From the Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada
Received for publication, October 25, 2002
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ABSTRACT |
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Human tissue factor pathway inhibitor-2 (hTFPI-2)
is a 32-kDa serine protease inhibitor that is associated with the
extracellular matrix. hTFPI-2 inhibits several extracellular
matrix-degrading serine proteases and may play a role in tumor invasion
and metastasis. To study the signal transduction pathway that leads to
the activation of the hTFPI-2, we cloned the potential promoter region
of this gene adjacent to a heterologous luciferase reporter gene.
Phorbol 12-myristate 13-acetate (PMA) induced the luciferase reporter gene in HEK293 cells and other epithelial cell lines, such as the human lung carcinoma A549 cells, the breast carcinoma MCF7 cells,
and the cervical HeLa cells. This PMA induction was blocked with the
MEK inhibitor UO126, suggesting that the PMA-induced activation of the
hTFPI-2 promoter is mediated through MEK. Furthermore, epidermal
growth factor induced the luciferase reporter gene in HeLa cells.
Cotransfection of the luciferase construct with constitutively active
components of the Ras/Raf/MEK/ERK pathway in EcR-293 cells lead to a 7- to 92-fold induction of the luciferase reporter gene, indicating that
regulation of hTFPI-2 is mediated through this pathway. A series of
luciferase reporter gene constructs with progressive deletions of the
5'-flanking region suggested that the minimal basal promoter activity
is located between nucleotide positions Growth hormones and the tumor promoting agent
PMA1 initiate diverse
intracellular signaling pathways that lead to the phosphorylation of
transcription factors and ultimately to the regulation of target genes.
Among the pathways often used to transduce signals are the
mitogen-activated protein kinase (MAPK) cascades. These cascades consist of a three-kinase module that includes an MEK kinase (MEKK), which activates an MAPK/ERK kinase (MEK), which in turn activates a
MAPK (1). Three well characterized MAPKs have been described in
mammalian cells: the mitogen-responsive ERK, the stress-responsive JNK/SAPK, and the p38 MAPK. The Ras/Raf/MEK/ERK signaling
cascade regulates cell proliferation and differentiation (2).
Components of this pathway are often activated in human tumors and
oncogenic Ras, and constitutively activated ERKs have been found in a
large variety of malignancies (3-5). We have used transcript profiling to identify genes that are differentially regulated by this pathway. Among the many activated genes, we have identified the human tissue factor pathway inhibitor-2 (hTFPI-2) as a gene that is highly up-regulated by the ERK/MAPK pathway.
hTFPI-2 is a 32-kDa serine proteinase inhibitor with three tandem
Kunitz-type domains (6, 7) and has high homology to hTFPI-1, a
regulator of the extrinsic blood coagulation pathway. The second
Kunitz-type domain of hTFPI-1 binds to factor Xa, and this complex
inhibits the activity of the factor VIIa-tissue factor complex through
interaction of the first Kunitz-type domain in hTFPI-1 and the active
site of VIIa/TF (8). Despite the high homology of hTFPI-2 to hTFPI-1,
hTFPI-2 is a weak inhibitor of the activation of factor X (9) and
hTFPI-2 poorly inhibits tissue factor. However, hTFPI-2 inhibits the
tissue factor-factor VIIa complex and a variety of serine proteases,
including trypsin, plasmin, plasma kallikrein, chymotrypsin, and
cathepsin G, but it does not inhibit thrombin, urokinase-type
plasminogen activator, and tissue-type plasminogen activator (6, 9).
Most of the hTFPI-2 expressed in dermal fibroblasts and
endothelial cells localizes within the extracellular matrix, probably
bound to heparan sulfate (10-12). hTFPI-2 can prevent the conversion
of Pro-MMP-1 (matrix metalloprotease 1, interstitial collagenase) and
Pro-MMP-3 (matrix metalloprotease 3, stromelysin-1/transin-1) into
MMP-1 and MMP-3 by plasmin and trypsin (13) and therefore might
indirectly regulate matrix proteolysis and connective tissue turnover.
The role of hTFPI-2 in cancer progression is not completely elucidated.
On one hand, hTFPI-2 has an anti-invasive effect that might be mediated
via inhibition of plasmin that activates proteases promoting
degradation of the extracellular matrix and tumor invasion. Several
tumor cell lines were less invasive when they were stably transfected
with hTFPI-2 cDNA (14-17). On the other hand, hTFPI-2 has been
shown to have a pro-invasive effect in hepatocellular carcinoma cells
(18).
In this study, we investigated the signaling pathway and
transcriptional elements that regulate the expression of the
hTFPI-2 gene in epithelial cells. Although it has been shown
that PMA can stimulate hTFPI-2 gene expression in glioma cells and that the promoter region Materials--
The Phorbol 12-myristate 13-acetate was ordered
from Sigma-Aldrich Chemicals Co. (St. Louis, MO) and recombinant human
epidermal growth factor from Austral Biologicals (San Raman CA). The
LipofectAMINE Plus reagent as well as the Ecdysone-Inducible Mammalian
Expression system, including EcR-293 cells, zeocin, and pronasteron A,
and the expression vector pIND were purchased from Invitrogen Corp. (Carlsbad, CA). The MEK inhibitor UO126 and the Luciferase assay system
were obtained from Promega Corp. (Madison, WI). The Phospho-p44/42 MAPK
(Thr202/Tyr204) antibody was purchased from
Cell Signaling Technology (Beverly, MA). The c-Myc (9E10) monoclonal
antibody and the MEK-1 (C-18) and Raf-1 (C-12) polyclonal antibodies
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The
QuikChangeTM site-directed mutagenesis kit was from
Stratagene (La Jolla, CA). The FuGENE6 transfection reagent was
obtained from Roche Diagnostics Corp.
Cell Culture--
The HEK293, HeLa, A549, and MCF7 cell lines
were obtained from American Type Culture Collection (ATCC, Rockville,
MD). All the cell lines were cultured in Dulbecco's modified Eagle's
medium (DMEM) (Wisent Inc., St. Bruno, Quebec, Canada) with 10% fetal bovine serum (HyClone Laboratories, Mississauga, Ontario, Canada), except for the A549 cell line, which was cultured in DMEM supplemented with 5% fetal bovine serum.
Plasmids--
The plasmid pRK5 containing the myc-RasV12 gene
with a glycine to valine mutation at amino acid position 12 and a
sequence (EQKLISEEDLGS) containing a myc epitope inserted between a
methionine and a threonine (amino acid positions one and two) was a
generous gift from Nathalie Lamarche-Vane, McGill University,
Montreal, Canada. The plasmid was digested with the restriction
enzyme ClaI and the 5'-overhang filled in with Klenow. The
myc-RasV12 fragment was subsequently released by ApaI and
cloned into the EcoRV/ApaI site of pIND.
pAN130-containing Raf-1 (20) served as a template to amplify the
carboxyl-terminal, catalytic domain of Raf-1 (RafCT) using the
oligonucleotides O-1 and O-2 listed in Table
I. At the same time an EcoRI
site (printed in boldface) was created to facilitate the cloning
of the amplified 962-bp fragment into the
EcoRI/XhoI sites of pcDNA3. A Kozak sequence
(printed in italic) was inserted in-frame by cloning the
phosphorylated, annealed oligonucleotides O-3 and O-4 (Table I) into
the EcoRI site of RafCT, creating an AflII site
(printed in boldface). The nucleotide sequence was verified by
sequencing. The Kozak-RafCT fragment was released by
AflII/XhoI and cloned in the corresponding sites
of pIND.
MEK-1 was released from pAN104 (20) by BamHI/XhoI
restriction digest and cloned into the corresponding sites of
Bluescript KS. MEK-1SD was created by site-directed mutagenesis
(QuikChangeTM site-directed mutagenesis kit, Stratagene)
using two complementary oligonucleotides. The nucleotides that were
changed to obtain a serine to aspartic acid mutation in MEK-1SD are
underlined in O-5 in Table I. The nucleotide sequence was verified by
sequencing, and the MEK-1SD was released from Bluescript with
BamHI/XhoI and cloned into the corresponding
sites of pIND.
The plasmid mycCMV5-ERK2-MEK1-LA was kindly provided by Melanie H. Cobb, University of Texas Southwestern, Dallas, TX. The plasmid was
digested with HindIII, the 5'-overhand was filled in with
Klenow fragment, and ERK2-MEK1-LA was subsequently released by
KpnI and cloned into the KpnI/EcoRV
sites of pIND.
Amplification of the hTFPI-2 Promoter Region--
Genomic DNA
was isolated from 293 cells. The cells were washed in PBS, lysed in a
buffer containing 10 mM NaCl, 10 mM EDTA, 0.5%
Sarkosyl, and 10 mM Tris, pH 8, and incubated with
proteinase K (10 mg/ml) at 50 °C overnight. After two phenol and two
chloroform extractions, the genomic DNA was ethanol-precipitated and
dissolved in TE, pH 8. A 1.5-kb fragment of the 5'-flanking region of
the hTFPI-2 was amplified with an Expand High Fidelity PCR system (Roche Molecular Biochemicals) using the oligonucleotides O-7 and O-8
(Table I) to create a KpnI and a BglII
restriction site (printed in boldface). This
KpnI/BglII fragment was cloned into the plasmid
pXP2, which contains the firefly luciferase reporter (21) and was a
generous gift from Mark Featherstone, McGill University, Montreal,
Canada. The resulting construct was named p-1511-luc. The sequence
was found identical to the one published by Kamei (22) except for a C
to A change at nucleotide position Promoter Deletion Constructs--
The hTFP-2
promoter/luciferase reporter plasmids p-1293-luc, p-1055-luc,
p-881-luc, p-733-luc, p-384-luc, p-222-luc, and p-89-luc were created
by restriction digest on the original p-1511-luc plasmid. The
p-1511-luc plasmid was digested with DraI, EarI, NarI, MscI, EcoNI, EcoRI,
and SmaI, respectively, and the DNA fragments were released
by BglII digest. The 5'-overhang created by EarI,
NarI, EcoNI, and EcoRI restriction
digest was filled in with Klenow fragment, and the restriction
fragments were cloned into the SmaI/BglII site of pXP2.
Mutagenesis of Single Potential Transcription Binding
Sites--
The putative Sp1 transcription factor binding site
GGGGCGG between nucleotide positions Transient Transfection--
For inhibitor studies, the HEK293
cells (150,000 cells/well) were seeded the day prior to transfection.
The cells were cotransfected with 1 µg of p-1511-luc and 70 ng of a
GFP-spectrin control plasmid (23). 24 h after transfection, the
cells were serum-starved for 24 h. Where indicated, the
MEK1/2-specific inhibitor UO126 was added 15 min prior to PMA
induction. The cells were harvested in phosphate-buffered saline (PBS)
and divided into two tubes. Cells in one tube were lysed using cell
culture lysis reagent (Promega) and centrifuged at 12,000 × g for 5 min, and the cell extract was assayed for firefly
luciferase activity using the luciferase reporter assay system
(Promega). Light intensity was measured by using a microtiter plate
luminometer (DYNEX Technologies, Inc., Chantilly, VA). The cells
in the second tube were trypsinized and washed, and the percentage of
cells that express GFP was determined by cytofluorimetry (EPICS XL-MCL)
to control for transfection efficiency. Determination of protein by
the Bradford assay (Bio-Rad) was carried out, controlling for
harvesting efficiencies. Luciferase activity was expressed as firefly
light units/µg of protein and normalized for transfection efficiency.
HEK293, HeLa, A549, and MCF7 cells were seeded into six-well plates at
150,000, 250,000, 250,000, and 400,000 cells/well, respectively, 1 day
prior to transfection. All cells were transfected with 1 µg of the
reporter gene constructs. HEK293 and HeLa cells were transfected with
FuGENE6 at a 3 µl of FuGENE/1 µg of DNA ratio, and A549 and MCF7
cells were transfected with LipofectAMINE Plus according to the
manufacturer's protocol. 24 h after transfection, the cells of
each well were split into six wells of a 24-well plate. 7 h later,
the cells were serum-starved overnight and subsequently treated with
PMA (250 nM) or EGF (50 ng/ml) as indicated. Cell extracts
were lysed and analyzed as described above. The -fold stimulation of
luciferase was calculated as firefly light units/µg of protein of
PMA- or EGF-treated cells divided by the firefly light units/µg of
protein of nontreated cells.
Two days prior to transfection, EcR-293 cells cultivated in DMEM
supplemented with 10% fetal bovine serum and Zeocin (400 µg/ml) were
seeded into six-well plates at 200,000 cells/well. EcR-293 cells stably
express the modified ecdysone receptor. A gene of interest, cloned into
a pIND-based inducible expression vector, can be induced with the
ecdysone analog pronasteron A that binds to the ecdysone receptor.
Cells were transfected with 6 µl of FuGENE6 and 1.07 µg of total
DNA (0.9 µg of RasV12, RafCT, MEK-1SD, ERK2-MEK1-LA, or pIND control
vector, 0.1 µg of reporter plasmid, and 70 ng of GFP control
plasmid). The medium containing fetal bovine serum was replaced 24 h after transfection with DMEM without serum, pronasteron A was
added 4 h later to the medium to induce protein expression at a
concentration of 6 µM unless indicated differently, and
cells were harvested 20 h later. Luciferase activity was expressed
as firefly light units/µg of protein and normalized for transfection efficiency.
Western Blots--
Cells were harvested and washed with PBS. The
cells were lysed in PBS containing 0.1 mM sodium vanadate,
10 mM sodium pyrophosphate, 1.5% Triton X-100, and
the proteinase inhibitors aprotinin (10 µg/ml), leupeptin (10 µg/ml), and phenylmethylsulfonyl fluoride (1 mM). 15 µg
of total protein was separated by SDS-PAGE and transferred to
nitrocellulose membranes. The membranes were blocked with 1% bovine
serum albumin in TBST (150 mM NaCl, 0.05% Tween 20, 20 mM TrisCl, pH 7.5) and subsequently incubated with the
anti-myc (9E10) at a dilution of 1:250 at 4 °C overnight, or with
anti-Raf-1 (C12), or anti-MEK1 (C18) at dilutions of 1:1000 for 1 h at room temperature. The Phospho-p44/42 MAPK
(Thr202/Tyr204) antibody was diluted 1:1000 in
TBST containing 4% skin milk powder and incubated for 1 h. After
incubation with the secondary antibodies conjugated to horseradish
peroxidase (1:1000) for 1 h, the bands were visualized by ECL
(Roche Diagnostics Corp., Indianapolis, IN).
We have observed that the hTFPI-2 gene was highly up-regulated on
microarrays that were probed with cDNAs originating from HEK293
cells treated with 250 nM PMA for 4 h (data not
shown). Recently, PMA induction of a hTFPI-2-luciferase reporter gene has also been shown in glioma cells (19), and mRNA of hTFPI-2 is
up-regulated in BeWo and JEG-3 trophoblast cells treated with PMA
(24).
To study the signaling pathway that leads to the induction of hTFPI-2
gene expression by PMA and to investigate important promoter elements
involved in its transcriptional regulation, we PCR-amplified a 1511-bp
fragment of the 5'-flanking region of the hTFPI-2 gene from genomic DNA
isolated from HEK293 cells and cloned the 1511-bp fragment adjacent to
the firefly luciferase reporter gene (p-1511-luc).
hTFPI-2 Promoter Activity Induction by Phorbol Esters and
Inhibition of the PMA-dependent Induction by the MEK
Inhibitor UO126--
To assess whether this potential promoter region
allows transcription of luciferase, we transiently transfected HEK293
cells with the reporter plasmid p-1511-luc and monitored changes in luciferase activity of PMA-treated and untreated cells. Cells incubated
with 250 nM PMA showed a 10-fold stimulation of luciferase compared with cells without PMA treatment (Fig.
1A). Induction of the
luciferase reporter gene was decreased by 90% if cells were
preincubated with the MEK1 inhibitor UO126 at a concentration of 10 µM. UO126 completely inhibited PMA induction at a
concentration of 100 µM, suggesting that PMA activates
the hTFPI-2 promoter through the MEK signaling pathway (Fig.
1A).
As shown in the Western blot in Fig. 1B, PMA
activated p44/p42 MAPK in HEK293 cells and the phosphorylation of
p44/p42 could be inhibited by the MEK1-inhibitor UO126 in a
dose-dependent manner. This result indicates that MEK
activation is necessary for the induction hTFPI-2 promoter activity by
PMA.
Phorbol Esters and the Growth Factor EGF Can Stimulate the
Luciferase Reporter Gene in Epithelial Carcinoma Cell
Lines--
HEK293 cells have very recently been reported as atypical
epithelial cells and may originate from neuronal cells (25). To determine whether the PMA induction of the hTFPI-2 gene observed in
HEK293 cells was unique to this cell line or whether PMA promotes up-regulation of the hTFPI-2 gene in other epithelial cells lines as
well, we transiently transfected human lung carcinoma A549 cells,
breast carcinoma MCF7 cells and cervical carcinoma HeLa cells with the
luciferase reporter plasmid p-1511-luc. A 3.7-fold PMA-dependent induction of the hTFPI-2 promoter activity
was observed in A549 cells, whereas PMA induced the hTFPI-2 promoter
activity in MCF7 and HeLa cells 30- or 20-fold, respectively (Fig.
2). Because PMA has been reported to
transactivate the epidermal growth factor receptor (26), we tested
whether the hTFPI-2 gene could be induced by the growth factor EGF.
Although EGF up-regulated the hTFPI-2 promoter activity in HeLa cells
10-fold, no substantial stimulation of the luciferase reporter gene was
obtained in A549 and MCF7 cells (Fig. 2).
Ras, Raf, MEK, and ERK Can Induce the hTFPI-2 Promoter
Activity--
To assess the importance of the components of the
ERK/MAPK signaling pathway in the regulation the hTFPI-2 promoter
activity, we cotransfected EcR-293 cells with the plasmid p-1511-luc
and vectors containing constitutively activated signaling components, such as RasV12, RafCT, MEK-1SD, ERK2-MEK1-LA, or the empty control vector (pIND). The -fold stimulation of luciferase was calculated as
normalized luciferase activity obtained in cells expressing active
signaling components divided by the luciferase activity of samples
originating from vector-transfected control cells (Fig. 3A). Protein expression of
RasV12, RafCT, MEK-1SD, and ERK2-MEK1-LA in transiently transfected
EcR-293 cells is shown on Western blots in Fig. 3B. All the
constitutively activated signaling components were well expressed.
Expression of RasV12 stimulated the luciferase reporter gene 37-fold
compared with the control vector. Expression of constitutively active
signaling MAPK components further downstream of Ras, such as RafCT,
MEK-1SD, and ERK2-MEK1-LA, induced the luciferase reporter the gene 7-, 92-, or 39-fold, respectively (Fig. 3A), indicating that the
hTFPI-2 gene expression can be regulated by the Ras/Raf/MEK/ERK pathway
in EcR-293 cells. The highest activation of the hTFPI-2 promoter was
obtained by MEK-1SD containing aspartic acids at amino acid positions
218/222 (27), whereas the RasV12 and ERK2-MEK1-LA fusion protein (28)
activated the hTFPI-2 promoter to a similar extent. In conclusion,
these results demonstrate that the Ras/Raf/MEK/ERK signaling pathway mediates regulation of the hTFPI-2 gene.
The Minimal Inducible Promoter Activity Is Located within the
The minimal basal promoter activity includes the Identification of an AP-1 Site as a Specific Inducible DNA Response
Element--
To determine potential cis-acting elements
responsible for the PMA and Ras inducibility of the
To assess the importance of these sites, we mutated the presumptive Sp1
site (GG at position
Taken together these results provide evidence that the AP-1 site at
position A schematic summary of the results presented in this study is
shown in Fig. 7. We showed that the
hTFPI-2 gene is up-regulated in several epithelial cells following
stimulation by PMA (Fig. 2). Additionally, EGF, a growth factor, was
able to stimulate promoter activity of the hTFPI-2 gene as shown in
HeLa cells (Fig. 2). In HEK293 cells, induction of the promoter
activity by PMA could be blocked by the MEK-specific inhibitor UO126
(Fig. 1), suggesting that PMA induction of hTFPI-2 is mediated through
a pathway that involves MEK. Indeed, activated Ras, Raf, MEK, and ERK
were able to promote gene transcription (Fig. 3), indicating that the
Ras/Raf/MEK/ERK signaling pathway is necessary for promoter activation.
The ERK/MAPK pathway activates Fos proteins, which dimerize with
Jun proteins and bind as the AP-1 complex to a consensus DNA
sequence 5'-TGA(G/C)TCA-3' (29). We located an AP-1 consensus site at position 89 and
384, whereas the
minimal inducible promoter activity is between
89 and
222. We have
used the computer program TFSEARCH and mutagenesis to analyze potential
transcription factor binding sites. We identified an AP-1 binding site
at nucleotide position
156 (inducible activity) and a Sp1 site at
position
134 (basal activity) as potential cis-acting
elements in the promoter region of the hTFPI-2.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
312 to +1 is critical for minimal and inducible promoter activity of hTFPI-2 (19), the signal transduction pathway by
which PMA induces gene expression of hTFPI-2 and the promoter elements
involved in hTFPI-2 regulation have not been studied in detail. Here we
show that the hTFPI-2 gene expression is regulated by the ERK/MAPK
signaling pathway and that the activity of this pathway is directed to
an AP-1 site in the promoter of the hTFPI-2 gene.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Oligonucleotides
47 relative to the translation
start site.
190 and
184 was
changed to GGGGCAA, the putative AP-1 site
TGAATCA between nucleotide positions
162 and
156 was altered to GCTAGCA, and the
overlapping Sp1/AP-2 and GC box
GGCTCCGCCCCGGCGGGGG between nucleotide
positions
144 and
126 was modified to
GGCTTTGCCCCAACGGGGG. Double-stranded oligonucleotides containing the corresponding nucleotide changes were
phosphorylated, annealed, and cloned as
HindIII/EagI fragments into the corresponding
sites of p-1055-luc. Each mutation was confirmed by DNA sequencing. The
oligonucleotides used for p-198MSP1A-luc were O-9 and O-10, for
p-198MAP1-luc O-11 and O-12, and for p-198MSP1B/MAP2-luc O-13 and O-14
(Table I). p-222MAP1/MSP1B-luc and p-222MAP1/MAP2-luc were created by
PCR using the Expand High Fidelity PCR system using the
oligonucleotides O-15 and O-16 or O-17, respectively. In both
constructs the putative AP-1 site TGAATCA
between nucleotide positions
162 and
156 was altered to
GCTAGCA, and either the putative Sp1 site
between nucleotide positions
140 and
134 CCGCCCC altered to TTGCCCC or the putative AP-2 site between
nucleotide positions
136 and
126 CCCGGCGGGGG was
altered to CCCGGCAAGGG. The sequence for the
HindIII and EagI restriction sites are printed in
boldface, and the nucleotide changes are underlined (Table I). The PCR
products were verified by sequencing and subcloned as
HindIII/EagI fragments into the corresponding
sites of p-1055-luc.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Induction of the hTFPI-2 promoter activity by
PMA is inhibited by the MEK inhibitor UO126. A, HEK293
cells were transiently cotransfected with the luciferase reporter
plasmid p-1511-luc and a control vector containing GFP. 24 h
later, the cells were serum-starved for 24 h. UO126 was added at a
concentration of 10 or 100 µM 15 min prior to 250 nM PMA treatment for 4 h. Luciferase activity was
calculated as light units/µg of protein and normalized for
transfection efficiency. Data are shown as mean ± S.D. from one
representative experiment performed in duplicate transfections.
B, inhibition of Phospho-p44/42 MAPK by UO126. HEK293 cells
were incubated with 250 nM PMA for 4 h (lane
1) and treated with 0.1, 1, or 10 µM UO126 prior to
PMA treatment (lanes 2-4). Proteins were separated on a
12% SDS-PAGE and subjected to Western blotting with the Phospho-p44/42
MAPK (Thr202/Tyr204) antibody.
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Fig. 2.
Stimulation of hTFPI-2 promoter activity by
PMA and EGF in various epithelial carcinoma cell lines. A549,
MCF7, and HeLa cells were transiently transfected with
p-1511-luc. The cells were serum-starved for 16 h and
subsequently treated with 250 nM PMA or 50 ng/ml EGF for
6 h. The -fold stimulation of luciferase was calculated as firefly
light units/µg of protein of PMA or EGF-treated cells divided by the
firefly light units/µg of protein of nontreated cells. Data are shown
as mean ± S.D. from at least three independent transfection
experiments performed in duplicates.
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Fig. 3.
Induction of the hTFPI-2 promoter activity by
RasV12, RafCT, MEK-1SD, and ERK2-MEK1-LA. A, EcR-293
cells were cotransfected with RasV12, RafCT, MEK-1SD, ERK2-MEK1-LA,
or pIND control vector and reporter plasmid p-1511-luc. A vector
containing GFP was included to control for transfection efficiencies.
The cells were serum-starved 4 h prior to induction of protein
expression with pronasteron A for 20 h. The -fold stimulation of
luciferase was calculated as normalized firefly light units/µg of
protein of cells expressing signaling components divided by the
normalized firefly light units/µg of protein of cells transfected
with the control vector pIND. Data are shown as mean ± S.D. from
one representative experiment performed in duplicate
transfections. B, EcR-293 cells were transfected with 1 µg
of pIND (lanes 1, 3, and 5), RasV12
(lane 2), RafCT (lane 4), MEK-1SD (lane
6), or ERK2-MEK1-LA (lane 7). 24 h later,
the cells were serum-starved 4 h prior to induction of protein
expression with pronasteron A at a concentration of 10 µM
for 20 h. Proteins were separated on 15% (lanes 1-4)
or 12% (lanes 5-7) SDS-polyacrylamide gels and subjected
to Western blotting with anti-myc (9E10) (lanes 1 and
2), anti-Raf-1 (C12) (lanes 3 and 4),
or anti-MEK1 (C18) antibodies (lanes 5-7).
89/
222-bp Region of the hTFPI-2 Promoter--
We
transiently cotransfected EcR-293 cells with a series of luciferase
reporter gene constructs containing progressive deletions of the
5'-flanking region with either a vector containing RasV12 or the empty
control vector pIND. The -fold stimulations of the Ras
versus control vector are shown in Fig.
4A. 35- to 51-fold stimulations of luciferase were obtained with constructs p-1511-luc through p-222-luc, whereas no inducible luciferase activity was obtained with construct p-89-luc, suggesting that the minimal inducible
promoter activity is located between nucleotide positions
222 and
89 (Fig. 4A). Similar results were obtained in PMA-treated HEK293 cells expressing the deletion constructs. PMA
stimulated p-1511-luc through p-222-luc 5- to 8-fold, whereas p-89-luc
was not induced by PMA (Fig. 4B). Therefore, we decided to
investigate the promoter elements in the
222/
89 region that are
responsible for the 51-fold Ras and 8-fold PMA stimulations as compared
with control cells.
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Fig. 4.
Deletion constructs of the hTFPI-2
promoter. A, RasV12 or control vector pIND and
luciferase reporter gene constructs with 5'-ends between nucleotides
1511 and
89 and a common 3'-end at
1 were transiently
cotransfected into EcR-293 cells. A vector containing GFP was included
to control for transfection efficiency. The cells were serum-starved
4 h prior to induction of protein expression with pronasteron A
for 20 h. The -fold stimulation of luciferase was calculated as
normalized firefly light units/µg of protein of cells expressing
signaling components divided by the normalized firefly light units/µg
of protein of cells transfected with the control vector pIND. Data are
shown as mean ± S.D. from at least three independent transfection
experiments. B, HEK293 cells were transiently transfected
with the deletion constructs. The cells were serum-starved for 16 h prior to treatment with 250 nM PMA for 4 h. The
-fold stimulation of luciferase was calculated as firefly light
units/µg of protein of PMA-treated cells divided by the firefly light
units/µg of protein of mock treated cells. Data are shown as
mean ± S.D. from at least three independent experiments performed
in triplicates.
89/
384-bp region,
because p-384-luc showed similar basal luciferase activity as longer
constructs, whereas no basal activity was obtained with construct
p-89-luc (Table II). As shown in
Table II, the luciferase activity dropped by 74% in vector
(pIND)-transfected cells expressing p-222-luc compared with cells
expressing p-384-luc, indicating that the region,
384 to
222 bp,
contains transcription factor binding sites important for basal
activity. Similarly, luciferase light units were higher in samples
originating from cells transfected with RasV12 and a reporter gene
construct containing 384 bp of an upstream promoter segment compared
with cells transfected with RasV12 and a reporter construct containing
only a 222-bp promoter segment (Table II). This loss of activity is
similar to the loss of basal activity in the
222/
384 region and
therefore may be due to the absence of transcription factor binding
sites important for basal activity. However, we can not rule out that
the region,
384/
222 bp, may contain additional enhancer elements
that can contribute to the inducible activity.
Luciferase activity of the deletion constructs described in Fig. 4
(normalized firefly units/µg of protein)
89/
222-bp
promoter region, we used the computer program TFSEARCH version 1.3 that
searches highly correlated sequence fragments versus the
TFMATRIX transcription factor binding site profile data base by E. Wingender, R. Knueppel, P. Dietze, and H. Karas (GBF-Braunschweig).
Several putative transcription factor binding sites were identified,
including a potential Sp1 site (CGGGGGCGGCGGGG)
between nucleotide positions
192 and
179, a potential AP-1 site
(ATGAATCA) between positions
163
and
156, and an overlapping Sp1 (underlined)/AP-2 and GC box
(GGCTCCGCCCCGGCGGGGG) between
positions
144 and
126 (Fig. 5).
View larger version (22K):
[in a new window]
Fig. 5.
Potential transcription factor binding sites
in the minimal inducible promoter of hTFPI-2. Potential
cis-acting elements as determined by TFSEARCH are
underlined. Sites that were mutated are shown in
boldface, and the positions are indicated by
numbers.
185/
184 mutated to AA, printed in boldface
above), mutated the presumptive AP-1 site (TGAAT at positions
162 to
158 mutated to GCTAG), or altered the presumptive Sp1 and AP-2 sites
(CC at positions
140/
139 mutated to TT, and GG at positions
133/
132 mutated to AA; printed in boldface above). The resulting
reporter gene plasmids p-198MSP1A-luc, p-198MAP1-luc, and
p-198MSP1B/MAP2-luc lacked the consensus
184 Sp1A site, the consensus
156 AP-1 site, or the consensus
134/
126 Sp1B/AP-2 sites,
respectively. As illustrated in Fig. 6,
mutation of the putative Sp1A site in a 198-bp 5'-flanking promoter
region resulted in a 40% reduction in the basal and inducible activity
as compared with p-222-luc. The reporter construct p-198MSP1A was still
52-fold induced by RasV12 as compared with the control vector (Table
II). The loss of some basal activity might be due to the mutation of the consensus Sp1A site or the absence of the
198 to
222
region. Mutation of the consensus AP-1 site caused a
considerable decrease in inducible activity, whereas substantial basal
activity was still retained, indicating that this putative AP-1 site is
important for inducible activity. Furthermore, mutation of the
overlapping consensus Sp1B/AP-2 site affected basal and inducible
activity severely (Fig. 6). Deletion of the consensus
156 AP-1 and
the consensus
126 AP-2 site caused only a minor decrease in inducible activity as compared with p-198MAP1-luc, a construct that had only the
consensus
156 AP-1 site mutated, suggesting that the putative
126
AP-2 site may have a subtle effect on inducible promoter activity.
Substantial basal activity, however, was still retained comparable to
p-198MAP1-luc (Fig. 6). Deletion of the consensus
156 AP-1 and the
consensus-134 Sp1 sites in the
222-bp 5'-flanking region of the
hTFPI-2 promoter strengthened our previous observation.
p-222MAP1/MSP1B-luc showed no basal activity above background and was
also not stimulated by RasV12, suggesting that these sites are
cis-acting elements critical for basal and inducible activity (Fig. 6). Similar results were obtained with another MAPK
signaling pathway component MEK-1SD or with PMA (data not shown).
View larger version (14K):
[in a new window]
Fig. 6.
Mutagenesis of potential transcription factor
binding sites. EcR-293 cells were transiently cotransfected with
RasV12 or control vector pIND and luciferase reporter gene constructs
containing modified potential transcription factor binding sites.
Cotransfection with a vector containing GFP controlled for transfection
efficiency. The cells were serum-starved 4 h prior to induction of
protein expression with pronasteron A for 20 h. Luciferase
activity was calculated as light units/µg of protein and normalized
for transfection efficiency. Data are shown as mean ± S.D. from
at least four independent transfection experiments.
156 to
162 represents a cis-acting element that
is critical for induction of the hTFPI-2 promoter activity by the MEK
signaling pathway and the Sp1 site at position
134 to
140 is
essential for basal promoter activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
156 to
162 as a cis-acting element
essential for inducible promoter activity. Additionally, a Sp1
consensus site at positions
134 to
140 of the hTFPI-2 promoter was
essential for basal promoter activity (Fig. 6).
View larger version (22K):
[in a new window]
Fig. 7.
Schematic summary of MAPK pathway that
regulates the activity of the hTFPI-2 promoter. The MAPK signaling
components involved in the activation of the hTFPI-2 gene are indicated
in boldface, and cis-acting elements essential
for basal and inducible promoter activity are indicated in
boxes.
Our study in HEK293 cells indicates that the hTFPI-2 gene is regulated by the Ras/Raf/MEK/ERK signaling pathway. Each constitutively active signaling component of this pathway was able to induce high transcriptional activity of the hTFPI-2 gene. In agreement with these reporter gene studies, we observed up-regulation of the hTFPI-2 gene in preliminary microarray experiments comparing EcR-293 cells expressing RasV12, RafCT, MEK-1SD, or ERK2-MEK1-LA to nontransfected cells (data not shown). RasV12 is a strong activator of the Raf/MEK/ERK pathway, because it contains a glycine to valine mutation at amino acid position 12 and therefore remains in the active GTP-bound state (30). In contrast, RafCT, the carboxyl-terminal part of Raf that lacks the regulatory amino-terminal domain, is a moderate activator of the MEK/ERK pathway (31). Consistent with this, we observed a 37-fold induction of hTFPI-2 promoter activity by Ras and a 7-fold induction by RafCT (Fig. 3). Similarly, the stronger activator Raf-CAAX, a full-length Raf containing a CAAX-box (32) induces higher (15-fold) induction of hTFPI-2 promoter activity than RafCT (data not shown).
The tumor-promoting agent PMA often serves as a model agent to study
the mechanism by which growth factors regulate growth and
differentiation of the cells. PMA mimics the action of diacylglycerol, the endogenous activator of protein kinase C (PKC) (33, 34), and PKC
activates Raf by direct phosphorylation (35). However, activation of
Raf by PKC is Ras-dependent in several cell lines (36, 37).
Not all the phorbol ester responses can be explained by the PKC action,
and some responses may be attributed to nonkinase phorbol ester
receptors such as the - and
-chimaerins or to Ras-GRP, a
phorbol ester receptor that plays a role in PMA activation of Ras (38).
Furthermore, PMA has been shown to transactivate the epidermal growth
factor receptor in mouse epidermal JB6 cells, and PMA-induced tumor
promotion may be partially mediated through this receptor (26). PMA can
activate distinct groups of MAPKs, such as the mitogen-responsive ERKs
(extracellular signal-regulated kinases), the stress-responsive
JNK/SAPKs (c-Jun amino-terminal kinase/stress-activated protein
kinases), and p38 MAPKs. The pathways induced by PMA are cell
line-dependent; for example PMA activates the JNK/MAPK
pathway in normal oral keratinocytes but not in
immortalized/transformed keratinocytes, HeLa cells, or HEK293 cells
(39, 40). In this report we provide evidence that hTFPI-2 gene
expression induced by PMA is regulated through the Ras/Raf/MEK/ERK
signaling pathway. The specific MEK inhibitor UO126 could block PMA
induction, indicating that MEK/ERK activation is essential for
induction of hTFPI-2 promoter activity by PMA. Because UO126 inhibited
PMA promoter activation at the commonly used concentration of 10 µM, we hypothesize that the ERK/MAPK pathway may be
sufficient for PMA induction of hTFPI-2. Besides affecting the
PMA-dependent hTFPI-2 promoter activation, UO126 had a
minor negative effect on basal levels of the hTFPI-2 gene
transcription, probably due to the low level of activation of hTFPI-2
by endogenous MAPK signaling components.
Activation of the ERK/MAPK causes induction of fos genes
through phosphorylation of ternary complex factors (41). Indeed, we
have observed up-regulation of FosB when PMA-induced HEK293 cells were probed on microarrays (data not shown). Fos heterodimerizes with Jun to form the AP-1 complex, which activates gene transcription by binding to the AP-1 element. Furthermore, Jun/Fos heterodimers can
lead to increased c-jun transcription through binding to the AP-1 sites in the c-jun promoter (41). In agreement with
this, we located a putative AP-1 site at position 156 to
162 as a cis-acting element essential for inducible promoter activity.
Our results suggest that basal promoter activity is located in the
region, 384 to
89 bp, of the hTFPI-2 promoter consistent with a
previous promoter deletion study in human transformed bone marrow
microvascular endothelial cells (22), identifying an 85-bp fragment
(corresponding to the
299 to
214 region in our study) that
contained most of the basal activity. In addition to this, we have
identified a consensus Sp1 site at
134 to
140 that is essential for
the basal activity in the promoter region,
222 to
89 bp.
The region, 222 to
89 bp, is sufficient for a 52-fold Ras and an
8-fold PMA induction of the hTFPI-2 promoter activity as compared with
basal level (Fig. 4). We therefore investigated the candidate
transcription factor binding sites in this promoter region and found
that the consensus AP-1 site at position
156 to
162 is essential
for induction of promoter activity by Ras (Fig. 6). Recently, Konduri
(19) identified a 231-bp region between
312 and
81 in the hTFPI-2
promoter region that is responsive to PMA. Investigating hTFPI-2
promoter regulation in glioma cells, Konduri found a strong repressor
in the region between
927 to
1181 and enhancer elements between
1511 and
1181. These repressor and enhancer elements found in
glioma cells might be tissue-specific regulatory elements, because we
did not find such elements in HEK293 cells. Similar luciferase
activities were observed in constructs of various lengths between 1511 and 384 induced by Ras (Table II) or PMA (data not shown). Konduri
suggested that three potential AP-1 sites (corresponding to
309 to
299,
213 to
203, and
162 to
156 in our study) may be involved
in PMA-related gene induction (19). Although we identified the
156 to
162 consensus AP-1 site as essential for transcription of the
hTFPI-2 gene, our results suggest that the
213 to
203 promoter
segment may not be essential for Ras-related gene induction in EcR-293
cells, because the promoter segment of construct p-198MSP1A-luc, which
lacks the
213/
203 segment, is sufficient for a 52-fold Ras
induction similar to longer constructs (Table II).
The ERK/MAPK pathway stimulated by growth factors and phorbol esters is
associated with cellular proliferation and differentiation. The
biological consequences of hTFPI-2 induction by agents stimulating the
ERK/MAPK pathway and the role of hTFPI-2 in tumor progression is not
fully understood. On one hand, several cell lines stably overexpressing
hTFPI-2, such as glioma SNB19 cells, choriocarcinoma JAR cells,
amelanotic melanoma cells, and prostate cancer cells, were less
invasive than their parental cells (14-17) and addition of recombinant
hTFPI-2 inhibited the invasiveness of SNB19 glioma cells as measured in
a Matrigel assay (42). On the other hand, hTFPI-2 has been shown to
have a pro-invasive effect in cancer. Recombinant hTFPI-2 has been
shown to be a mitogen for vascular smooth muscle (43), and
hTFPI-2 potentiates hepatocyte growth factor-induced invasion of human
hepatocellular carcinoma cells and is capable of inducing
invasion of human hepatocellular carcinoma on its own (18). Bimodal
function has also been described for tissue inhibitors of
metalloproteinases, another family of tissue inhibitors. Tissue
inhibitors of metalloproteinases inhibit the activity of matrix
metalloproteinases by binding to their zinc binding site, but they are
also involved in the cell surface-targeted MMP activation cascade,
stimulate growth, and are anti-angiogenic (44). Similarly, the TFPIs
may also perform a very wide variety of biological activities, and
transcriptional regulation of TFPIs may play an important role in the
balance of protease activities resulting in the metastatic/invasive phenotype.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. M. Cobb for the generous gift of the plasmid mycCMV5- MEK1-Erg2-LA, Dr. N. Lamarche-Vane for the plasmid pRK5 myc-RasV12, and Dr. M. Featherstone for the luciferase containing vector pXp2. We also thank Dr. M. Featherstone, Dr. M. Jaramillo, and Dr. U. Oberholzer for constructive comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the National Research Council Genomics and Health Initiative program. This is National Research Council of Canada publication number 44862.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. Tel.: 41-31-632-8323;
Fax: 41-31-632-8966; E-mail: Christina.Kast@insel.ch.
Published, JBC Papers in Press, November 24, 2002, DOI 10.1074/jbc.M210935200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; AP-1, activating protein-1; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; ERK, extracellular signal regulated kinase; hTFPI-2, human tissue factor inhibitor-2; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MEKK, MEK kinase; PBS, phosphate-buffered saline; PKC, protein kinase C; JNK, c-Jun amino-terminal kinase; SAPK, stress-activated protein kinase; CMV, cytomegalovirus; GFP, green fluorescence protein; MMP, matrix metalloprotease.
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