From the Department of Obstetrics and Gynecology,
Technische Universität München, D-81675 München,
Germany, the ¶ ABL-Basic Research Program, NCI, National
Institutes of Health, Frederick Cancer Research and Development Center,
Frederick, Maryland 21702, and the
Division of Basic Sciences,
NCI, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Hepatocyte growth factor/scatter
factor (HGF/SF) is a pleiotropic effector inducing invasion and
metastasis of tumor cells that express the Met tyrosine kinase
receptor. One of the effectors of HGF/SF is the urokinase-type
plasminogen activator, a serine protease that facilitates tumor
progression and metastasis by controlling the synthesis of the
extracellular matrix degrading plasmin. Stimulation of NIH 3T3 cells
that were stably transfected with the human Met receptor (NIH
3T3-Methum) with HGF/SF induced a trans-activation of
the urokinase promoter and urokinase secretion. Induction of the
urokinase promoter by HGF/SF via the Met receptor was blocked by
co-expression of a dominant-negative Grb2 and Sos1 expression
construct. Further, the expression of the catalytically inactive
mutants of Ha-Ras, RhoA, c-Raf, and Erk2 or addition of the
Mek1-specific inhibitor PD 098059 abrogated the stimulation of the
urokinase promoter by HGF/SF. A sequence residing between Hepatocyte growth factor/scatter factor
(HGF/SF)1 is a multipotent
growth factor affecting motility, morphogenesis, growth, and
angiogenesis (1). Aberrant expression of HGF/SF is associated with
enhanced tumor invasion and metastasis (2) and has been shown to be a
strong negative prognostic factor in human breast cancer (3). The
receptor for HGF/SF encoded by the c-Met protooncogene (4) is
synthesized as a single polypeptide 170-kDa precursor. After
glycosylation and proteolytic cleavage, the resulting dimer has a
50-kDa One signal transduction pathway involved in signaling through Ras or
small G-proteins of the Rho family is the mitogen-activated protein
kinase (MAPK) cascade (8, 9) that after activation assembles in
sequential kinase cascades undergoing reversible phosphorylation. The
best known pathway, the Ras Because HGF/SF has been shown to be involved in tumor cell invasion and
metastasis, it has been hypothesized that tumor-associated proteases
might be one target of HGF/SF-Met signaling. Indeed two groups of
proteases, matrix metalloproteinases (12) and the serine protease
urokinase (13, 14), are up-regulated by HGF/SF in different cell lines.
Urokinase (15) converts plasminogen into plasmin, a serine protease
with broad substrate specificity toward components of the extracellular
matrix including laminin, vitronectin, and fibronectin (16-18).
Together, these proteolytic functions facilitate the migration of tumor
cells through the extracellular matrix and basement membrane barriers.
Membrane attachment of urokinase to the urokinase receptor increases
the rate of plasmin formation at the plasma membrane (19) and focuses proteolytic activity at the leading edge of the tumor (20). High
urokinase expression is correlated with a poor prognosis of patients
suffering from a variety of different types of cancer including that of
the breast, ovary, and lung (21, 22).
Studies on the regulation of urokinase expression have shown that the
urokinase gene is regulated at the transcriptional level (23-25). The
urokinase promoter contains functional binding sites for the
transcription factors AP-1, PEA3, and NF- Because the HGF/SF precursor protein shares a very high sequence
homology with the kringle and serine protease domains of plasminogen,
it was discussed whether urokinase is a putative activator of the
precursor molecule HGF/SF precursor protein. Indeed, Naldini et
al. (29) showed that urokinase activates HGF/SF precursor protein
to the active form, which is a disulfide-linked heterodimer consisting
of a 69-kDa Cell Culture--
NIH 3T3 cell lines were obtained from the
American Type Culture Collection and stably transfected with the human
Met cDNA or the empty vector (pMB1) that contains the long terminal
repeat promoter from Moloney murine sarcoma virus and the
polyadenylation signal of simian virus 40 by the calcium phosphate
method (31). The cells were cultured in Dulbecco's modified Eagle's
medium supplemented with 10 mM HEPES, 272 mM
asparagine, 550 mM arginine, penicillin-streptomycin, and
10% fetal calf serum (all from Life Technologies, Inc.). Collection of
conditioned medium was performed using the same medium without fetal
calf serum (serum-free medium). Purified human HGF/SF (kindly provided
by R. Schwall Genentech, Inc., South San Francisco, CA) represents the
activated, heterodimeric form and was used, if not otherwise specified,
at a final concentration of 200 ng/ml.
Vectors--
The dominant-negative Erk1 and Erk2 constructs
contain the coding region of these MAPKs in which the conserved codon
71 and 52 of Erk1 and Erk2 involved in phosphate transfer was mutated from lysine to arginine (32), thus impairing catalytic activity. The
dominant-negative form of Sos1 (33, 34) lacks the guanine nucleotide
exchange domain. Grb2 Zymography--
Conditioned media collected from equal numbers
of cells were denatured and electrophoresed in a 10% SDS-PAGE gel
containing 0.2% (w/v) casein with or without 5 µg/ml plasminogen.
The gel was incubated at room temperature for 2 h in the presence
of 2.5% Triton X-100 and subsequently overnight at 37 °C in a
buffer containing 10 mM CaCl2, 0.15 M NaCl and 100 mM Tris-HCl, pH 7.5. The gel was
stained for protein with 0.25% Coomassie.
Plasminogen-dependent proteolysis was detected as a white
zone in a dark field. As a control, the same samples were run in a gel
without plasminogen.
Western Blot Analysis--
Cells were lysed in buffer containing
50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mM
sodium fluoride, 1 mM sodium orthovanadate, 10 µg/ml
aprotinine, 1 mM phenylmethylsulfonyl fluoride (buffer A),
cleared by centrifugation, and electrophoresed in a 10% SDS-PAGE under
reducing condition. The resolved proteins were transferred to a
nitrocellulose membrane (BA-S85, Schleicher & Schuell). The filter was
subjected to a buffer containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris, 0.25% gelatin (w/v), and
0.5% Triton X-100 and incubated sequentially with a rabbit polyclonal
antibody against the human Met receptor (SC-161, Santa Cruz
Biotechnology, Santa Cruz, CA) followed by a horseradish peroxidase-conjugated anti-rabbit IgG. Reactive proteins were visualized by ECL according to the manufacturer (Amersham Pharmacia Biotech).
MAPK Assays--
Cells were stimulated with indicated amounts of
HGF/SF for 24 h, lysed in buffer A, and cleared by centrifugation.
After normalization for protein, extracts were incubated for 3 h
at 4 °C with protein A-agarose and antibody to Erk (C16, Santa Cruz
Biotechnology, Santa Cruz, CA). The beads were washed three times in
HNTG buffer (20 mM HEPES, 150 mM NaCl, 10%
glycerol, 10 mM sodium pyrophosphate) and then incubated in
kinase buffer (1 µg/µl myelin basic protein, 50 µM
ATP, 1 µCi of [ Transfections--
Cells were transfected by the calcium
phosphate method (41, 42) with chloramphenicol acetyltransferase (CAT)
reporter constructs fused to the wild type or deleted fragments of the human urokinase promoter (23). To correct for transfection
efficiencies, all transient transfections were performed in the
presence of 4 µg of a Mobility Shift Assays--
Cells at 80% confluency were
stimulated with 100 ng/ml HGF/SF for 12 h, and nuclear extracts
were prepared as described by Dignam et al. (43). NIH
3T3-Methum (7.5 µg) were incubated in a buffer containing
20 mM HEPES, 0.2 mM EDTA, 0.25 mM
dithiothreitol, 50 mM NaCl, 10% glycerol, and 1 µg
poly(dI·dC). To each reaction 5 fmol of a Klenow end-labeled ([ Met-HGF/SF Signaling Activates the Urokinase Promoter and
Stimulates Urokinase Secretion--
To study the regulation of the
urokinase-type plasminogen activator by HGF/SF, we used a cell clone
(called NIH 3T3-Methum) that is derived from an NIH 3T3
cell line stably transfected with the human Met receptor (31). The NIH
3T3-neo cell clone is stably transfected with the empty vector and was
used as a control. NIH 3T3-Methum cells overexpress the
human Met receptor as shown by Western blotting with an antibody
recognizing human Met (Fig.
2A) that does not interact
with the mouse Met receptor. Transient transfection of a CAT reporter
driven by the wild type urokinase promoter in NIH
3T3-Methum cells shows basal activity (Fig. 2B)
compared with NIH 3T3-neo cells, which show almost no basal activity
(data not shown). Addition of increasing amounts of human HGF/SF to the
NIH 3T3-Methum cells 24 h prior to harvesting induces
the urokinase promoter in a dose-dependent manner (Fig. 2,
B and C). Human HGF/SF does not activate the
endogenous mouse Met receptor. To determine whether NIH
3T3-Methum cells can be stimulated to secrete urokinase, we
collected conditioned media from HGF/SF-stimulated NIH
3T3-Methum cells and performed plasminogen zymography. A
weak activity (Mr = ~55 kDa), which
co-migrated with authentic mouse urokinase (data not shown), was
secreted by untreated NIH 3T3-Methum cells, which could be
stimulated substantially after addition of human HGF/SF (Fig.
2D). Addition of an antibody against urokinase prior to
zymography abolished the induction of proteolytic activity by HGF/SF
(data not shown). Deletion of plasminogen from the gel abolished the
band, which indicated that the proteolytic activity could be ascribed
to a plasminogen activator (Fig. 2E). These data are in
accordance with previous results showing that HGF/SF-Met signaling
up-regulates urokinase (2, 20, 44) and additionally suggest that the
increased synthesis of urokinase after stimulation with HGF/SF is most
likely a reflection of trans-activation of the urokinase promoter.
The HGF/SF-dependent Stimulation of Urokinase Promoter
Activity Is Inhibited by the Co-expression of Plasmids Encoding
Dominant-negative Grb2 and Sos1--
We were interested in following
the signaling pathway involved in activation of the urokinase promoter
by HGF/SF from the Met receptor to the nucleus and for this purpose
made use of dominant-negative forms of key signal transduction
proteins. In general, after activation of receptor tyrosine kinase
receptors, adaptor proteins like Grb2 bind to the cytoplasmatic part of
the protein and recruit Sos1 to the plasma membrane (35). This places
Sos1 in the vicinity of Ras-GDP, leading to the exchange of GDP to GTP
and the activation of Ras signaling. NIH 3T3-Methum cells
were co-transfected with the urokinase promoter and an expression
vector encoding a dominant-negative Grb2 (35). Expression of the
mutated Grb2 (Grb2 Involvement of Ha-Ras, RhoA, and c-Raf in Regulation of the
Urokinase Promoter by Human HGF/SF--
To determine whether Ha-Ras is
also involved in the induction of urokinase by Met-HGF/SF signaling,
NIH 3T3-Methum cells were cotransfected with a
dominant-negative Ha-Ras protein, RasN17, and the wild type urokinase
promoter. The inducible activation of the urokinase promoter by HGF/SF
was completely abrogated by co-expression of the RasN17 construct,
whereas the vector control (pSV2neo) had no effect (Fig.
4A).
Because c-Raf binds to activated Ha-Ras (8), we determined the
sensitivity of urokinase expression to a dominant-negative c-Raf
expression vector (RafC4) (38). NIH 3T3-Methum cells were
transiently transfected with the urokinase promoter-driven CAT reporter
and RafC4 or the empty vector control (pBGC4) and stimulated with 200 ng/ml of human HGF/SF. Expression of RafC4 led to a reduction in the
ability of HGF/SF to stimulate the urokinase promoter (Fig.
4A). The empty vector control failed to repress the
induction of the CAT reporter by HGF/SF. Similarly co-expression of a
dominant-negative RhoA (RhoN19) (37) inhibited basal and HGF/SF-mediated activation of the urokinase promoter (Fig.
4B).
RhoA can be part of c-Raf-dependent and -independent signal
transduction pathways (46, 47). To determine the localization of RhoA
in the c-Raf-Erk signaling cascade, a constitutively activated c-Raf
(Raf BXB) (38) and the dominant-negative RhoA was co-transfected with
the urokinase promoter in NIH 3T3-Methum cells. Expression
of the constitutively activated serine-threonine kinase caused an
activation of the urokinase promoter that could be abolished by the
dominant-negative RhoN19 construct (Fig. 4C). Thus,
regulation of urokinase by RhoA is an event downstream of c-Raf.
Repression of the Urokinase Promoter by a MEK1-specific Inhibitor
(PD 098059) and by a Dominant-negative Erk2 Expression
Construct--
Because Mek1 is stimulated by the serine-threonine
kinase c-Raf (9), we tested the possibility that the urokinase promoter was regulated through this MAPK activator. A specific inhibitor of
Mek1, PD 098059, (48) was added to NIH 3T3-Methum cells
immediately after transfection with the urokinase promoter, and the
cells were stimulated with HGF/SF. The inhibitor abrogated the
induction of the urokinase promoter by HGF/SF (Fig.
5A).
We were interested in determining which MAPK is involved in the
regulation of the urokinase promoter by HGF/SF and did three sets of
experiments in this regard. First, we cotransfected a dual activity
phosphatase (CL 100) that inactivates multiple MAPK members (49) and
found that it down-regulated basal as well as HGF/SF-induced activation
of the urokinase promoter (data not shown). We therefore concluded that
member(s) of the MAPK family are involved in regulation of the
urokinase promoter. We next tested each MAPK family member
individually. NIH 3T3-Methum were transiently cotransfected
with the urokinase promoter-driven CAT reporter and expression vectors
encoding either a dominant-negative Erk1 or Erk2 (32). Expression of
the dominant-negative Erk2 but not Erk1 (data not shown) caused a
repression of HGF/SF-mediated urokinase promoter activation (Fig.
5B). Cotransfection of a dominant-negative Jnk1 expression
vector (data not shown) or incubation of the NIH 3T3-Methum
cells with the highly specific p38 inhibitor SB 203580 (Fig. 5A) did not affect induction of the urokinase promoter by
HGF/SF. If Erk2 is involved in the regulation of the urokinase promoter by HGF/SF, then this ligand should be able to stimulate Erk activity in
NIH 3T3-Methum cells. To test this hypothesis we performed
an in vitro kinase assay to measure Erk-MAPK activation.
After addition of increasing amounts of HGF/SF to NIH
3T3-Methum cells, we found a sustained activation of Erk
activity after the 24-h stimulation period, a time at which EGF had
lost its activation potential (Fig. 5C). Stimulation of NIH
3T3-Methum cells with EGF for 20 min strongly induced
Erk-MAPK activity (data not shown). In contrast HGF/SF did not affect
Jnk or p38 activity (data not shown).
Induction of AP-1 DNA Binding Activity by HGF/SF--
The previous
experiments were performed with the urokinase promoter containing 2109 nucleotides upstream of the translation start site. To determine the
region of the urokinase promoter required for its stimulation by
HGF/SF, NIH 3T3-Methum cells were transfected with CAT
reporters driven by different 5' deletion fragments (
Because mobility shift assays indicate specific binding of NIH
3T3-Methum nuclear extract to AP-1 sites in the urokinase
promoter, we determined the functional role of the AP-1 family of
transcription factors in regulating urokinase expression. With this
objective, NIH 3T3-Methum cells were transfected with an
expression vector encoding a dominant-negative protein termed A-Fos
that inhibits DNA binding of AP-1 proteins in an equimolar
concentration (39). NIH 3T3-Methum were transiently
cotransfected with the urokinase promoter and 2.5 µg of A-Fos (Fig.
6D). Transfection of the expression vector encoding A-Fos
reduced HGF/SF-inducible activity of the urokinase promoter. By
contrast, co-transfection of the urokinase construct with equivalent
amounts of empty vector (pCMV 500) did not reduce urokinase promoter
activity. The AP-1 family member c-Jun has been shown to be involved in
the mitogenic response of various growth factors, cytokines, and tumor
promoters (51), so we questioned whether it is also involved in the
induction of the urokinase promoter by HGF/SF. Indeed, cotransfection
of a dominant-negative c-Jun mutant (10) in NIH 3T3-Methum
cells prevented the induction of the promoter by HGF/SF (data not
shown), suggesting that c-Jun is one of the transcription factors
involved in the activation of the urokinase promoter by HGF/SF. These
results suggest that in NIH 3T3-Methum cells, basal and
HGF/SF-inducible activity of the urokinase promoter requires
trans-acting factor(s) that bind to AP-1 sites.
Induction of the Urokinase Promoter by HGF/SF Involves
Down-regulation of JunD--
JunD is a member of the AP-1 family that
has been shown to negatively regulate growth and antagonize
transformation by activated Ha-Ras in mouse cells (11). Because the
induction of the urokinase promoter by HGF/SF involves Ha-Ras (Fig.
4A), we explored the possibility of whether JunD is affected
by HGF/SF in NIH 3T3-Methum cells and whether it would
affect urokinase gene regulation. To investigate this possibility, we
carried out Western blotting with an antibody against JunD employing
nuclear extracts from stimulated and unstimulated NIH
3T3-Methum cells. After stimulation with HGF/SF, the
content of JunD protein in nuclear extract from NIH
3T3-Methum cells was lower compared with unstimulated cells
(Fig. 7A). The JunD band was
not detected upon preabsorption of the antibody with the immunizing
peptide (Fig. 7A, lanes 3 and 4). In
view of the requirement of the two AP-1-binding sites at
Because mobility shift assays indicated that JunD binding to both AP-1
sites in the urokinase promoter is lower after HGF/SF stimulation, we
determined the function of this transcription factor in regulating
urokinase expression. We reasoned that up-regulation of JunD prior to
stimulation of NIH 3T3-Methum cells with HGF/SF should
abrogate the induction of the urokinase promoter by HGF/SF. To this
end, NIH 3T3-Methum cells were transfected with the
urokinase promoter and an expression vector encoding wt JunD (Fig.
8A) and then stimulated with
HGF/SF. Indeed, expression of 2.5 µg of wt JunD reduced the induction of the urokinase promoter by 50% relative to the empty expression vectors. However, the repression of HGF/SF induction of urokinase promoter activity was reduced in a concentration-dependent
manner at higher JunD concentrations (10 µg). To exclude the
possibility that the observed effect of wt JunD on the urokinase
promoter is not exclusively an indirect effect caused by
heterodimerization with another member of the AP-1 family of
transcription factors, we repeated the experiment with an artificial
derivative of wt JunD, denoted JunD/EB1 (Fig. 8B). In the
JunD/EB1 construct the naturally occurring dimerization domain has been
replaced by an heterologous homodimerization domain from the
Ebstein-Barr virus transcription factor EB1 and forms only JunD/JunD
homodimers in the cell (40). The inductive effect of HGF/SF on the
urokinase promoter could also be repressed with JunD/EB1, confirming
that JunD is involved in the regulation of urokinase by HGF/SF. All together these results suggest that the induction of urokinase by
HGF/SF can be inhibited by overexpression of JunD.
HGF/SF is a pleiotropic growth factor that promotes cell
proliferation and survival and stimulates cell motility. Increased invasiveness of cancer cells after HGF/SF stimulation is accompanied by
up-regulation of various proteases (2, 52). Although we and others (13,
14, 53) showed that HGF/SF up-regulates one important
invasion-associated protease, urokinase, the signal transduction
pathways, and transcriptional regulatory mechanisms are still unknown.
Using dominant-negative expression vectors of signal transduction
proteins, we show here that up-regulation of the urokinase promoter by
HGF/SF is mediated via activation of a
Grb2/Sos1/Ha-Ras/c-Raf/RhoA/Mek1/Erk2/c-Jun pathway. Moreover JunD is
overexpressed in unstimulated cells and down-regulated after
stimulation of the urokinase promoter with HGF/SF.
Much attention has been given to identifying the adaptor proteins
associated with the Met receptor following stimulation by HGF/SF. In
this context, Grb2 (34), Gab1 (54), PI 3-kinase (55), and Cbl (6) have
been identified as being activated by HGF/SF stimulation involving two
phosphorylation sites, Tyr1349 and Tyr1356,
located at the carboxyl terminus of Met. Rahimi et al. (55) used dominant-negative constructs of p85 and wortmannin to show that PI
3-kinase mediates the proliferative effect of HGF/SF. These authors
show that the inhibitory effect of PI 3-kinase on proliferation is
mediated by S6 kinase and does not involve c-Jun expression. In our
experiments we did not observe any involvement of PI 3-kinase in
regulation of the urokinase promoter. Neither the PI 3-kinase inhibitor
wortmannin nor a dominant-negative PI 3-kinase (45) inhibited the
induction of the promoter by HGF/SF, whereas Grb2/Sos1 and c-Jun were
important for the basal and inducible activity of the urokinase
promoter. Therefore binding of different adaptor proteins mediate
distinct biological activities of HGF/SF. Binding of Grb2/Sos1 to the
Met receptor mediates effects like branching morphogenesis (52),
transformation (6), and as shown here, urokinase regulation, whereas
binding of PI 3-kinase induces S6 kinase and proliferation (55).
Our previous work (42) showed that Ha-Ras regulates the urokinase
promoter, but at that time we did not look at Rho family members,
because their role in transformation was not recognized then (56). The
results presented here show that Ha-Ras and RhoA, both members of a
superfamily whose activities are controlled by GDP/GTP cycling, are
necessary for the induction of the urokinase promoter by HGF/SF. RhoA,
a Rho family member, is involved in control of cell shape but also
cooperates with Ras and Raf in oncogenic cell transformation and
anchorage-independent growth (37, 47). The general role of RhoA in
regulation of HGF/SF responses is not clear because inhibition of RhoA
does not prevent HGF/SF-induced membrane ruffling and cell spreading
(57). Our results indicate a role for Rho in HGF/SF signaling by
affecting the regulation of urokinase. The effect of RhoA on urokinase
is not general to all transforming Rho family members, because a dominant-negative Rac1 mutant had no effect on urokinase promoter activity (data not shown). Although we showed involvement of RhoA in
the regulatory response by HGF/SF-Met receptor signaling, this effect
might not be restricted to HGF/SF alone. RhoA might regulate urokinase
expression in general, because a constitutive active RhoA mutant
transforms NIH 3T3 cells to grow and invade the surrounding tissue in
nude mice (47). This would suggest that the invasive phenotype of RhoA
transformed cells is mediated at least in part through up-regulation of urokinase.
The observation that dominant-negative mutants of Ha-Ras, RhoA, and
c-Raf inhibit inducible and basal activation of the urokinase promoter
by HGF/SF suggests a role for RhoA in the Erk-MAPK pathway. Involvement
of RhoA in the Erk-MAPK pathway is in agreement with the inhibitory
effect of C3 transferase, a RhoA inhibitor, on Erk2 activation (58).
Although RhoA can activate the Jnk-MAPK pathway (59), in the case of
HGF/SF-induced urokinase regulation we did not observe any effect of a
dominant-negative Jnk expression plasmid, nor could HGF induce Jnk
activity in NIH 3T3-Methum cells. This argues against an
involvement of Jnk downstream of RhoA for urokinase regulation.
Additionally we identified Erk2 as the MAPK involved in up-regulation
of urokinase by HGF/SF. RhoA is located downstream of c-Raf in the
signal transduction pathway from the Met receptor to the urokinase
promoter. This is consistent with previous reports (37, 59) showing
that transformation by c-Raf or Ha-Ras can be inhibited by
co-expression of a dominant-negative RhoA (RhoN19), suggesting
involvement of RhoA somewhere between c-Raf and Erk.
The NIH 3T3 cell clone used in this study, NIH 3T3-Methum,
is stably transfected with the Met receptor and has a higher basal urokinase promoter activity, compared with the vector transfected NIH
3T3-neo cell clone, which has no detectable promoter activity. Therefore, even without ligand stimulation, overexpression of human Met
activates the urokinase promoter and the MAPK signaling pathway.
Overexpression of dominant-negative components of the MAPK pathway in
NIH 3T3-Methum repressed constitutive urokinase promoter
activity, which indicates that the same signaling mechanism is used as
in HGF/SF-Met-induced urokinase expression.
On the basis of the results reported in this paper, we propose a model
(Fig. 9) addressing different signaling
molecules important in the regulation of the urokinase promoter by
HGF/SF-Met signaling, providing insights regarding the mechanisms by
which a protein-tyrosine kinase regulates a certain cellular gene.
Activation of the Met receptor by HGF/SF leads to recruitment of Grb2
and Sos1 to the receptor, thereby activating Ha-Ras and c-Raf. The
signal is transduced to the nucleus through a
c-Raf/Mek1/Erk2-dependent cascade and activates AP-1 family
members. PI 3-kinase, Rac, Jnk, p38, S6-kinase, and ATF-2 are not
involved in this response. Despite the apparent linear nature of the
Ras 2109 and
1870 base pairs (bp) was critical for stimulation of the urokinase
gene by HGF/SF. Mobility shift assays with oligonucleotides spanning an
AP-1 site at
1880 bp or a combined PEA3/AP-1 site at
1967 bp showed
binding of nuclear factors from NIH 3T3-Methum cells.
Expression of an expression plasmid that inhibits DNA binding of AP-1
proteins (A-Fos) abrogated inducible and basal activation of the
urokinase promoter. Nuclear extract from unstimulated NIH
3T3-Methum cells contained more JunD and showed a stronger
JunD supershift with the AP-1 oligonucleotides, compared with
HGF/SF-stimulated cells. Consistent with the levels of JunD expression
being functionally important for basal expression of the urokinase
promoter, we found that overexpression of wild type JunD inhibited the
induction of the urokinase promoter by HGF/SF. These data suggest that
the induction of urokinase by HGF/SF is regulated by a
Grb2/Sos1/Ha-Ras/c-Raf/RhoA/Mek1/Erk2/c-Jun-dependent mitogen-activated protein kinase pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-chain exposed on the cell surface. The 145-kDa
-chain
spans the plasma membrane and has a kinase domain within the
cytoplasmatic region (5). Upon HGF/SF binding, the Met receptor
dimerizes and becomes phosphorylated on tyrosines located in the
cytoplasmatic region of the
-chain. These tyrosine residues then act
as specific binding sites for adaptor proteins (6) with Src homology 2 domains, which transmit signals intracellularly, one effector being Ras
(7). The exact targets of HGF/SF-Met signaling downstream of Ras have
not been elucidated, and it is unclear which signal transduction
pathways mediate the diverse biological signals of HGF/SF.
Raf
Mek
Erk-MAPK cascade, is
typically stimulated by growth factors and mitogens such as HGF/SF,
EGF, and fibroblast growth factor (8). Two other MAPK pathways (p38-
and Jnk-MAPK) are stimulated primarily by stress, cytokines, or
hormones and require activation of a member of the Rho family, although
Ras can participate. Phosphorylation of MAPKs by both tyrosine and
threonine residues leads to activation of various transcription
factors, including members of the AP-1 family encompassing Jun and Fos
family members that can homodimerize and heterodimerize after
activation (10). Fine tuning of the transcriptional response is
dependent upon which member of the AP-1 family is activated. Although
c-Jun has been shown to have activating properties on gene expression,
binding of JunD inhibits promoter activity (11).
B (24, 25), which are
important for both constitutive and regulated urokinase expression
(26). An additional important enhancer sequence of the urokinase
promoter is a combined PEA3/AP-1 element (24) homologous to the
polyoma virus enhancer A element (27) that has been shown to
be a target for transcriptional activation by oncogenes in several
promoters (28).
-subunit and a 34-kDa
-subunit. Activation involves
the formation of a complex between HGF/SF precursor protein and
urokinase binding to the Met receptor (30). Considering that urokinase
has been previously shown to be up-regulated by HGF/SF (13, 14), we
determined the signal transduction pathway connecting the Met receptor
with the transcription factors regulating urokinase gene expression.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
N is an amino-terminal Src homology 3 domain
deletion mutant of Grb2 (35). RasN17 is a dominant-negative Ha-Ras
construct in which amino acid 17 is substituted by asparagine (36). The
dominant-negative RhoN19 mutant displays a mutation at amino acid 19 where serine is replaced by asparagine (37). The RafC4 expression
plasmid encodes the c-Raf protein lacking the carboxyl-terminal kinase
domain. Raf BXB is an in-frame deletion of amino acids 26-302 of c-Raf
rendering the serine-threonine kinase constitutively active (38). The
TAM 67 vector encodes a c-Jun protein lacking the trans-activation
domain between amino acid 3-122 but retains the leucine zipper and
DNA-binding domains (10). The A-Fos construct has an amphipathic acidic
extension appended to the amino terminus of the Fos leucine zipper that binds the basic region of Jun, thus preventing binding of the basic
region of Jun/Fos to DNA (39). In the JunD/EB1 vector, the dimerization
domain of wt JunD is replaced by the heterologous homodimerization
domain of the Ebstein-Barr virus transcription factor EB1 and binds DNA
exclusively as a homodimer (40).
-32P]ATP) for 15 min at 30 °C. The
reaction was terminated by adding 2× reducing sample buffer and
heating to 95 °C for 3 min. The beads were removed by
centrifugation, and the supernatant was separated in a 15% SDS-PAGE
gel. The gel was dried and autoradiographed for 6 h at
80 °C.
-galactosidase expression vector. A solution
containing 124 mM calcium chloride, 15 mM
HEPES, pH 7.1, 280 mM NaCl, 1.5 mM
Na2HPO4 was added dropwise to plasmid DNA with
continuous stirring and added to 60% confluent cells. After 6 h
the cells were rinsed twice with phosphate-buffered saline, changed to
fresh 10% fetal bovine serum-containing medium, and cultured for an
additional 42 h. The cells were harvested and lysed by repeated
freeze-thaw cycles in 0.25 M Tris-HCl, pH 7.8. Transfection
efficiencies were determined by assaying for
-galactosidase
activity. After normalization for transfection efficiency, CAT activity
was measured by incubating cell lysates at 37 °C with 4 µM [14C]chloramphenicol and 1 mg/ml acetyl
coenzyme A. The mixture was separated by extraction with ethyl acetate,
and acetylated products were separated on thin layer chromatography
plates using chloroform/methanol as the mobile phase. Reactions were
visualized by autoradiography, and radioactivity was quantified using a
Molecular Dynamics 445 SI PhosphorImager.
-32P]ATP) oligonucleotide were added in the absence
or presence of a 100-fold excess of the wild type or mutated competitor
sequence, and binding was allowed for 15 min. Subsequently, 1 µg of
the indicated antibody was added, and incubation was continued for 1 h at 4 °C. The reaction mixture was electrophoresed in a 5% polyacrylamide gel using 0.5× TBE (89 mM Tris, 89 mM boric acid, 1 mM EDTA) running buffer. The
gel was dried and exposed to x-ray film overnight at
80 °C. The
sequences are shown in Fig. 1.
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Fig. 1.
Sequences used in mobility shift assays.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
HGF/SF induces the urokinase promoter and
urokinase secretion in NIH 3T3-Methum cells.
A, cell lysates were resolved on a 10% PAGE and human Met
(h-Met) detected by Western blot analysis using an antibody
to human Met. The band at ~170 kDa represents the single chain
precursor form of Met, whereas that at ~140 kDa represents the
-chain of the mature Met heterodimer. The experiment was repeated
twice. B, NIH 3T3-Methum cells at 70%
confluency were transiently transfected with 5 µg of a CAT reporter
driven by the wild type urokinase promoter (uPA CAT). After
6 h, the medium was changed, and cells were cultured for 16 h, after which they were stimulated with the indicated amounts of
HGF/SF for 24 h. The cells were lysed and assayed for CAT activity
(B). The conversion of [14C]chloramphenicol to
acetylated derivatives was determined using a PhosphorImager and
expressed as fold induction relative to basal urokinase promoter
activity (C). The data shown represent the average values
and standard deviations of three independent experiments. D
and E, NIH 3T3-Methum cells, were grown to 80%
confluency in the presence of 10% fetal calf serum changed to
serum-free medium and 2 h later stimulated with the indicated
amounts of HGF/SF. After an additional 24 h, the conditioned
medium was harvested and clarified by centrifugation, and the cells
were counted. Conditioned medium, normalized to cell number, was
denatured and electrophoresed in a 10% SDS-PAGE containing 0.2% (w/v)
casein in the presence (D) or absence (E) of 5 µg/ml plasminogen. The gels were incubated in the presence of 2.5%
Triton X-100 for 2 h and overnight in a buffer containing 10 mM CaCl2. The gels were stained for protein
with 0.25% Coomassie. The experiment was repeated four times.
N) antagonizes the induction of the urokinase
promoter by HGF/SF as well as basal promoter activity, whereas the Grb2
(pCGN) vector does not show an effect (Fig.
3). Overexpression of a dominant-negative
Sos1 (33) also reduced inducible and constitutive activation of the
urokinase promoter by HGF/SF (Fig. 3). In contrast, a dominant-negative
PI 3-kinase (p110*
kin) (45) did not show any effect on basal or
inducible urokinase gene regulation (data not shown), and wortmannin, a known inhibitor of PI 3-kinase, showed no inhibition of urokinase promoter activity (see Fig. 5A). Together these data
indicate a role for Grb2/Sos1 but not for PI 3-kinase in the regulation of urokinase expression by HGF/SF-Met signaling.
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Fig. 3.
The HGF/SF-dependent stimulation
of the urokinase promoter activity is inhibited by the co-expression of
plasmids encoding dominant-negative Grb2 and Sos1. NIH
3T3-Methum cells were co-transfected with 5 µg of a CAT
reporter driven by the wild type urokinase promoter (uPA-CAT), a
-galactosidase-expressing vector, and 2.5 µg of an expression
vector encoding a dominant-negative Grb2 (Grb2
N) or 5 µg of a
dominant-negative Sos1 (
m Sos1) or the wt Sos1. The cells were
stimulated with 200 ng/ml HGF/SF for 24 h, lysed, and assayed for
CAT activity. The conversion of [14C]chloramphenicol to
acetylated derivatives was determined using a PhosphorImager and
expressed as fold induction relative to basal urokinase promoter
activity. The data shown represent the average values and standard
deviations of four independent experiments.
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Fig. 4.
Involvement of Ha-Ras, c-Raf, and RhoA in
regulation of the urokinase promoter by HGF/SF. A, NIH
3T3-Methum cells were transiently transfected using 5 µg
of a CAT reporter driven by the wild type urokinase promoter
(uPA-CAT), a -galactosidase-expressing vector, and, where
indicated, 2 µg of a dominant-negative Ha-Ras (RasN17) or
5 µg of a dominant-negative c-Raf (RafC4). B,
2.5 µg of a dominant-negative RhoA (RhoN19) and an
equimolar amount of each control vector (pSV2neo,
pBGC4, and pCMV5). The cells were stimulated with
200 ng/ml HGF/SF. C, NIH 3T3-Methum cells were
co-transfected with 5 µg of a urokinase promoter-regulated CAT
reporter (uPA-CAT), a
-galactosidase-expressing vector, 6 µg of a vector bearing a constitutively active c-Raf (Raf
BXB), or the vector (pMNC), and 3 µg of a
dominant-negative RhoA (RhoN19), or the vector
(pCMV5). Both vectors were added in equimolar amounts. For
A-C the cells were cultured for 48 h, and cell
extracts were assayed for
-galactosidase activity, and equivalent
amounts (after correcting for transfection efficiencies) were incubated
with [14C]chloramphenicol. The mixture was extracted with
ethyl acetate and subjected to thin layer chromatography. The
conversion of [14C]chloramphenicol to acetylated
derivatives was determined using a PhosphorImager and expressed as fold
induction relative to basal urokinase promoter activity. The data shown
represent the average values and standard deviations of three to five
independent experiments.
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Fig. 5.
Abrogation of the
HGF/SF-dependent induction of urokinase promoter activity
by a MEK1-specific inhibitor (PD 098059) and a dominant-negative Erk2
expression vector. A, NIH 3T3-Methum cells
were transiently transfected with the urokinase CAT reporter
(uPA-CAT) and stimulated with 200 ng/ml HGF/SF.
Concomitantly the inhibitors PD 098059 (20 µM), SB 203580 (20 µM), and wortmannin (100 nM) dissolved in
Me2SO were added, or an equivalent amount of
Me2SO without inhibitor was applied. B, NIH
3T3-Methum cells were co-transfected with the urokinase CAT
reporter, a -galactosidase-expressing vector, and 5 µg of an
expression vector encoding a dominant-negative Erk2 (mt
Erk2). For both A and B, the cell extracts,
corrected for differences in transfection efficiency, were assayed for
CAT activity. The conversion of [14C]chloramphenicol to
acetylated derivatives was determined using a PhosphorImager and
expressed as fold induction relative to basal urokinase promoter
activity. The data shown represent the average values and standard
deviations of three independent experiments. C, effect of
HGF/SF and EGF on Erk-MAPK activity. Cells were stimulated with the
indicated amounts of HGF/SF and EGF for 24 h, and equal amounts of
protein were subjected to immunoprecipitation with antibody to Erk, and
in vitro kinase assays were performed using myelin basic
protein as the substrate. Transfer of the upper gel part to
nitrocellulose and Western blotting with an Erk antibody demonstrated
the same level of Erk in all samples (data not shown). The results
shown are representative of three experiments.
2345,
2109,
and
1870 bp) of the urokinase promoter, and the cells were stimulated
for 24 h with HGF/SF (Fig. 6,
A and B). A strong activation was evident with
2109 bp of 5'-flanking sequence, which was not further augmented with
longer stretches of the urokinase promoter (
2345 bp). The
1870 bp
urokinase CAT construct showed only a low activity and could not be
stimulated by HGF/SF. We have previously shown that an AP-1-binding
site at
1880 bp (AP-1B) and a combined PEA3/AP-1 site at
1967
(AP-1A) residing in the region between
2109 and
1870 is critical
for the stimulation of the urokinase promoter by Ha-Ras (42, 50). Because HGF/SF regulates urokinase through Ha-Ras (Fig. 4A)
we performed mobility shift assays with nuclear extracts (43) of either
HGF/SF-stimulated or unstimulated NIH 3T3-Methum cells and
incubated them with 32P-labeled oligonucleotides spanning
the AP-1A (Fig. 6C) or AP-1B (data not shown) site. The
mobility of the AP-1A containing oligonucleotide was reduced and showed
one retarded complex in the presence of NIH 3T3-Methum
nuclear extracts (Fig. 6C, asterisk), whereas
nuclear extract from HGF/SF-induced NIH 3T3-Methum cells
showed two different retarded complexes (Fig. 6C,
arrows). The specificity of the AP-1 binding interaction was
indicated by the ability of an excess of unlabeled oligonucleotide to
compete for the binding (Fig. 6C, lanes 2 and
7). Because the AP-1A site is a combined PEA3/AP-1 site, we
were interested in determining which part of this combined site is
important for binding of transcription factors of HGF/SF-induced and
uninduced NIH 3T3-Methum nuclear extracts. Mutation of the
AP-1 part of the PEA3/AP-1 site failed to specifically compete the
retarded complex (Fig. 6C, lanes 3 and
8), whereas mutation of the PEA3 site still showed specific
competition (Fig. 6C, lanes 4 and 9).
These results indicate that nuclear factors from HGF/SF-stimulated and
unstimulated NIH 3T3-Methum cells have specific DNA binding
activity toward the AP-1 part but not to the PEA3 part of the combined
PEA3/AP-1 site at
1967 bp. Mobility shift assays employing an
oligonucleotide spanning the AP-1B site (a consensus AP-1-binding site)
showed a similar result (data not shown).
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Fig. 6.
Induction of AP-1 DNA binding activity by
HGF/SF. A, NIH 3T3-Methum cells were
transiently transfected with 5 µg of a CAT reporter driven by the
indicated 5'-deleted fragment of the urokinase promoter and a vector
encoding the -galactosidase gene, and the cells were stimulated with
200 ng/ml HGF/SF, lysed, and assayed for CAT activity. B,
the conversion of [14C]chloramphenicol to acetylated
derivatives was determined using a PhosphorImager and expressed as fold
induction relative to the
2109 bp urokinase promoter. The data shown
represent the average values and standard deviations. C,
nuclear extracts (n.e.) from NIH 3T3-Methum
cells treated with or without 100 ng/ml of HGF/SF for 12 h were
incubated with 2 × 104 cpm of a Klenow
-32P-end-labeled oligonucleotide spanning the AP-1A site
(combined PEA3/AP-1 site at
1967) in the absence or presence of a
100-fold excess of the indicated competitors, and electrophoresed in a
5% polyacrylamide gel. The AP-1A mt competitor oligonucleotide has
point mutations in the AP-1 part, whereas the Ets mt competitor has
point mutations in the PEA3 part of the combined PEA3/AP-1 site,
respectively (for sequences see Fig. 1). Arrows indicate
retarded complex of HGF/SF-stimulated NIH 3T3-Methum cells,
and the asterisk indicates unstimulated cells. D,
NIH 3T3-Methum cells were transiently transfected with the
urokinase CAT reporter (uPA-CAT) and 2.5 µg of an
expression vector that inhibits DNA binding of AP-1 proteins
(A-Fos) or equimolar amounts of the empty expression vector
(pCMV 500) and stimulated with 200 ng/ml HGF/SF, and the
cells were lysed and assayed for CAT activity. The data shown represent
the average values and standard deviations. For A-D the
results shown are representative of three or more separate
experiments.
1967 and
1880 bp for stimulation of the urokinase promoter by HGF/SF, we
carried out mobility shift assays to investigate the binding of JunD to
these sites. Two oligonucleotides (AP-1A and AP-1B) spanning either of
the AP-1 sites were incubated with nuclear extracts from
HGF/SF-stimulated or unstimulated NIH 3T3-Methum cells and
a polyclonal antibody raised against the carboxyl-terminal part of
JunD, which also harbors the DNA-binding domain (Fig. 7B).
The JunD-specific antibody produced a supershift in stimulated and
unstimulated cells, suggesting that this protein is a component of the
DNA-binding complex. However, the intensity of the retarded band was
greater for unstimulated (Fig. 7B, lanes 4 and
11) than for stimulated (Fig. 7B, lanes
5 and 12) NIH 3T3-Methum cells, and binding
of the JunD antibody to nuclear extracts from unstimulated cells
blocked DNA-protein complex formation markedly. This is consistent with
the higher JunD protein content found in nuclear extracts from
unstimulated NIH 3T3-Methum cells (Fig. 7A).
Overexpression of a dominant-negative JunD, which lacks amino acids
1-162 corresponding to the trans-activation domain, reduced basal
activity of the urokinase promoter in uninduced NIH
3T3-Methum cells (Fig. 7C), indicating that JunD
is required for basal urokinase activity in NIH 3T3-Methum
cells.
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Fig. 7.
Stimulation of NIH 3T3-Methum
cells with HGF/SF reduces nuclear JunD content. A,
nuclear extracts (n.e.) from NIH 3T3-Methum
cells treated with or without 100 ng/ml of HGF/SF were resolved on a
10% SDS-PAGE and analyzed by Western blot analysis using an antibody
to JunD in the presence (+) or the absence ( ) of the immunizing
peptide. The product at ~39 kDa represents JunD. B,
nuclear extracts were prepared from the NIH 3T3-Methum cell
clone treated with or without 100 ng/ml of HGF/SF and incubated with
Klenow
-32P-end-labeled oligonucleotides spanning the
AP-1B (lanes 2-7) or the AP-1A (lanes 9-14)
sites of the urokinase promoter. After 15 min, antibody to JunD or IgG
as control (1 µg) was added, and the reaction mixture was
subsequently subjected to gel electrophoresis. The data are
representative of three experiments. C, NIH
3T3-Methum cells were co-transfected with a CAT reporter
driven by the wild type urokinase promoter and 3 µg of an expression
vector encoding a trans-activation domain lacking JunD
(RSV
JunD) or the empty expression vector
(pLMVP) and stimulated with 200 ng/ml HGF/SF, and the cells lysed and
assayed for CAT activity. The data shown represent the average values
and standard deviations of three independent experiments.
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Fig. 8.
Induction of the urokinase promoter by HGF/SF
involves down-regulation of JunD. A, NIH
3T3-Methum cells were transiently transfected using 5 µg
of a CAT reporter driven by the wild type urokinase promoter
(uPA-CAT) and varying amounts of wt JunD cDNA (vector
pLMVP) or 5 µg of the expression vector JunD/EB1
(B), an artificial JunD construct that can form only JunD
homodimers, and stimulated with HGF/SF. For A and
B, the cell extracts, corrected for differences in
transfection efficiency, were assayed for CAT activity.
[14C]Chloramphenicol conversions were determined with a
PhosphorImager and expressed as fold induction relative to basal
urokinase promoter activity. The data shown represent the average
values and standard deviations of three or more separate
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Raf
Mek
Erk-MAPK signaling cascade, there is evidence
that it represents a subset of a complex array of signaling
interactions at several levels (46). This might also apply for the
regulation of the urokinase promoter. Each component of the signaling
cascade identified here as being involved in the regulation of the
urokinase promoter by HGF/SF may be activated by other than the
identified upstream components.
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Fig. 9.
Proposed model of urokinase promoter
activation by HGF/SF.
The transcriptional requirements for urokinase promoter stimulation by
HGF/SF, 12-O-tetradecanoylphorbol-13-acetate, or Ha-Ras are
similar (42, 60). Mutation of both AP-1 sites at 1967 and
1880 bp
in the urokinase promoter substantially impaired the ability of HGF/SF
as well as 12-O-tetradecanoylphorbol-13-acetate (60) to
stimulate the promoter. The AP-1 site at
1967 bp is a combined
PEA3/AP-1 site, and a remarkable difference between 12-O-tetradecanoylphorbol-13-acetate or Ha-Ras (42) and
HGF/SF induction of the urokinase promoter is that the PEA3 part of
this site is not required for HGF/SF induction. Addition of HGF/SF to
the NIH 3T3-Methum cell clone shows a change from one
complex (containing JunD) that had bound to the AP-1 sites to two
complexes. Although we did not identify every single protein in these
complexes, we are certain that they involve AP-1 family members.
Transient overexpression of A-Fos, a hybrid protein that inhibits DNA
binding of all endogenous AP-1 family members (39), completely
down-regulated inducible urokinase expression, and a dominant-negative
c-Jun construct inhibited the induction by 70%. Thus the induction of
the urokinase promoter is mediated at least partially by c-Jun.
Several of our observations are consistent with a role for JunD in the constitutive activity of the urokinase promoter. Unstimulated NIH 3T3-Methum cells have a higher JunD content than HGF/SF-stimulated cells, and the amount of JunD bound to oligonucleotides spanning the AP-1A and AP-1B sites of the urokinase promoter was higher for untreated cells. The high JunD expression of untreated NIH 3T3-Methum cells accounts for part of the constitutive activity of the urokinase promoter, because a dominant-negative JunD reduced the basal activity of the urokinase promoter by 60%. When stimulated with HGF/SF, the JunD content of the cell decreases, and less JunD is bound to the urokinase promoter so that other AP-1 family members, including c-Jun can mediate the activation of the urokinase promoter by HGF/SF. Similarly, Pfarr et al. (11) showed a high JunD level in resting fibroblasts and a degradation of JunD following serum stimulation, whereas in parallel the level of c-Jun increased. Overexpression of JunD partially reversed Ha-Ras transformation and growth in soft agar. Together with other studies (11, 40), this lead to the assumption that JunD has an inhibitory effect on cell growth and anti-mitogenic effects. To test this idea further, we overexpressed wt JunD in NIH 3T3-Methum cells and showed that the induction of the urokinase promoter by HGF/SF could be reduced by 50%. This indicates that JunD can inhibit growth factor-induced activation of a tumor-associated protease. The inhibition of HGF/SF-mediated urokinase activation by JunD could result from antagonizing the c-Jun function, by forming JunD/c-Jun heterodimers. Interestingly, higher amounts of wt JunD caused a decrease in the repression of HGF/SF induction of urokinase promoter activity. It is thus possible that the ratio of JunD to c-Jun is an important feature for the regulation of urokinase by HGF/SF. Alternatively, high level expression of JunD could result in titration of corepressors for JunD from the urokinase promoter and impair transcriptional regulation.
In conclusion, using an NIH 3T3 cell line that has been stably
transfected with the Met receptor, we have demonstrated that HGF/SF
induces the urokinase promoter by a
Grb2/Sos1/Ha-Ras/c-Raf/RhoA/Mek1/Erk2/c-Jun-mediated signal
transduction pathway. Considering the strong evidence implicating urokinase (20, 21, 61) and HGF/SF-Met signaling (2, 3) in tumor cell
invasion and metastasis, these findings raise the possibility that
interfering with this signaling cascade may reduce HGF/SF-induced
urokinase synthesis and extracellular matrix degradation in cancer cells.
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ACKNOWLEDGEMENTS |
---|
We are very grateful to the following
researchers for providing expression constructs: Drs. Y. Nagamine
(Friedrich Miescher Institute, Basel, Switzerland) and M. Sakaue (Kobe
University School of Medicine, Kobe, Japan) for the Sos1 constructs,
Dr. U. Knaus (Scripps Institute, La Jolla, CA) for the RhoN19 and the
Rac1 construct, Dr. U. Rapp (Institut für Strahlenbiologie, University of Würzburg, Germany) for the RafC4 and Raf BXB
expression constructs, Dr. M.-C. Hung (M. D. Anderson Cancer Center,
Houston, TX) for the Grb2N vector, Drs. J. Frost and M. Cobb
(Southwestern Medical Center, Dallas, TX) for the Erk1 and Erk2
expression vectors, and Dr. M. Castellazzi (INSERM-U412, Lyon, France)
for the JunD/EB1 construct. We thank H. Daub and E. Zwick (Max-Planck
Institut, Martinsried, Germany) for the RasN17 construct and for
technical help with the MAPK assays. We are indebted to Dr. A. Saltiel
(Parke-Davis Inc., Ann Arbor, MI) for the generous gift of
MEK-inhibitor (PD 098059) and Dr. R. Schwall (Genentech Inc., South San
Francisco, CA) for purified human HGF/SF. The work would not have been
possible without the urokinase promoter-driven CAT reporters kindly
provided by Dr. F. Blasi (DIBIT, H. S. Raffaele Scientific
Institute, Milan, Italy). Finally, we thank Dr. D. Bohmann (EMBL,
Heidelberg, Germany) for helpful discussions and Dr. D. Boyd (M. D.
Anderson Cancer Center, Houston, TX) for comments on the manuscript and
continuous support.
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FOOTNOTES |
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* This work was supported by Wilhelm-Sander Stiftung Grant 96.041.1 (to E. L. and M. S.) and by the NCI, DHHS, National Institutes of Health under contract with Advanced Bioscience Laboratories (to M. J. and G. F. V. W.).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.
§ This study was performed in partial fulfillment of a Ph.D. thesis.
** To whom correspondence should be addressed: Dept. of Obstetrics and Gynecology, Technische Universität München, Klinikum rechts der Isar, Ismaninger Str. 22, D-81675 München, Germany. Tel.: 49-89-4140-2429; Fax: 49-89-4140-4846; E-mail: ernst.lengyel{at}lrz.tum.de.
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ABBREVIATIONS |
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The abbreviations used are: HGF/SF, hepatocyte growth factor/scatter factor; MAPK, mitogen-activated protein kinase; Erk, extracellular regulated kinase; CAT, chloramphenicol acetyltransferase; bp, base pair; AP-1, activation protein 1; EGF, epidermal growth factor; wt, wild type; PAGE, polyacrylamide gel electrophoresis; PI, phosphatidylinositol; uPA, urokinase-type plasminogen activator.
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REFERENCES |
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