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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3811-3823
Vascular Endothelial Cell Growth Factor-Induced Tissue Factor
Expression in Endothelial Cells Is Mediated by EGR-1
By
Diana Mechtcheriakova,
Alexander Wlachos,
Harry Holzmüller,
Bernd R. Binder, and
Erhard Hofer
From the Department of Vascular Biology and Thrombosis Research at
VIRCC, University of Vienna, Vienna, Austria.
 |
ABSTRACT |
Vascular endothelial cell growth factor (VEGF) is a major regulator
of angiogenesis. We report here that treatment of endothelial cells
with VEGF leads to upregulation of tissue factor mRNA and protein
expression on the cell surface. Reporter gene studies show that
transcriptional activation of the tissue factor gene by VEGF is
mediated by a GC-rich promoter element containing overlapping binding
sites for Sp1 and EGR-1. As shown by immunofluorescence and
electrophoretic mobility shift assays, upon VEGF treatment EGR-1
rapidly accumulates in the nucleus and binds to its respective recognition site in the tissue factor promoter. Sp1 occupies this element in unstimulated cells and seems to be partially displaced by
increasing amounts of EGR-1. Transfection of endothelial cells with an
EGR-1 expression plasmid mimics the upregulation of tissue factor
transcription observed after VEGF treatment. In contrast, NF B, the
major transcription factor involved in tissue factor upregulation by
inflammatory stimuli, is not activated by VEGF. These data show that
VEGF induces a response in endothelial cells largely distinct from
inflammatory stimuli, and suggest that EGR-1 is a major mediator of the
activation of the tissue factor and possibly other VEGF-responsive genes.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
TISSUE FACTOR (TF) is a member of the
cytokine-receptor superfamily. It functions as the high-affinity
receptor/cofactor for plasma factor VII/VIIa1,2 and is the
primary cellular initiator of blood coagulation. Consistent with a
protective role in the hemostatic response, TF is expressed
constitutively in several extravascular cell types surrounding blood
vessels and at boundaries of organs, but is normally absent in vascular
endothelial cells. However, it is rapidly induced in response to
inflammatory stimuli including lipopolysaccharide (LPS), tumor necrosis
factor- (TNF-), and interleukin-1 (IL-1).3,4
On the other hand, the constitutive expression of TF was shown to be a
characteristic feature of various neoplastic cells. Data obtained from
experimental tumor models and clinical studies suggest that TF may
facilitate tumor growth in vivo by enhancing angiogenesis. Indeed,
tumor cells transfected to overexpress TF established larger and more
vascularized tumors.5 Moreover, TF has been reported to be
detected on vascular endothelial cells and tumor cells within human
breast cancer tissue, but is absent on vessels of benign fibrocystic
breast disease.6 Additional evidence indicates that the
same tumor cells that express TF also produce vascular endothelial cell
growth factor (VEGF), and that TF may regulate VEGF production by those
cells.7 Taken together, these data are consistent with a
highly complex interaction between tumor cells and endothelial cells in
the tumor microenviroment, and suggest that tumor cells activate nearby
endothelial cells and regulate blood vessel growth in vivo. It is not
clear whether vessel growth and expression of TF by endothelial cells
is mediated by the same angiogenic protein, eg, VEGF, or by some other
cytokine produced by the tumor. However, VEGF is a likely candidate
because more vascularized, TF-positive tumors secrete increasing
amounts of VEGF,5,7 and VEGF from murine fibrosarcoma
induces expression of TF in endothelial cells.8
To date, five angiogenic and endothelial cell-specific growth factors
of the VEGF family have been isolated: VEGF-A, VEGF-B, VEGF-C, VEGF-D,
and placental growth factor, PlGF.9,10 Three high-affinity
tyrosine kinases receptors for VEGF-A, VEGF-C, and PlGF have been
identified on endothelial cells: VEGF receptor-1, encoded by the flt-1
gene; VEGF receptor-2, encoded by the flk-1/KDR gene; and VEGF
receptor-3, encoded by the flt-4 gene. Some intracellular signaling
pathways triggered by these receptors have been described recently.11-13 However, the transcription factors involved
in the angiogenic response to VEGF have not yet been defined.
TF gene expression has been characterized for different cell types and
appears to be controlled by transcription factors that are
constitutively active as well as factors induced by external signals.
In case of inflammatory stimuli, such as LPS, TNF- or IL-1 ,
induction of the TF promoter in human monocytic cells and endothelial
cells is mediated by a single NFB site binding c-Rel/p65 heterodimers in combination with two AP-1 sites.3,4
DNA-binding motifs for NFB were found in the promoters of more than
50 genes that are known to be activated upon
inflammation.14 NFB is kept in a premade inactive form
in the cytoplasm and rapidly translocates to the nucleus after
stimulation. In contrast, serum and phorbol ester induction of the TF
gene in epithelial cells is controled by a GC-rich promoter region
containing overlapping Sp1/EGR-1 sites.15 Recently, the
same region was shown to be responsible for increased expression of TF
in hypoxic mononuclear phagocytes and epithelial cells.16
Sp1 is constitutively expressed and mediates a basal promoter activity.
EGR-1, also known as Zif268, NGFI-A, Krox 24, or TIS8,17-19
is expressed with the kinetics of an immediate-early transcription
factor. The egr-1 gene can be induced by diverse signals that initiate
growth and differentiation without requirements for de novo protein
synthesis.20,21 Putative nucleotide recognition elements
for EGR-1, which usually overlap with Sp1 binding sites, appear in the
promoters of a number of pathologically important genes, including
transforming growth factor-1 (TGF-1),22
TF,3 urokinase-type plasminogen activator,23 PDGF-A and PDGF-B,24 as well as in the EGR-1 promoter
itself.25
We have now investigated the mechanism(s) responsible for TF
upregulation by VEGF. A VEGF-responsive region in the TF
promoter has been characterized. This region is distinct from the
promoter elements involved in TNF-- or LPS-induced TF gene
expression. The transcription factor EGR-1 has been identified as a
major component of a VEGF-inducible complex binding to this region. Furthermore, EGR-1 overexpression has been found to mimic TF gene activation by VEGF. The data presented define for the first time a
transcription factor induced by VEGF that may be generally important for genes upregulated by this cytokine.
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MATERIALS AND METHODS |
Cell culture.
Human umbilical vein endothelial cells (HUVEC) and human skin
microvascular endothelial cells (HSMEC) were prepared as previously described.26,27 HUVEC and HSMEC were cultured at 37°C
and 5% CO2 in medium 199 containing 20% iron-supplemented
bovine calf serum (SCS) (HyClone, Logan, UT), 1 U/mL
heparin, 50 µg/mL ECGS, 2 mmol/L glutamine, 100 U/mL penicillin, and
0.1 mg/mL streptomycin. Cells were used for experiments up to passage
number 5. Recombinant human VEGF was obtained from PromoCell
(Heidelberg, Germany) or was a gift of Dr Matthias Clauss
(Max-Planck-Institut für Physiologische und Klinische Forschung,
Bad Nauheim, Germany). Induction of cells was performed by
addition of VEGF165 or VEGF121 at 1.25 nmol/L and TNF- (Genzyme Inc, Cambridge, MA) at 100 U/mL to
confluent cells for 4 hours (clotting assay) or 6 hours (reporter gene assay).
Clotting assay.
Cells were seeded in 6-well plates at 80% to 90% confluency and grown
overnight. Cells were scraped from the plates and analyzed for TF
activity as described.4,8 Briefly, after induction for 4 hours with VEGF or TNF-, cells were washed twice and then scraped in
1mL clotting buffer (12 mmol/L sodium acetate, 7 mmol/L diethylbarbitate, and 130 mmol/L sodium chloride; pH 7.4). One hundred
microliters of resuspended cells were mixed with 100 µL of citrated
plasma (Sigma, St Louis, MO), and clotting times were measured after recalcification with 100 µL of 20 mmol/L
CaCl2 solution at 37°C. TF equivalents were determined
by using a standard curve obtained from rabbit brain thromboplastin
(Sigma). For studies with neutralizing antibodies (American Diagnostica
Inc, Greenwich, CT; no. 4508) cells were incubated for 30 minutes before starting the clotting assay. Mithramycin A,
a DNA-binding drug known to preferentially bind to GC-rich
DNA sequences,28-30 was used to estimate the functional
importance of a GC-rich region within the TF promoter. For this
purpose, cells were preincubated with medium containing mithramycin
(Sigma) for 30 minutes before addition of VEGF or TNF-.
Northern blot analysis.
Total cellular RNA was isolated from confluent cell cultures by
scraping cells in TRIzol reagent (GIBCO, Grand Island,
NY), extracting DNA and proteins with chloroform, and
precipitating the RNA by isopropanol. Twenty micrograms of total RNA
was fractioned on a 1.3% agarose-6.4% formaldehyde gel, transferred
to a nylon membrane (Amersham Life Science Inc, Little Chalfont,
UK), and covalently linked by UV irradiation.
Membranes were prehybridized in 5X NET (20X NET: 3 mol/L sodium
chloride, 300 mmol/L TRIS/chloride, 20 mmol/L EDTA, pH 7.5), 5X
Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), and 100 µg/mL salmon sperm DNA at 56°C for 4 hours. Then
-32P-radiolabeled probe equal to
106 cpm/mL was added and hybridization was allowed to
proceed overnight. A 641-bp fragment of the human TF cDNA was used as a
specific probe. The probe was labeled by random priming using the
Prime-it II Random Primer Kit from Stratagene Cloning Systems (La
Jolla, CA). The membrane was washed at 56°C twice
with 2X NET, 0.2% SDS for 30 minutes, once with 1X NET, 0.2% SDS, and
again with 2X NET, 0.2% SDS. Bound radioactivity was visualized by
exposure to XAR-5 films at  70°C using intensifying screens
(Eastman Kodak Co, Rochester, NY).
Electrophoretic mobility shift assays.
Nuclear extracts from confluent endothelial cell cultures were prepared
as previously described,4,31 except that a modified buffer
C was used during nuclear extraction [20 mmol/L HEPES-KOH, pH 7.9, 420 mmol/L NaCl, 400 mmol/L (NH4)2SO4,
1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L
dithiothreitol (DTT), 25% glycerol]. Protease inhibitors (0.5 mmol/L
phenylmethylsulfonyl fluoride, 25 µmol/L Na-p-tosyl-L-lysine
chloromethyl ketone, 50 µmol/L N-tosyl-l-phenylalanine chloromethyl
ketone) were added at all steps during extract preparation. For the
electrophoretic mobility shift assay, 3 µg of nuclear proteins was
incubated with radioactively labeled oligonucleotide equal to
105 cpm in binding buffer (20 mmol/L HEPES-KOH, pH 7.9, 1 mmol/L EDTA, 5 mmol/L MgCl2, 50 mmol/L KCl, 1 mmol/L DTT,
10% glycerol) and 2 µg poly (dI/dC) (Boehringer Mannheim, Mannheim,
Germany), giving a total volume of 20 µL, for 30 minutes at room
temperature. The double-stranded synthetic oligonucleotides were
radioactively labeled by filling in the overhangs with Klenow enzyme in
the presence of (-32P)dATP and subsequently purified
over a 10% polyacrylamide gel. The sequences of the probes used were
as follows: hTF AP-1,
5'-aattGCGGTTGAATCACTGGGGTGAGTCATCCCTTGCAGG-3'; hTF NFB,
5'-aattCCCGGAGTTTCCTACCGG-3'; hIg NFB,
5'-aattCAGAGGGGGATTTCCCAGAG-3'.
The oligonucleotides spanning the GC-rich region of the human TF
promoter are listed in Figure 5. Where indicated, 2 µg of polyclonal
rabbit antibodies, all obtained from Santa Cruz Biotechnology (Santa
Cruz, CA) [Egr-1: sc-189x, AP-2: sc-184x, AP-1 (c-Fos): sc-52x], were
added to the binding reactions for 30 minutes at 4°C before the
addition of the radioactive probe. Protein-DNA complexes were resolved
by native 5.5% polyacrylamide gel electrophoresis in 0.5X TBE.
Transfection of HUVEC.
A fragment of the porcine TF promoter (330 to +118) designated
as wild-type promoter was cloned into a luciferase expression vector,
pUBT-luc.4,32 The NFB, AP-1, and SP1/Egr-1 deletion fragments were obtained from polymerase chain reaction (PCR) fragments of the 330/+118 wild-type TF promoter, resulting in the
substitution of the sequences from position 161 to 137,
from 209 to 156 or from 134 to 43 with an
XbaI linker sequence, respectively.4 The minimal
promoter construct was created from an XbaI/HindIII restriction enzyme fragment of the  NFB construct containing the
sequences from 160 to +118 of the TF promoter, which was then
ligated into a luciferase expression vector pUBT-luc. All constructs
were sequenced to show the fidelity of PCR and subcloning procedures.
One of the 330 to +118-bp constructs was found to contain a
spontaneous point mutation (G  A) at the position 51 before the consensus EGR-1 site 51(G/A)GCGGGGGCG 42 and
was included in the experiments. The expression vectors pCB.EGR-1 (containing the full-length murine EGR-1 cDNA coding sequence) and the
pCB.EGR-1331-374 (lacking the first and part of the second EGR-1
zinc finger domains) were generously provided by Dr Vikas P. Sukhatme (Harvard Medical School, Boston, MA).33
Twenty-four hours before transfection, HUVEC were seeded in 6-well
tissue-culture plates to reach 80% to 90% confluency the next
morning. Transient transfections were performed by using the
Lipofectamine reagent (GIBCO) according to the protocol. Cells were
incubated with transfection mixture containing 3 µg DNA (including a
cytomegalovirus [CMV]--gal construct as internal
control) and 8 µL Lipofectamine in a total volume of 1 mL medium 199 per well for 2 hours and 10 minutes. Induction by VEGF was performed
the next day for 6 hours. Luciferase and -gal assays were performed with cellular lysates of transfected cells as previously
described.34,35 All experimental values were determined
from duplicate wells, and several identical experiments were performed
with plasmids from different DNA preparations to assess the influence
of DNA quality. Relative promoter activities were calculated relative to the wild-type promoter activity (100%), and results from
three independent experiments were presented as means ± SD.
Cell enzyme-linked immunosorbent assay (ELISA).
Cells were seeded into a 96-well plate to reach confluency the next
day. Cells were treated with TNF- (100 U/mL) or VEGF (1.25 nmol/L) for various periods of time before fixation with 0.1% glutaraldehyde in phosphate-buffered saline (PBS). The antibodies specific for VCAM-1, ICAM-1, and E-selectin (R&D Systems, Minneapolis, MN; VCAM-1: BBA-5, ICAM-1: BBA-3, ELAM-1: BBA-1)
were diluted in PBS, 0.1% Tween 20, 1% skimmed milk, and incubated
with the fixed cells for 1 hour at 37°C. Goat anti-mouse IgG
conjugated to peroxydase (Amersham) was used at a dilution of 1:2,000
and incubated for 1 hour at 37°C. For development, o-phenylene
diamine (Sigma) was added and incubated for 10 to 20 minutes in the
dark at room temperature. The reaction was stopped by 3 mol/L
H2SO4 and the optical density
(OD) was measured at 492 nm.
Immunofluorescence.
Immunofluorescence assay was performed mainly as previously
described.36 Briefly, cells were grown in LabTek
tissue-culture chamber slides (Nunc, Inc, Naperville, IL)
for at least 24 hours before fixation. After appropriate stimulation,
cells were washed twice with PBS, fixed for 10 minutes at room
temperature with 3.7% formaldehyde, 2% sucrose in PBS, and
permeabilized for 5 minutes with 0.5% Triton X-100 (Serva, Heidelberg,
Germany) in PBS. Primary antibodies (polyclonal rabbit
IgG from Santa Cruz Biotechnology; Egr-1: sc-189x and sc-110x; SP1:
sc-59x; p65: sc-109x) were diluted in PBS, 1% bovine serum albumin
(BSA) and incubated with the cells for 1 hour at room temperature. To
confirm the specificity of anti-EGR-1 antibodies, the immunizing
peptide (sc-189) was preincubated with antibodies for 15 minutes before
addition to the cells. Cells were washed in PBS and incubated with
rhodamine- or fluorescein isothiocyanate (FITC)-labeled goat
anti-rabbit IgG (Accurate Scientific, Westbury, NY) for 1 hour at room temperature. To visualize cell nuclei, a fluorescent
groove-binding probe for DNA (4',6-Diamidino-2-Phenylindole, DAPI;
Sigma) was added to the secondary antibody solution at 100 ng/mL.
Slides were washed in PBS and mounted with mounting fluid (Difco,
Detroit, MI). The immunofluorescence results were
analyzed with a Bio-Rad MRC 600 confocal laser scanning microscope
(Bio-Rad, Hercules, CA).
Statistical analysis.
The results obtained were analyzed by one-way analysis of variance and
the Student's paired t-test.
| |
RESULTS |
VEGF upregulates TF activity and TF mRNA in endothelial cells.
VEGF has been shown to modulate procoagulant activity on the surface of
human monocytes and endothelial cells.8 However, the
mechanisms of VEGF action have not been defined. Therefore, we tested
the effect of VEGF on TF expression in comparison to a known inducer of
TF, TNF-. TF activity, as monitored by a one-stage clotting assay,
was very low in unstimulated HUVEC, and VEGF165 caused a
dose-dependent increase in TF activity reaching plateau values at 1.25 nmol/L (Fig 1A). Accordingly, all of the
following experiments were performed in the presence of this
concentration of VEGF165. Depending on the experiment, TF
activity was upregulated from 10- to 80-fold, which is comparable to
the induction observed with TNF- (100 U/mL). HUVEC induced by
VEGF165 or TNF- reached maximal TF activity at 4 hours
(Fig 1B). To confirm that the procoagulant activity detected in the
clotting assay is indeed mediated by TF, anti-TF monoclonal antibodies
were preincubated with the cells before the clotting reaction. These
antibodies completely abolished VEGF-induced procoagulant activity
(data not shown).
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| Fig 1.
VEGF upregulates TF activity and mRNA in HUVEC. (A) VEGF
induces TF activity in a dose-dependent manner. TF activity equivalents
were determined in a one-stage clotting assay. Incubation of cells with
increasing concentrations of VEGF in the range of 0.125 to 5 nmol/L for
4 hours. (B) Incubation with 1.25 nmol/L VEGF for 0.5 , 1, 2, 4, and 6 hours. For comparison, kinetics of TF induction by TNF- (100 U/mL)
is shown. Each value is the mean ± SD of triplicate in one experiment
representative of five performed with similar results. (C) VEGF
upregulates TF mRNA in HUVEC: Northern blot analysis of total RNA (20 µg) extracted from unstimulated cells and cells treated with VEGF for
2 and 4 hours. This experiment is representative of two experiments
done with similar results.
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It is known that the human VEGF165 isoform binds to both
VEGF receptor-1 (Flt-1) and VEGF receptor-2 (Flk-1/KDR), whereas VEGF121 interacts selectively only with
Flk-1/KDR.10,37 Because HUVEC express both types of VEGF
receptors,13,38 we investigated whether both VEGF isoforms
would induce TF. Treatment of endothelial cells with
VEGF121 or VEGF165 at 1.25 nmol/L resulted in
comparable TF upregulation (data not shown). Thus, VEGF binding to the
Flk-1/KDR receptor seems to be sufficient to mediate TF activation. In
all of the following experiments VEGF165 was used.
To evaluate whether the upregulation observed on the protein level is
based on induction on the mRNA level, total RNA was isolated from HUVEC
exposed to 1.25 nmol/L VEGF for 2 and 4 hours, and TF mRNA levels were
determined by Northern blotting (Fig 1C). TF mRNA was not detectable in
unstimulated HUVEC and was strongly upregulated by VEGF treatment at
both time-points.
Furthermore, to show that the effect of VEGF on endothelial TF
expression is not restricted to umbilical-vein-derived endothelial cells, we tested HSMEC after treatment with increasing concentrations of VEGF. A similar dose-response curve and kinetics of TF induction were detected by clotting assay (data not shown). Thus, our data clearly demonstrate that TF is generally induced on vascular
endothelial cells by the angiogenic factor VEGF to an extent comparable
to the inflammatory stimulus TNF-.
VEGF does not upregulate adhesion molecule expression on the surface
of endothelial cells.
Because TNF- treatment of endothelial cells results in upregulation
of a number of genes involved in the inflammatory response, including
the endothelial-leukocyte adhesion molecules VCAM-1, E-selectin, and
ICAM-1, we tested whether VEGF induces expression of these proteins.
Using a cell ELISA, we failed to detect any increase of VCAM-1,
E-selectin, or ICAM-1 expression on the surface of VEGF-treated HUVEC,
while TNF- used in parallel caused a strong upregulation
(Fig 2).
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| Fig 2.
VEGF does not upregulate adhesion molecule expression on
the surface of endothelial cells. Cell ELISA was performed with
anti-VCAM-1, anti-E-selectin, and anti-ICAM-1 antibodies. HUVEC were
treated by TNF- (100 U/mL) or VEGF (1.25 nmol/L) for 1, 3, and 6 hours. Each value is the mean ± SD of triplicate in one experiment
representative of three performed with similar results.
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NFB is not involved in VEGF-induced TF upregulation.
Mackman et al39 and our group4 have previously
analyzed the regulation of the TF gene by inflammatory
mediators. These studies showed that the NFB-like site
within the TF promoter is required for transcriptional upregulation
during the inflammatory response. Therefore, we investigated whether
NFB would play an important role in the induction of the TF gene by
VEGF. The observation that VEGF does not induce adhesion molecule
expression already suggested that NFB may not be involved, because
NFB activation is known to be a major trigger of adhesion molecule
induction. By using immunofluorescence staining with anti-p65
antibodies, we first analyzed whether VEGF induces NFB translocation
from the cytoplasm into the nucleus. Unstimulated HUVEC showed a clear cytoplasmic localization of the p65 subunit of NFB
(Fig 3A, I). One-hour treatment with
TNF- (100 U/mL) resulted in translocation of p65 into the nucleus
(Fig 3A, II), while VEGF treatment (1.25 nmol/L) from 10 minutes to 4 hours had no detectable effect (Fig 3A, III).
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| Fig 3.
NFB is not induced by VEGF. (A) Immunofluorescence
staining of p65 (NFB subunit) in unstimulated (I), TNF--treated
(II), or VEGF-treated (III) HUVEC. TNF- (100 U/mL) and VEGF (1.25 nmol/L) were added for 1 hour before cell fixation. Scale bar, 25 µmol/L. (B) Electophoretic mobility shift assay with oligonucleotides
containing the NFB recognition sites from the TF and Ig promoters.
Nuclear extracts were prepared from unstimulated HUVEC and cells
induced for 1 hour with VEGF (1.25 nmol/L) or TNF- (100 U/mL). These
data are representative of three experiments performed with similar
results.
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Taking into account the possibility that VEGF could induce activation
of NFB subunits other than p65, we performed in addition electrophoretic mobility shift assays with the TF NFB as well as the
consensus NFB sites that have been shown to bind c-Rel/p65 and
p50/p65 complexes, respectively. Nuclear extracts from VEGF-treated cells failed to form protein-DNA complexes with the respective binding
sites. In contrast, NFB binding activity was significantly activated
by TNF- (Fig 3B). These results show that VEGF treatment of
endothelial cells does not lead to NFB activation.
Functional importance of a GC-rich region for VEGF-induced expression
of TF: Mithramycin strongly inhibits TF activity.
Mithramycin is a DNA-binding drug known to preferentially bind to
GC-rich DNA sequences. It has been shown to specifically inhibit the
transcription of genes highly dependent on GC-rich regulatory elements
within their promoters such as c-myc, H-ras, dihydrofolate reductase,
and VEGF.28-30 We examined whether mithramycin could
inhibit VEGF- or TNF--induced upregulation of TF
(Fig 4). Treatment of HUVEC with
mithramycin at 1 µmol/L resulted in complete inhibition of
VEGF-induced TF activity. At the same time, induction by TNF- was
blocked by only 50% at the highest concentration of mithramycin used
(10 mmol/L). At concentrations of mithramycin ranging from 10 nmol/L to
10 µmol/L, cell morphology and viability were not altered (data not
shown). To show that the observed inhibition of TF upregulation is not
due to a more general effect of this drug on DNA synthesis, we tested
whether mithramycin would block the induction of E-selectin by TNF-.
The E-selectin promoter does not contain GC-rich regions functionally
important for TNF- regulation. We could not detect any decrease of
TNF--induced E-selectin expression in presence of mithramycin.
Thus, these data suggest that the VEGF-responsive element of the TF
promoter is located within a GC-rich region that is separate from the
NFB recognition site.
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| Fig 4.
Mithramycin strongly inhibits VEGF-induced TF activity.
HUVEC were preincubated for 30 minutes with increasing concentrations
of mithramycin (M) before stimulation with VEGF (A) or TNF- (B) for
4 hours. TF activity equivalents were measured in a one-stage clotting
assay. (C) Mithramycin has no effect on TNF--induced E-selectin
expression measured by ELISA. X-axis: concentration of mithramycin.
Each value is the mean ± SD of triplicate in one experiment
representative of two performed with similar results.
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A VEGF-inducible DNA binding complex containing EGR-1 interacts with
the GC-rich region at 85 to 70 of the human TF promoter.
The results presented thus far show that NFB is not involved in
VEGF-mediated TF upregulation. Moreover, the inhibition of VEGF effects
by mithramycin points to the importance of a GC-rich promoter region in
TF upregulation by VEGF. To identify possible VEGF-induced proteins
interacting with this region we used several overlapping
oligonucleotides, covering the entire GC-rich region of the human TF
promoter (171 to 52) (Fig
5A). Nuclear extracts from unstimulated cells and cells treated with
VEGF for 1 hour were incubated with each of the six radiolabeled
oligonucleotides (Figs 5B and 6). OL1, OL3, and OL5 exhibited a similar
pattern of protein binding of three distinct complexes, that were
specific, constitutive, and not altered by treatment with VEGF.
Complexes I and II were attributed to Sp1 binding and were supershifted by anti-Sp1 antibodies4 (and data not shown). Complex III
was not characterized in detail because it was detected in both
unstimulated and VEGF-stimulated extracts.
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| Fig 5.
(A) Transcription factor binding elements within the
region of 230 to 50 bp of the human TF promoter. The start site
of transcription is indicated by an arrow. Numbering is from the human
TF sequence as given in Mackman et al.59 Oligonucleotides
spanning the EGR-1/Sp1 sites (designated OL1 to OL6) were used in
electrophoretic mobility shift assays. (B) Binding of nuclear proteins
to the GC-rich region. Oligonucleotides OL1 to OL6 were radiolabeled
and incubated with nuclear extracts from uninduced cells () and
cells stimulated with VEGF (+) for 1 hour. Protein-DNA complexes I,
II, III, and IV are indicated; ns, nonspecific binding. OL4 and OL6
showed identical protein/DNA complexes. Shown is one experiment that is
representative of four experiments with similar results.
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OL4 and OL6, a shorter version of OL4, showed an identical pattern of
protein binding: in addition to complexes I and II described above, a
single inducible complex (complex IV) was detected with VEGF-stimulated
cells. Sp1 binding (complexes I and II) seemed to decrease upon
induction of complex IV. OL6 includes Sp1 binding sites as well as an
overlapping putative EGR-1 recognition site GCGGGGGCG21
overlapping the Sp1 sites. A similar inducible complex, but of lower
intensity, was observed with OL2, which also contains in addition to an
Sp1 recognition element an EGR-1 site that differs from the consensus
sequence at positions 1 and 9.
Competition studies were performed using various unlabeled
oligonucleotides to probe the binding specificity of complexes I, II,
and IV with OL6 (Fig 6). All three
complexes were competed by excess of cold OL6 (lane 3). Competition
with OL1 (Sp1 consensus) blocked formation of complexes I and II,
leaving complex IV intact (lane 4). An oligonucleotide covering AP-1
sites from the TF promoter did not influence the formation of any of
the three complexes (lane 5).
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| Fig 6.
Identification of the VEGF-induced factor binding to OL6.
Radioactively labeled OL6 was incubated with nuclear extracts from
unstimulated cells (lane 1) and cells treated with VEGF (lanes 2 to 8)
in the absence or presence of unlabeled oligonucleotides and specific
antibodies. The Sp1 (complexes I and II), VEGF-induced (complex IV),
and nonspecific (ns) protein/DNA complexes are indicated.
VEGF-stimulated extracts were analyzed by competition with a 100-fold
molar excess of unlabeled oligonucleotides OL6 (lane 3), OL1 (lane 4),
or AP-1 (see Materials and Methods, lane 5). Supershift experiments
were performed using 3 µL of anti-EGR-1 (lane 6), anti-AP-1 (lane
7), or anti-AP-2 (lane 8) antibodies. Shown is one experiment that is
representative of three experiments with similar results.
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The constitutive expression of Sp1 in endothelial cells suggests that
this transcription factor occupies the binding sites in the absence of
a VEGF-inducible factor. Our electrophoretic mobility shift data
confirm Sp1 binding in unstimulated cells. Upon exposure to VEGF,
however, the intensity of Sp1 binding decreased substantially and
seemed to be replaced by a VEGF-inducible protein interacting with the
same GC-rich element of the TF promoter.
To identify the proteins present in the VEGF-inducible complex IV, we
used antibodies directed against EGR-1, AP-1, and AP-2 in supershift
experiments (Fig 6, lanes 6 to 8). AP-2 is a zinc-finger transcription
factor that also interacts with a GC-rich nucleotide sequence very
similar to the EGR-1 site and has been shown to play a significant role
in the upregulation of the VEGF gene by TGF-.40
Preincubation with anti-EGR-1 antibodies completely abolished
formation of complex IV (lane 6), whereas antibodies to AP-1 and AP-2
(lanes 7 and 8) failed to produce supershifts. Therefore, we conclude
that VEGF-inducible complex IV represents binding of EGR-1. We assume
that the identity of the OL6 sequence to the EGR-1 consensus binding
site and possible additional influences of surrounding nucleotides may
explain the preferential binding of EGR-1 to OL6.
Analysis of the VEGF-responsive region within the TF promoter by
reporter gene studies.
To confirm the role of EGR-1 elements in the transcriptional activation
of the TF gene by VEGF, a series of deletion constructs based on the
330/+118 basal TF promoter construct were transiently transfected into HUVEC. Six plasmids were analyzed: wt, covering the
wild-type TF promoter from 330 to +118 bp; AP-1, NFB, and Sp1/EGR-1, which contain substitutions of the respective transcription factor binding sites; min, a minimal promoter construct, which is limited to the GC-rich promoter region including Sp1 and EGR-1
sites; mut, a mutated promoter construct containing a single point
mutation (G A) at the position 5' to the consensus EGR-1 site. This point mutation is located within the region to which
VEGF-induced EGR-1 was found to bind in the electrophoretic mobility
shift assay. Values of all other reporter constructs are given in
percent of wt activity. Deletion of AP-1 or NFB sites as well as the
point mutation did not affect basal promoter activity
(Fig 7A). However, deletion of the entire
GC-rich region reduced basal promoter activity by more than 60%. The
basal expression of the minimal promoter construct was slightly
reduced, although this difference was not statistically significant.
These results are in good agreement with previously published data
showing that Sp1 mainly regulates basal activity of the TF
promoter.15
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| Fig 7.
Identification of the VEGF-responsive region within
the TF promoter. Reporter plasmids used in this study are shown
schematically (see also Materials and Methods). Luciferase activity of
reporter constructs transiently transfected in HUVEC in the absence (A)
or presence (B) of VEGF was calculated relative to wild-type (wt)
promoter activity designated as 100%. In each experiment values were
determined from duplicate wells and normalized for transfection
efficiency. Results from three independent experiments are shown as
mean values ± SD. *P < .01 versus wt activity.
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Luciferase activity of the wild-type reporter was induced twofold to
threefold by VEGF. This may not reflect the full degree of TF gene
upregulation in vivo, because we observe a partial activation of TF
expression in primary endothelial cells by the transfection procedure
leading to increased basal levels. Deletion of NFB and AP-1 sites
had no influence on the level of upregulation (Fig 7B). However,
deletion of all Sp1 and EGR-1 sites abolished VEGF induction
completely. In contrast, the minimal promoter construct covering all
Sp1 and EGR-1 sites showed a level of upregulation comparable to the
wild-type promoter. Interestingly, the point mutation immediately
5' to the consensus EGR-1 significantly reduced promoter
responsiveness to VEGF. These results show that the VEGF-responsive site is located within the GC-rich region of the TF promoter and suggest that the consensus EGR-1 site in the 3' part of this
region is functionally important for maximal induction of the TF promoter.
A point mutation affects DNA binding of EGR-1.
The influence of nucleotides surrounding an EGR-1 recognition element
on the affinity of EGR-1 binding has not been subject to detailed
analysis. However, based on the results of our reporter studies, we
expected the point mutation 5' to the EGR-1 consensus site to
interfere with EGR-1 binding to DNA. We performed electrophoretic mobility shift assays with oligonucleotide mOL6, containing the same G
A substitution present in the reporter construct. It may be
of importance to note that this position is conserved in the sequences
of human and porcine TF promoters.4 mOL6 formed Sp1
complexes identical to the wild-type oligonucleotide OL6. However,
VEGF-inducible EGR-1 binding was no longer detectable with the mutant
oligonucleotide (Fig 8). Competition
studies showed that this mutated oligonucleotide was able to compete
the formation of the EGR-1 complex, but at significantly higher
concentration than the wild-type (data not shown). These results
confirm that a single nucleotide substitution immediately 5' of
the consensus EGR-1 site significantly decreases the affinity of EGR-1
binding to its recognition site and, thus, interferes with VEGF
induction of TF expression.
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| Fig 8.
A point mutation 5' to the EGR-1 consensus site
abrogates EGR-1 binding. Sequences of the wild-type (OL6) and mutant
(mOL6) are shown at the top. The consensus EGR-1 site is underlined and
the G A substitution is indicated by a bold letter. Nuclear
extracts from unstimulated () and VEGF-stimulated (+) cells were
incubated with radiolabeled OL6 or mOL6. The position of the EGR-1
complex is indicated. Shown is one experiment that is representative of
three experiments with similar results.
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Over-expression of EGR-1 results in trans-activation of the
TF promoter in HUVEC.
To further confirm the functional importance of EGR-1 in the
transcriptional regulation of TF gene expression, we performed cotransfection experiments. Expression vectors encoding full-length EGR-1 or a transcriptionally inactive mutant, mEGR-1, lacking the first
and part of the second zinc finger domains,33 were transiently transfected together with the wild-type TF reporter or the
Sp1/EGR-1 construct (Fig 9).
Cotransfection of full-length EGR-1 markedly increased the activity of
the wild-type promoter in a manner comparable to VEGF induction.
However, the mutant EGR-1 did not lead to upregulation of luciferase
expression. In addition, the Sp1/EGR-1 construct did not display any
EGR-1-mediated response. Taken together, these findings show that
EGR-1 is able to mediate transcriptional activation of the TF gene via
the VEGF-responsive element, and substantiate the functional role of
EGR-1 in the transcriptional regulation of the TF gene by VEGF.
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| Fig 9.
Overexpression of full-length EGR-1, but not a
transcriptionally inactive mutant, results in activation of the TF
promoter in HUVEC. EGR-1-mediated activation of the TF promoter is
prevented by deletion of the Sp1/EGR-1 sites. Luciferase activity of
wild-type (wt, white bars) or deleted ( Sp1/EGR-1, black bars)
reporter constructs in the presence of the EGR-1 expression plasmid or
the inactive mutant (mEGR-1) was calculated relative to the wt promoter
activity (mean ± SD from three independent experiments performed in
duplicates; *P < .01 versus basal wt activity).
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Subcellular localization of EGR-1 protein in endothelial cells.
To substantiate the importance of EGR-1 for the VEGF response, we
analyzed the subcellular localization and expression of EGR-1 protein
in unstimulated and VEGF-induced endothelial cells (Fig 10A through G). The great majority
of unstimulated cells under normal growth conditions had diffuse
staining in the cytoplasm, as well as weak staining in the nucleus (Fig
10C). Fifteen minutes of treatment with VEGF were sufficient to trigger
a clear increase in nuclear staining (Fig 10D). Maximal nuclear
staining was seen between 1 and 2 hours of stimulation with VEGF. Six
hours after the addition of VEGF to the cells, the signal distribution
returned to the level seen in uninduced cells. At 8 hours, weak
cytoplasmic staining was detectable in most cells (Fig 10G). We tested
two anti-EGR-1 antibodies (Santa Cruz Biotechnology) and obtained comparable results. After preincubation of anti-EGR-1 antibodies with
the peptide to which the antibodies were raised (Fig 10A and B), only a
very low level of nuclear staining was still visible and cytoplasmic
staining was completely abolished, confirming the specificity of the
antibodies used. Nuclear localization of induced EGR-1 protein was
confirmed by costaining with DAPI (data not shown). In contrast,
staining for Sp1 was constitutively present in the nucleus and is not
influenced by VEGF treatment (Fig 10H through L). These data combined
with other data described above show that VEGF treatment of endothelial
cells results in rapid accumulation of EGR-1 transcription factor in
the nucleus, thereby activating expression of the TF gene.
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| Fig 10.
Subcellular localization of EGR-1 and Sp1 in HUVEC.
Immunofluorescence staining of EGR-1 in unstimulated endothelial cells
(C) and cells stimulated with VEGF for 15 minutes (D), 1 hour (E), 6 hours (F), and 8 hours (G). Preabsorbtion of the anti-EGR-1 antibodies
with an appropriate peptide abolished both cytoplasmic and nuclear
staining of unstimulated (A) and VEGF-stimulated cells (B). Sp1
staining was exclusively nuclear and identical for all time points (H
through L). The picture is representative of four experiments with
similar results.
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DISCUSSION |
Several recent findings suggest that TF may have function(s) distinct
from the activation of the coagulation cascade. This protein has a
capacity to transmit intracellular signals41 and appears to
participate in embryonic blood vessel development,42 metastasis,43,44 and tumor-associated
angiogenesis.5,45
Detection of TF on the endothelium of tumor-induced vessels raised the
question whether tumor cells per se stimulate the production of TF. One
possibility would be that expression of TF is mediated by the direct
angiogenic factor VEGF, because many tumor cells produce VEGF and
endothelial cells express its high-affinity receptors. For this reason
we were interested to understand the molecular mechanisms that
would lead to TF activation in endothelial cells in response to VEGF.
Our data show that TF expression induced by VEGF was as strong as the
expression induced by TNF-. However, whereas inflammatory cytokines
are known to trigger the TF gene in endothelial cells mainly via
activation of NFB, the present report shows that the mechanisms
leading to induction of TF expression by the angiogenic factor VEGF are
different from those of inflammatory stimuli and do not involve NFB.
Several lines of evidence indicate that the main factor involved in
VEGF-mediated TF expression is the transcription factor EGR-1. Firstly,
strong inhibition of TF activity by mithramycin, a drug that binds to
GC-rich DNA sequences, initially suggested the functional importance of
the GC-rich region within the TF promoter in response to VEGF. This
region is separate from the NFB recognition site and spans several
Sp1/EGR-1 overlapping binding sites. Secondly, a VEGF-inducible DNA
binding complex was detected with an oligonucleotide covering the
sequence of the human TF promoter from 85 to 70. In
unstimulated endothelial cells this element is occupied by Sp1.
Supershift data using anti-EGR-1 antibodies clearly show that the
VEGF-inducible complex contains the transcription factor EGR-1. It is
intriguing that Sp1 binding to this region decreased substantially and
seems to be replaced by EGR-1 upon exposure of the cells to VEGF. Thus,
it appears that the induction of the TF gene by VEGF in endothelial
cells involves the interplay of the zinc-finger transcription factors Sp1 and EGR-1 with the indicated promoter element. A similar
competition for binding was proposed for the regulation of PDGF-A and
PDGF-B expression in endothelial cells by PMA.24 Thirdly,
reporter gene studies confirmed that the VEGF-responsive site is
localized within the GC-rich region of TF promoter. The consensus EGR-1 site in the 3' part of this region seems to be functionally
important for maximal induction of the TF gene by VEGF because a point
mutation 5' to the consensus EGR-1 site significantly diminished
upregulation by VEGF. This position which is conserved in the sequences
of the human, porcine, and murine promoters seems to be important for
the optimal interaction of VEGF-induced EGR-1 with its recognition site, because in electrophoretic mobility shift assays VEGF-inducible EGR-1 binding was no longer detectable with the corresponding mutant
oligonucleotide. Another observation based on the reporter gene studies
concerns the basal expression of the TF promoter in endothelial cells.
Deletion of the entire GC-rich region reduced basal promoter activity
by about 60%. As mentioned above, in unstimulated endothelial cells
this element is occupied by constitutively expressed Sp1 factors. These
results are in accordance with data from Cui et al15
showing that Sp1 plays a role for basal activity of the TF promoter in
the epithelial-like HeLa cells.
Finally, overexpression of full-length EGR-1, but not of an inactive
mutant, results in trans-activation of the TF promoter comparable to VEGF induction. Addition of VEGF to cells overexpressing functional EGR-1 did not result in further activation of the TF promoter (data not shown). These findings substantiate the significant role of EGR-1 in VEGF-induced transcriptional regulation of the TF gene
in endothelial cells.
A multitude of data published on EGR-1 supports its central role as a
multifunctional transcription factor important for growth and
differentiation. EGR-1 mRNA is induced within 15 to 30 minutes by a
wide range of extracellular signals, including growth factors, and does
not require de novo protein synthesis. Some data suggest that multiple,
independent, possibly additive pathways for the induction of this gene
must exist. When neural PC12 cells were exposed simultaneously to
maximally inducing levels of PMA and EGF, EGR-1/TIS induction was
synergistically elevated.46 Similarly, when endothelial
cells were treated with a combination of VEGF and TNF-, TF activity
was more than additively upregulated46 (and D.M., E.H.,
unpublished data, 1998). However, the specific signaling
pathways and their possible cross-talk leading to EGR-1 activation in
endothelial cells still need to be defined. Another aspect of EGR-1 is
the cell-type-specific variation of its function. For example, the
activity of the PDGF-A promoter is reduced by EGR-1 in NIH3T3 cells but
activated in human embryonic kidney-derived 293 cells.47
Second, the dramatic enhancement in transcriptional activity seen in
HeLa cells with the EGR-1 construct from which the repressor domain was
deleted (284-330) was not detected in 3T3 cells.33
Importantly, EGR-1 is one of only a small number of bifunctional
transcription factors that contain both activation and repression
domains. Signal-specific posttranslational modification of EGR-1,
interaction with other regulating factors, and/or presence of a
transcription factor inhibitor may activate or repress different target
genes in a cell-type-specific manner.
In addition to the results discussed above, we have further tested the
subcellular localization and expression of EGR-1 protein in endothelial
cells by immunofluorescence. Its subcellular localization has been
described for serum-starved fibroblasts after serum
stimulation.33,48 Using immunostaining, we found that the
protein starts to accumulate in the nucleus within 15 to 30 minutes,
reaches a peak level at about 1 to 2 hours, and decreases gradually
thereafter within a few hours. The time course of nuclear accumulation
is in agreement with its proposed function as the primary inducer of TF
by VEGF. Thus far, the mechanisms that mediate rapid egr-1 gene
activation by growth factors are only partially understood. Our data
suggest the presence of low levels of the protein in the cytoplasm of unstimulated endothelial cells. It is not clear at the moment whether
that means that low amounts of EGR-1 are continuously synthesized in
endothelial cells in culture or if EGR-1 protein is retained in the
cytoplasm of untreated cells. It is well established that
many transcription factors can be selectively relocalized from th |
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ABSTRACT
INTRODUCTION