone (lane 2),
or Beas2B cells transfected uPA cDNA in eukaryotic expression
vector pRc/CMV (lane 3) were assayed for uPA expression.
Proteins were separated on 8% SDS-PAGE, transferred to nitrocellulose
membrane, and developed by Western blotting using anti-PAI-1 monoclonal
antibody. b, the cells were subjected to metabolic labeling
using [35S]methionine followed by immunoprecipitation
with anti-PAI-1 monoclonal antibody. Lanes 1-3
are as described in a.
-actin
loading controls. As shown by the composite data in Fig. 3b,
resting Beas2B cells express a small amount of PAI-1 mRNA.
Following addition of tcuPA, the level of PAI-1 mRNA was increased
by 3 h and remained elevated over 24 h.
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Fig. 3.
Time-dependent induction of PAI-1
mRNA by tcuPA treatment of Beas2B cells. a, the
cells were treated as described in the legend to Fig. 1. Total RNA (20 µg/lane) was isolated using TRI-reagent, separated by
agarose-formaldehyde gel electrophoresis, and subjected to Northern
blotting using 32P-labeled PAI-1 and -actin cDNAs.
b, the line graph portrays the integrated data of
four individual experiments.
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Fig. 4.
Effect of tcuPA on the rate of transcription,
decay of PAI-1 mRNA, and protein in Beas2B cells.
a, nuclei isolated from Beas2B cells treated with PBS or
tcuPA for 12 h as described in the legend to Fig. 1 were subjected
to the transcription reaction in the presence of [32P]UTP
at 30 °C for 30 min. 32P-Labeled nuclear RNA was
hybridized with uPA cDNA immobilized on nitrocellulose membrane.
-Actin and pcDNA3 cDNAs were used as positive and negative
loading controls, respectively. b, effect of tcuPA on PAI-1
mRNA stability. Beas2B cells were treated with PBS or tcuPA for
12 h, after which
5,6-dichloro-1-
-D-ribofuranosylbenzamidazole (10 µg/ml) was added for various periods of time. PAI-1 mRNA was
analyzed by Northern blotting. c, effect of tcuPA on PAI-1
protein stability. Beas2B cells were treated with PBS or tcuPA for
24 h, after which cycloheximde (10 µg/ml) was added and the
cycloheximide-treated samples were then assayed at different times over
a 0-24-h period. PAI-1 protein was measured by metabolic labeling
using [35S]methionine followed by immunoprecipitation
with anti-PAI-1 monoclonal antibody (upper panel). The same
samples were immunoprecipitated with a monoclonal antibody to
-actin
as loading controls (lower panel).
-D-ribofuranosylbenzimidazole for varying lengths of time. We analyzed PAI-1 mRNA by Northern blotting using 32P-labeled PAI-1 cDNA as shown in Fig. 4b.
PAI-1 mRNA of PBS-treated Beas2B cells has a very short half-life.
However, tcuPA treatment stabilized PAI-1 mRNA over 6 h. We
also sought to determine whether uPA increases PAI-1 protein stability.
To address this possibility we inhibited translation of PAI-1 by PBS or
uPA-treated cells with cycloheximde and then analyzed the stability of
the PAI-1 protein over varying time periods. Our results (Fig.
4c) show that PAI-1 protein is detectable in the control
PBS-treated cells and that basal levels were not appreciably changed
over 24 h. The low levels of basal PAI-1 expression are consistent
with our findings in unstimulated cells as illustrated in Fig. 1. In
the uPA-treated cells, PAI-1 protein expression was induced but then decreased to basal levels by 24 h, indicating that uPA treatment does not stabilize the induced PAI-1 protein over this interval.
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Fig. 5.
Effect of tcuPA concentration on PAI-1
protein expression. a, the cells grown to confluence
were treated with varying amounts of tcuPA (0-1 µg/ml) for 24 h
at 37 °C in basal medium containing 0.5% BSA. The total proteins
from cell lysates were subjected to immunoblotting as described in the
legend to Fig. 1. b, the figure shown illustrates the mean
band densities of four independent experiments.
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Fig. 6.
a, effect of tyrosine kinase and
phosphatase inhibitors on tcuPA-mediated PAI-1 expression. Cells grown
to confluence were treated with or without herbimycin A
(Herb), geneticin (Gen), and sodium orthovanadate
(Naor) for 2 h followed by two-chain (tcuPA,
1 µg/ml) for 24 h at 37 °C and subjected to immunoblotting
with anti-PAI-1 antibody as described in the legend to Fig. 1. The
bar graph illustrates the mean band densities of three
independent experiments. b, effect of tyrosine phosphatase
inhibitors on phosphotyrosine phosphatase 1C expression. Cells
grown to confluence were treated with PBS or tcuPA in the presence or
absence of sodium orthovanadate as described above. The cytosolic
extracts were subjected to Western blotting using an
anti-phosphotyrosine phosphatase antibody.
-Thrombin stimulated PAI-1
expression and appeared to augment PAI-1-inducing activity in the
presence of tcuPA (Fig. 7), whereas plasmin or the plasmin inhibitor
aprotinin had no effect (data not shown).
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Fig. 7.
Effect of inhibitors of tcuPA activity on
uPA-mediated PAI-1 by Beas2B cells. Confluent cells were treated
with or without B428 (0.02 mM), anti-uPA monoclonal
antibody (PAb) (2 µg/ml), -thrombin (Thr), or
chloromethyl ketone-inactivated uPA (Chl. uPA, 1 µg/ml)
for 24 h at 37 °C in basal medium containing 0.5% BSA. Total
proteins from cell lysates were isolated and subjected to
immunoblotting. The data are shown as the mean ± S.D. of four to
five independent experiments.
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Fig. 8.
Effect of different fragments of uPA on PAI-1
expression. a, cells grown to confluence were treated
with either the amino-terminal (1 µg/ml) or low molecular weight
(LMW, 1 µg/ml) fragments of uPA, two-chain
(tcuPA, 1 µg/ml) or single chain (scuPA) for
24 h at 37 °C in basal medium containing 0.5% BSA. Cellular
proteins were immunoblotted as described above with anti-PAI-1 or
anti- -actin antibody. The data illustrated are representative of the
findings of four independent experiments. b, Beas2B cells
were treated with PBS, growth factor domain deletion
(^GFD), kringle domain deletion (^kringle)
mutant, active uPA (tcuPA), chloromethyl ketone-inactived
uPA (Chl. uPA), or uPA in the presence of excess soluble
uPAR (suPAR). All proteins were used in a concentration of 1 µg/ml and suPAR was used in a 10-fold molar excess. PAI-1 expression
was measured by Western blotting, as described above. The data are
representative of four independent experiments. c, composite
densitometric analyses of the effect of deletion fragments of uPA on
PAI-1 induction in Beas2B cells. The cells grown to confluence were
treated with the same concentrations (1 µg/ml) of scuPA, ATF, growth
factor domain deletion (^GFD), or kringle domain
(^kringle) mutants, low molecular weight uPA, tcuPA alone,
or in the presence of excess amounts of soluble uPAR (suPAR, 10-fold
excess versus tcuPA) for 24 h at 37 °C. The cellular
proteins were subjected to immunoblotting using anti-PAI-1 and
anti-
-actin monoclonal antibodies. d, effect of growth
factor domain or kringle domain deletions from single chain uPA on
PTP1C expression. Cells grown to confluence were treated with PBS,
single chain uPA (scuPA), two-chain uPA, or single chain uPA
lacking either the GFD (^GFD) or kringle domain
(^kringle). The cytosolic extracts were subjected to
Western blotting using an anti-PTP1C antibody, as described above. The
figure represents the findings of two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
induces uPA in Beas2B cells (67). This mechanism could contribute to increased expression of uPA, which could
in turn increase uPA-mediated expression of PAI-1 by these cells.
Multiple other such interactions could likewise influence the
expression of PAI-1 and require further study.
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ACKNOWLEDGEMENTS |
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We are grateful to Kathy Johnson, Brad Low, and M. B. Harish for technical assistance.
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FOOTNOTES |
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* This work was supported by NHLBI National Institutes of Health Grants R01-HL71147-01, R01-HL-62453-01, and R01-45018-06, National Institutes of Health Grants HL60169, HL66442, and HL67381 (to D. B. C. and A. A. H.), and beginning Grant-in-aid 0060251U from the Mid-Atlantic Division of the American Heart Association (to K. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Lab C-6, The University of Texas Health Center, 11937 U.S. Highway 271, Tyler, TX 75708. Tel.: 903-877-7668; Fax: 903-877-7927; E-mail: sreerama. shetty{at}uthct.edu.
Published, JBC Papers in Press, March 17, 2003, DOI 10.1074/jbc.M207445200
2 S. Shetty, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: uPA, urokinase-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor; PAI-1, plasminogen activator inhibitor; GFD, growth factor like domain; BSA, bovine serum albumin; ATF, amino-terminal fragment; Beas2B, bronchial epithelial cells; PMSF, phenylmethylsulfonyl fluoride; CMV, cytomegalovirus; LPS, lipopolysaccharide; PTP1C, phosphotyrosine phosphatase 1C.
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