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-mediated Induction of Endothelial Tissue
Factor (*)
(Received for publication, March 7, 1995; and in revised form, July 31, 1995)
From the
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
This study examines the regulation of the human tissue factor
(TF) promotor in vitro and in vivo. Transient
transfections were performed in bovine aortic endothelial cells to
investigate the role of two fundamentally different AP-1 sites and a
closely located NF-
B site in the human TF promotor. The NF-B
site is functionally active, since overexpression of NF-B(p65)
resulted in induction of TF mRNA and activity. Promotor analysis showed
that NF-B induction was dependent on the integrity of the region
from base pair -188 to -181. Overexpression of Jun/Fos
resulted in TF induction of transcription and protein/activity.
Functional studies revealed that the proximal AP-1 site, but not the
distal, was inducible by Jun/Fos heterodimers. The distal AP-1 site,
which has a G 
A switch at position 4, was inducible by Jun
homodimers. Electrophoretic mobility shift assays, using extracts of
tumor necrosis factor 
(TNF)-stimulated bovine aortic
endothelial cells, demonstrated TNF-inducible binding to the
proximal AP-1 site, comprising JunD/Fos heterodimers. At the distal
AP-1 site, only minor induction of binding activity, characterized as
proteins of the Jun and ATF family, was observed. Consistently, this
site only marginally participates in TNF induction. Functional
studies with TF promotor plasmids confirmed that deletion of the
proximal AP-1 or the NF-B site decreased TNF-mediated TF
induction to a higher extend than loss of the distal AP-1 site.
However, integrity of both AP-1 sites and the NF-B site was
required for optimal TNF stimulation. The relevance of these in vitro data was confirmed in vivo in a mouse tumor
model. Expression plasmids for a dominant negative Jun mutant or
I-B were packaged in liposomes. When either mutated Jun or
I-B were injected intravenously 48 h before TNF, a reduction
in TNF-mediated TF expression in the tumor endothelial cells was
observed. Simultaneously, fibrin/fibrinogen deposition decreased and
free blood flow could be restored. Thus, TNF-induced up-regulation
of endothelial cell TF depends on a concerted action of members of the
bZIP and NF-B family.
Unstimulated endothelial cells express no tissue factor
(TF)
in vitro and in
vivo(1, 2, 3, 4, 5, 6) .
Recent studies show that TF expression in vitro can be induced
by
TNF(7, 8, 9, 10, 11) . In vivo, however, expression of TF has only been shown in
selected vascular beds: in the splenic endothelium in a septicemia
model (12) and in tumors such as Meth-A and Karposi
sarcomas(13, 14) . The human TF promotor has been
characterized, and its function has been extensively studied in
monocytes/macrophages(15, 16, 17, 18) .
The porcine TF promotor has been described in endothelial
cells(11) . The data available from the human TF promotor
suggest that two AP-1 and the NF-B site are central in the
endotoxin-dependent regulation of TF
expression(16, 17, 18) . In contrast, the
study of the porcine TF promotor shows that mainly the induction of
NF-B is responsible for lipopolysaccharide- and TNF-mediated
induction of endothelial TF expression(11) .
The discrepancy
in the role of AP-1 in the human and porcine TF promotor might be due
to differences in the sequence composition of the AP-1 binding sites of
the various species. Sequence alignments revealed important differences
between the AP-1 sites of the different species (11) . The
porcine TF promotor has two non-canonical AP-1 sites that differ from
the defined AP-1 consensus sequence in one central base (11) .
Non-canonical AP-1 sites have been reported of being weak binding
sites(19) . In contrast, the proximal AP-1 site in the human
(and the mouse) TF promotor contains the core of the consensus sequence (11, 15, 16) and thereby represents a high
affinity site for AP-1 binding. This indicates that different AP-1-like
proteins may be involved in the regulation of TF expression in
different species. If the porcine model (11) would be relevant
for human disease, then blocking of NF-B activation would provide
a powerful way to prevent excess TF expression in human disease. If the
human model (15, 16, 17, 18) is
relevant, then inhibition of NF-B activation might lead to
increased c-Fos transcription (20, 21) and thereby to
AP-1-mediated TF induction. To resolve this issue, we studied the role
of both AP-1 sites and their cooperative action with the NF-B site
in the human TF promotor.
A number of homo- and heterodimers can
recognize AP-1 sites(22, 23, 24, 25) but exhibit different affinities for different
motifs(26) . These proteins have been termed bZIP family (27) due to their ability to dimerize via an -helical
leucine ``zipper.'' The members of the Jun subfamily c-Jun,
JunB, and JunD are highly homologous in their dimerization and binding
domains and can compete for the same AP-1 sites(28) . The
transactivating capacities of c-Jun and JunD are dramatically increased
in combination with c-Fos(29, 30) . The functional
homologues of c-Fos, Fra-1, Fra-2, and FosB can also dimerize with Jun
proteins(30, 31, 32, 33) . In
addition, members of the ATF/CREB family like ATF-2, ATF-3, and ATF-4
(but not ATF-1) are capable of binding to proteins of the AP-1
transcription factor family (25, 26, 34) .
Thus, it was our hypothesis that the distal non-canonical AP-1 site in
the human TF promotor and the proximal canonical AP-1 site bind
different members of the AP-1/bZIP family.
-
P]dCTP (3000 Ci/mmol
at 10 Ci/ml), [
-P]dATP (3000 Ci/mmol at 10
Ci/ml), [-P]UTP (3000 Ci/mmol at 10 Ci/ml),
[
S]methionine (>10,000 Ci/mmol), Hybond-N
nylon filter, and Hyperfilm x-ray films were obtained from Amersham,
Braunschweig, Germany. Poly(dI-dC) was purchased from Sigma,
Deisenhofen, Germany. Rabbit reticulocyte lysate and recombinant AP-1
(c-Jun, human) were purchased from Promega. Anti-p50 (sc-114X),
anti-p65 (sc-109X), and anti-c-Rel (sc-70X) polyclonal antibodies,
anti-c-Jun (sc-45X; specific for c-Jun, non-cross-reactive with JunB or
JunD), anti-pan-Jun (sc-44X; recognizing the C termini of c-Jun, JunB,
JunD), anti-JunD (sc-74X; no c-Jun or JunB cross-reactivity),
anti-c-Fos (sc-52X; specific for c-Fos p62, non-cross-reactive with
FosB, Fra-1, Fra-2) and anti-ATF-2 (sc-187X; non-cross-reactive with
other ATF/CREB proteins) polyclonal antibodies, an anti-ATF-1 (sc-243X;
non-cross-reactive with other ATF/CREB proteins) monoclonal antibody,
and recombinant ATF-2-bZIP(350-505) were obtained from Santa Cruz
Inc., Santa Cruz, CA. TNF (10
units/mg) was a gift
from Knoll AG, Ludwigshafen, Germany.
-galactosidase control plasmid pSV--Gal, and the
chloramphenicol transferase control vector pCAT-control were obtained
from Promega. pSPT18 was purchased from Boehringer Mannheim, Germany.
The TF promotor mutants pHTF(-278)Luc (A1-A2-N),
pHTFM2(-278)Luc (A2-N), pHTFM3(-278)Luc (A1-N), and
pHTF(-111)Luc have been described previously ( (15) and (16) ; see Table 1). The plasmid pHTFO(-441)Luc
(A1-A2) was prepared by inserting the polymerase chain reaction product
of region bp -441 to bp -195 into the SmaI site of
pHTF(-111)Luc (Table 1). The clones A1, A2, and N were
constructed by subcloning oligonucleotides, spanning 24 bp of the
respective binding site into the SmaI site of
pHTF(-111)Luc (Table 1). All plasmids were characterized by
DNA sequencing. pSV-c-Jun(35) , pBK28(c-Fos)(35) ,
T7-Jun, and T7-Fos (36, 37) were generously provided
by Dr. I. Verma (San Diego, CA). NFB(p65) (RC-CMV-p65) and
I-B (RC/CMVMAD-3wt) have been described(38) . The mutated
Jun plasmid pDB7, a derivate of the point mutant Mut14(39) ,
was obtained from Dr. D. Bohmann (EMBL, Heidelberg, Germany). The human
TF cDNA probe 
HTF8 was generously provided by Dr. E. Sadler
(Washington University, St. Louis, MO)(40) . Meth-tRNA was a
gift of Dr. Xanthopoulus (Karolinska Institute, Stockholm, Sweden);
other plasmids mentioned were obtained from ATCC.
Methylcholanthrine-A-induced (Meth-A) sarcoma cells were a gift of Dr. D. Männel (DKFZ, Heidelberg, Germany) and were cultured in RPMI 1640, 10% FCS, 100 units/ml penicillin, 100 units/ml streptomycin as described elsewhere(14) .
solution (Behring, Marburg, Germany) the samples were
incubated at 37 °C. The time from addition of CaCl to
the first defined fibrin strand was determined. TF activity was
calculated by comparing the measured clotting time with a standard
curve made with known amounts of TF(1) . The measured amount of
TF was expressed as picograms of TF/10
cells ± S.D.
All experiments were performed at least three times, and each
experiment was done in triplicates.
. Aliquots (30 µl) were
added to 470 µl of 50 mM Tris, pH 7.9, 175 mM NaCl, 5 mM EDTA, and 0.5 mg/ml BSA. Factor Xa formation
was assessed using 100 µl of S2222 added to the entire 0.5-ml
sample. Hydrolysis was monitored at room temperature by measuring the
change in absorbance at 405 nm, using a Beckmann DU 7400
spectrophotometer (Beckmann, Dreieich, Germany). Factor Xa (final
concentration 20 µg/ml) (44) served as control. Each
experiment was repeated three times.
Figure 1:
Overexpression of
c-Jun/Fos(AP-1), NF-B(p65), or c-Jun/Fos(AP-1) and NF-B(p65)
induces TF transcription, activity, and antigen. a, nuclei
were extracted from BAEC transiently transfected with CAT (=
mock control), c-Jun, c-Fos, and/or NF-B(p65) overexpressing
plasmids. Nuclear run-on experiments were performed as described under
``Materials and Methods'' to allow in vitro synthesis of [-P]UTP-labeled mRNA.
This was hybridized against filters onto which cDNAs for TF (top), Meth-tRNA (bottom), pSPT18, and TNF
(negative controls, data not shown) had been fixed. b,
activation of factor X by BAEC transfected with CAT (= mock
control), c-Jun, c-Fos, and/or NF-B(p65). Cells were transiently
transfected with the respective plasmids, harvested after 42 h, and
assayed for factor X activation in the presence of factor VII (35
µg/ml). The generation of factor Xa was measured
spectrophotometrically over a period of 15 min. S2222 served as
substrate as described under ``Materials and
Methods.'' Two experiments were performed in triplicates with
identical results. One typical experiment is shown. c, BAEC
were transiently transfected with CAT (= mock control), c-Jun,
c-Fos, and/or NF-B(p65), harvested 42 h after transfection, and
assayed for procoagulant activity as described under
``Materials and Methods.'' TF activity of each
sample was determined based on comparison with a standard curve
established with known amounts of recombinant TF. The data ±
S.D. represent the mean of three independent experiments performed in
triplicate (p values: control versus Jun/Fos =
0.008; control versus NF-B = 0.009; control versus Jun/Fos/NF-B =
0.009)
10
cells after 42 h of
transfection. Run-on reactions were performed in 0.7 M KCl, 50
mM MgCl, 50 mM Tris-HCl, pH 8.0, 25
mM DTT, 1 mM EDTA in the presence of 250 µCi of
[-P]UTP (3000 Ci/mmol) and incubated for 30
min at 30 °C. The synthesized mRNA was incubated with DNase I for 5
min at 30 °C, treated with proteinase K (10 mg/ml), and extracted
with 0.45-µm Millipore filters (type HA). The RNA was collected by
EtOH precipitation and redissolved in 300 µl of
DEPC-HO, 1 µl was counted, and equal numbers of
Cerenkov counts were made up to 2 ml of hybridization solution and
added to the previously prepared slot blot filters. Hybridization was
performed without prehybridization in 50% formamide, 5 SSC, 5
Denhardt's solution, 1% SDS for 4 days at 42 °C.
Filters were washed three times at room temperature for 10 min in 2
SSC and once for 10 min at 60 °C in 1 SSC. Blots
were exposed to Amersham Hyperfilms for 1-4 weeks at -80
°C with intensifying screens. The density of autoradiographic
signals was quantitated using a Beckman DU 7400 densitometer (Beckman,
Dreieich, Germany)(46, 47, 48) . For
preparation of filters, 3 µg of human TF plasmid DNA or the
respective household and control plasmids (GAPDH, Meth-tRNA, pSPT18,
p19-Luc, TNF cDNA) were applied to Hybond-N membranes using a slot
blot apparatus (Schleicher & Schüll, Dassel,
Germany) as described
previously(46, 47, 48) .
-P]dCTP to a specific activity
>10 cpm/µg DNA by the random prime technique (51) . Filters were prehybridized for 1 h and hybridized for
12-16 h at 65 °C in 50 mM PIPES, pH 6.8, 200 mM NaCl, 20 mM NaPO
, 30 mM NaHPO4, 1 mM EDTA, 5% SDS, 50 µg of salmon sperm/ml,
50 µg of yeast tRNA/ml(47) , washed 3 15 min in 1.3
SSC, 5% SDS, and exposed to Hybond-N Hyperfilms (Amersham,
Braunschweig, Germany) for 5 days.
10 were
harvested in cold PBS, pH 7.4, and pelleted at 1500 rpm for 5 min. The
pellet was resuspended in 400 µl of cold buffer A (10 mM HEPES-KOH, pH 7.9 at 4 °C, 1.5 mM MgCl,
10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF), and
incubated for 10 min on ice. The samples were centrifuged for 10 s at
highest speed, the supernatant was discarded and the pellet was
resuspended in 100 µl of cold buffer C (20 mM HEPES-KOH pH
7.9, 25% glycerol, 420 mM NaCl, 1.5 mM
MgCl, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF) and incubated on ice for 20 min. After centrifugation (2
min, 4 °C, highest speed) the supernatant was quick-frozen at
-80 °C. Protein concentration was determined using a
colorimetric assay based on Bradford(53) . When organ tissue
was used, the above protocol was modified according to Deryckere and
Gannon(54) . Large pieces of tissue (0.1 0.2 cm) were
frozen in liquid nitrogen, broken mechanically with a hammer, and
transferred to a 50-ml Falcon tube containing 5 ml of cold buffer A (10
mM Hepes-KOH, pH 7.9, at 4 °C, 10 mM KCl, 1.5
mM MgCl, 0.5 mM DTT, 1 mM EDTA,
0.2 mM PMSF, 0.6% Nonidet® P-40). The tissue was
homogenized in an Ultrathurax (Wheaton, Millville, NJ) for 1 min,
transferred to an 15-ml tube, and centrifuged for 30 s at 2000 rpm, 4
°C to remove tissue debris. The supernatant was incubated on ice
for 10 min and centrifuged for 5 min at 8000 rpm at 4 °C. The
supernatant was discarded, and the nuclear pellet was resuspended in
100 µl of buffer B (25% glycerol, 20 mM Hepes-KOH, pH 7.9
at 4 °C, 420 mM NaCl, 1.5 mM MgCl,
0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 2
mM benzamidine, 5 mg/ml leupeptin) and incubated on ice for 20
min. Cellular debris was removed by 2 min of centrifugation at 4 °C
and the supernatant was quick-frozen at -80 °C. Protein
concentrations were determined as above.Oligonucleotides, listed in Table 2, were synthesized on a Gene Assembler Plus (Pharmacia,
Freiburg, Germany) and purified on histidine gels(55) . They
were labeled by kinasing to a specific activity >5 10 cpm/µg DNA. Binding of AP-1 was performed in 25 µl of 10
mM HEPES, pH 7.9, 0.1 mM EDTA, 75 mM NaCl, 4
mM MgCl, 2 mM DTT, 17.5% glycerol, 1
mg/ml BSA (DNase-free) in the presence of 0.01 µg/µl
poly(dI-dC)(47) . For organ preparations poly(dI-dC) was scaled
up to 0.15 µg/µl. When recombinant proteins produced in rabbit
reticulocyte lysates (Promega) were included in the reaction, the
poly(dI-dC) concentration was increased to 0.05 µg/µl. When
recombinant human c-Jun (Promega) was used, poly(dI-dC) was replaced by
0.01 µg/µl AP-3 oligonucleotides (Promega) according to the
manufacturer's instruction. NF-B binding was performed in 10
mM HEPES, pH 7.5, 0.1 mM EDTA, 100 mM NaCl,
1 mM ZnCl, 4 mM MgCl, 2
mM DTT, 17.5% glycerol, 1 mg/ml BSA (DNase-free), and 0.1
µg/µl poly(dI-dC) in a total of 25 µl(47) . For
organ preparations, the poly(dI-dC) concentration was increased to 0.3
µg/µl. 8-10 µg of nuclear extract were incubated on
ice for 20 min in the appropriate binding buffer before adding
approximately 1 ng of labeled oligonucleotide. A typical binding
reaction contained 50,000 cpm (Cerenkov). The samples were incubated at
room temperature for and additional 15 min. Protein-DNA complexes were
separated from the free DNA probe by electrophoresis through 4% (AP-1)
or 5% (NF-B) native polyacrylamide gels containing 2.5% glycerol
and 0.5 TBE buffer(47) . The gels run at room
temperature with 30 mA for approximately 2.5 h. Gels were dried under
vacuum on Whatmann D-81 paper (Schleicher and
Schüll, Dassel, Germany) and exposed for
12-48 h to Amersham Hyperfilms at -80 °C with
intensifying screens. Specificity of binding was ascertained by
competition with a 160-fold molar excess of cold consensus
oligonucleotides. For supershifting experiments 2.5 µg of the
respective antibody were applied to the reaction mixture at the time
the labeled oligonucleotide was added.
-S]Methionine as substrate. 0.3-10 µl
(approximately 0.15-5 µg) of in vitro translated
proteins were used in EMSA. Unprogrammed lysate served as control.
) method.
Cells were exposed to the precipitate for 6 h (HUVEC) to 8 h (BAEC).
Medium was changed, and cells were incubated for 42 h. Cells were
harvested in the appropriate buffers.Plasmid DNA used in
transfections was isolated by alkaline lysis, followed by CsCl
equilibrium centrifugation(50) . For promotor studies 0.5
µg of luciferase promotor constructs/ml of medium were transfected.
To correct for variability in transfection efficiency 0.15 µg of
pSV--Gal plasmid/ml of medium were included. For transactivation
experiments, 0.25 µg/ml pSV-c-Jun, pBK28(c-Fos), NF-B(p65),
I-B, or mutated Jun were cotransfected with 0.5 µg of
luciferase containing promotor constructs. Reactions were filled up
with pCAT-control (serving as mock control) to give the final DNA
concentration of 1.4 µg/ml medium. Cell extracts were prepared by
lysis in 25 mM Tris phosphate, pH 7.8, 2 mM DTT, 2
mM 1,2-diaminocyclohexane-N,N,N,N`-tetraacetic acid,
10% glycerol, 1% Triton X-100 and assayed directly for luciferase
activity (60) . -Galactosidase activity was determined in
the same lysis buffer(61) . Luciferase and -galactosidase
activity were determined for each sample. The ratio of luciferase
activity to -galactosidase activity served as a measure for
normalized luciferase activity. For each experiment the normalized
luciferase activity of the promotorless luciferase plasmid was
subtracted from this quotient. The result was multiplied by 1000. To
compare different transfections for each series of experiments, the
relative Luc units of the triplicate were divided by those of the
control construct pGL2-control. The quotient was multiplied by 100 and
expressed as percentage of pGL2-control. In addition relative
luciferase units were calculated as percentage of pHTF(-278)Luc
basal expression(47) . Each experiment was performed in
triplicate. The data presented are the mean of at least three
independent transfections performed. Standard deviations are given as
vertical error bars.
H
mice (18-20 g) at the day, Meth-A sarcoma (10 cells/animal) were implanted. A second DNA:DOTAP injection was
performed 2 days before application of TNF. As soon as the tumors
reached an average diameter of 0.5 cm (12-14 days after
planting), TNF (5 µg/animal) was injected intravenously. Mice
were sacrificed at the times indicated in the figure legend. Before
harvesting the tumor tissues, mice were perfused with 30-40 ml of
PBS by intracardiac injection of PBS into the left
ventricle(14) .
10
of colored microspheres (10 mM, E-Z-Trac
Ultraspheres(TM), E-Z-Trac Inc., Los Angeles, CA) were injected for
10-20 s into the left ventricle of the anesthetized mouse before
the mice was sacrificed. Tumors were harvested, weighted, and
hydrolyzed in sodium hydroxide-SDS solution (E-Z-Trac Inc., Los
Angeles, CA) at 80 °C overnight. Microbeads were isolated according
to the manufacturer's instructions, counted microscopically, and
expressed as microspheres per gram of tumor (±
S.D.)(14, 48) .
O(13, 14, 48) .
Negative controls included omission of first or second antibodies and
the substitution of the first antibody by nonspecific antibodies (data
not shown). For immunofluorescence staining anti-fibrin-fibrinogen
antibodies (fluorescine conjugate, Cappel, West Chester, PA) were used
in a 1:8 dilution(14) . Sections were incubated with the
antibody for 45 min at room temperature, followed by three washes with
PBS. 10 cuts from at least three different experiments were analyzed
independently.
Denhardt's, 50% formamide, 0.25% SDS,
100 mM DTT, 50 µg/ml salmon sperm DNA, 100 µg/ml tRNA,
and 5-10 ng of labeled RNA probe. At the end of hybridization,
samples were incubated for 30 min at 37 °C with RNase A (20
µg/ml) and washed once at 37 °C for 30 min with 2 SSC,
50% formamide and twice for 30 min at 50 °C with 0.2 SSC.
Detection was performed immunologically with the DIG nucleic acid
detection system according to the manufacturer's instruction. A
TF sense riboprobe served as negative control (data not shown).
B(p65)B(p65). Transient transfection with
c-Jun/Fos(AP-1), NF-B(p65), or both transcription factors together
resulted in enhanced expression of TF compared to mock transfected
cells (expressing the bacterial CAT). TF was studied at several levels. (i) Nuclear run-on assays (Fig. 1a) revealed that induction of transcription of the TF gene occurred.
(ii) The
biological activity was characterized by its factor VII-dependent
activation of factor X (synthetic substrate assay; Fig. 1b) and by a coagulant assay (one-stage clotting
time; Fig. 1c). Since almost no factor X activation was
observed in the absence of factor VII (data not shown), most of the
coagulant activity induced is TF. But it cannot be absolutely excluded
that other proteins involved in activation of coagulation are also
inducible by overexpression of AP-1 or NF-B(p65). When BAEC were
cotransfected with AP-1 and NF-B(p65), an additive effect in TF
induction was observed in all systems tested (Fig. 1).
Structural analysis of the human TF promotor has demonstrated two
linked AP-1 sites, a proximal canonical site at -210 (A2), a
distal non-canonical site at -223 (A1), and an NF-B site(N)
at position
-188(15, 16, 17, 18) . The
availability of promotor constructs (Table 1) allows for
definition of the areas involved in activation of TF by AP-1 or
NF-B(p65) (Fig. 2). The plasmids containing both AP-1 sites
responded to c-Jun/Fos overexpression in the presence or absence of the
NF-B site (Fig. 2a). However, deletion of the
NF-B binding site reduced the induction by c-Jun/Fos (Fig. 2a), suggesting that endogenously present
proteins (presumably p65/c-Rel) capable to bind to this site, act in
concert with AP-1. To test whether optimal induction by c-Jun/Fos was
dependent on the presence of endogenous NF-B, we cotransfected
BAEC with plasmids overexpressing I-B. Overexpression of I-B
reduced the c-Jun/Fos induction as long as the NF-B site was
present (Fig. 2c).
Figure 2:
Functional analysis of the TF promotor
demonstrates activation of TF expression by c-Jun/Fos(AP-1) and
NF-B(p65). BAEC cells were cotransfected with the TF promotor
plasmids [A1-A2-N], [A1-A2], or [N] (1
µg/ml medium) (see ``Materials and Methods'')
and c-Jun, c-Fos, NF-B(p65), mutated Jun, and/or I-B (0.5
µg each/ml medium) and cultivated for 42 h. After harvest
luciferase activity was determined in each sample and normalized for
transfection efficiency to the amount of -galactosidase expressed
by the plasmid pSV--gal (Promega). The normalized data are
expressed as relative Luc units and represent the mean of three
independent experiments ± S.D. performed in triplicate. a, transactivation of the TF promotor by Jun/Fos(AP-1);
Jun/Fos(AP-1) overexpression induced TF as long as AP-1 sites were
present in the TF promotor constructs. b, transactivation of
the TF promotor by NF-B(p65); overexpression of
NF-B(p65)-induced TF as long as the NF-B site was present in
the TF promotor constructs. c, the TF promotor plasmid
[A1-A2-N] was cotransfected with c-Jun, c-Fos, or
NF-B(p65) and the specific inhibitor of the opposite transcription
factor; the Jun/Fos(AP-1)-dependent transactivation could be partly
suppressed by the NF-B-specific inhibitor I-B, while the
AP-1-specific inhibitor mutated Jun partly suppressed transactivation
by NF-B(p65).
When BAEC were transfected with a
plasmid overexpressing NF-B(p65) (Fig. 2b),
induction was maximal, when both AP-1 sites and the NF-B site were
present. NF-B(p65) inducibility was reduced, but not lost, when
the AP-1 sites were deleted (Fig. 2b). Deletion of the
NF-B site resulted in loss of inducibility by NF-B(p65). To
test, whether optimal NF-B induction was equally dependent on the
presence of endogenous AP-1, we cotransfected BAEC with plasmids
overexpressing NF-B(p65) and the Jun-specific inhibitor mutated
Jun. This negatively dominant c-Jun point mutant (39) is able
to dimerize with other members of the AP-1/bZIP family; however, it is
unable to bind to the DNA recognition site. Since c-Fos does not bind
to DNA at all, due to its failure to homodimerize(31) ,
overexpression of mutated Jun leads to a significant reduction of
Jun/Fos heterodimer binding. When NF-B(p65) and mutated Jun were
cotransfected, a reduction of the NF-B(p65)-mediated stimulation
was seen (Fig. 2c). Thus AP-1 and NF-B(p65) act in
concert in inducing TF ( Fig. 1and Fig. 2), which seems
to be dependent on the presence of DNA sequences previously described
as binding sites for AP-1 and the NF-B subunits c-Rel and
p65(11, 15, 16, 17, 18, 62) .
Figure 3:
The proximal AP-1 site (A2) preferentially
binds Jun/Fos heterodimers (AP-1), while the distal AP-1 (A1) site is
only very weakly recognized and not induced by AP-1: a and b, radiolabeled oligonucleotide probes containing the
canonical proximal (a) or the non-canonical distal (b) AP-1 site were incubated with decreasing amounts of
Jun/Fos programmed rabbit reticulocyte lysate. The amount of programmed
lysate, added to the binding reaction, is given above the lanes. The
mobility of the formed complexes was analyzed on 4% nondenaturing
polyacrylamide gels. Arrows indicate the specific AP-1
binding. Nonspecific lysate reactions are marked by brackets. c, BAEC were cotransfected with the TF promotor plasmids
[A1], [A2], or [A1-A2] (1 µg) (Table 1) and a control plasmid (CAT = ``mock'')
or c-Jun and c-Fos (0.5 µg each) and cultivated for 42 h. After
harvest, luciferase activity was determined and normalized for
transfection efficiency to the amount of -galactosidase expressed
by the plasmid pSV--gal (Promega). The normalized data are
expressed as relative Luc units and represent the mean of three
independent experiments ± S.D. performed in triplicate. The
inducibility of the various TF promotor plasmids by c-Jun/Fos
heterodimers is shown. The level of basal expression (transfected with
CAT as control) is indicated with Basal.
Figure 4:
The distal AP-1 site (A1) preferentially
binds Jun homodimers, while the binding capacity for Jun homodimers is
lower at the proximal AP-1 site (A2). a and b,
radiolabeled oligonucleotide probes containing the proximal (a) or the distal (b) AP-1 site were incubated with
recombinant Jun, produced as inclusion body in E. coli. The
amount of recombinant Jun, included in the binding reaction, is shown
above the lanes (control lane 1 shows 2.5 µl
(approximately 1.25 µg) of c-Jun/Fos programmed lysate). The
mobility of the formed complexes was analyzed on 4% nondenaturing
polyacrylamide gels. Arrows indicate the specific AP-1
binding. Nonspecific reactions are marked by brackets. c, BAEC were cotransfected with the TF promotor plasmids
[A1], [A2], or [A1-A2] (1 µg) (Table 1) and a control plasmid (CAT = ``mock'')
or c-Jun (0.5 µg) and cultivated for 42 h. After harvest,
luciferase activity was determined and normalized for transfection
efficiency to the amount of -galactosidase expressed by the
plasmid pSV--gal (Promega). The normalized data are expressed as
relative Luc units and represent the mean of three independent
experiments ± S.D. performed in triplicates. The inducibility of
the various TF promotor plasmids by c-Jun homodimers is shown. The
level of basal expression (transfected with CAT as control) is
indicated with Basal.
-induced cells (Fig. 5a, lane 3 versus lane
4) and in cells cotransfected with Jun/Fos(AP-1) (Fig. 5a, lane 6 versus lane 7). Specificity
of binding was demonstrated, since binding of c-Jun/Fos programmed
lysate resulted in a similar shift (Fig. 5a, lane
8). In addition, cold consensus AP-1 oligonucleotides inhibited
the shift seen after TNF stimulation (Fig. 5a, lane 5).
Figure 5:
TNF induces binding of different
proteins to the proximal (A2) and the distal AP-1 site (A1) of the
human TF promotor: BAEC were transiently transfected with CAT (=
``mock''), mutated Jun, or c-Jun and c-Fos (AP-1)
overexpressing plasmids and cultivated for 42 h. Where indicated,
TNF (1 nM) was added 1 h before harvest. Nuclear extracts
(10 µg/binding reaction) were prepared as described under
``Materials and Methods'' and assayed in EMSA for binding to
the proximal (a) or the distal (b) AP-1 site. To
confirm AP-1 binding, TNF-induced nuclear extract was competed
with a 160-fold molar excess of cold consensus AP-1 (lane 5).
In addition, a parallel binding reaction was performed with 10 µl
of Jun/Fos programmed lysate (lane 8). a, the AP-1
complex binding to the canonical proximal AP-1 site is indicated with
an arrow. b, the complexes binding to the distal
noncanonical AP-1 site are termed I, II, and III and indicated by arrows (see
``Results'').
In contrast, in nuclear extracts of BAEC three complexes (marked I, II, and III) were observed at the non-canonical distal AP-1 (Fig. 5b). When exposition of the films was extended for up to 8 days, a weak band, marked as complex I, occurred (Fig. 5b). The weak binding and its migration in the gel confirmed the results shown in Fig. 3b, i.e. this band is due to weak binding of Jun/Fos heterodimers.
Complex II (Fig. 5b) does
not represent AP-1 heterodimers based on the following criteria. (i)
The bands in control extracts (Fig. 5b, lane
1) and extracts from cells overexpressing Jun/Fos (Fig. 5b, lane 6) were only weakly (Fig. 5b, lanes 2 and 7) inhibited in
the presence of mutated Jun. (ii) Complex II seen after TNF
induction (Fig. 5b, lane 3) was not competed
by a 160-fold molar excess of cold AP-1 consensus oligonucleotides (Fig. 5b, lane 5) or by mutated Jun (Fig. 5b, lane 4), suggesting differences
between control and TNF-stimulated cells. These data further
indicate that complex II does not contain c-Fos or Fos-related
proteins; however, they do not exclude the involvement of other members
of the bZIP family, i.e. members of the ATF family (see Fig. 6b and Table 2), which are able to bind DNA
and therefore are only minorly influenced by mutated Jun. (iii) Complex
II migrated more rapidly (Fig. 5b) than the complex
formed with c-Jun homodimers (Fig. 4b).
Figure 6:
Characterization of complexes I, II, and
III, formed at the distal AP-1 site (A1). a, initial
characterization of the nuclear complexes I, II, and III formed at the
distal AP-1 site (Table 2) derived from the human TF promotor
indicates that complex I and complex II contain members of the Jun and
ATF family; complex III did not react with the antibodies used and was
defined as nonspecific (see ``Results''). Characterization
was performed six times, using three different nuclear extract
preparations, with identical results. One typical experiment is shown.
10 µg of nuclear extract were included in each binding reaction: lane 1, 1 nM TNF (1 nM, 2 h); lane
2, 1 nM TNF + 2.5 µg of anti-pan-Jun
antibodies; lane 3, 1 nM TNF + 2.5 µg
of anti-c-Jun antibodies; lane 4, 1 nM TNF
+ 2.5 µg of anti-JunD antibodies; lane 5, 1 nM TNF + 2.5 µg of anti-c-Fos antibodies; lane
6, 1 nM TNF + 2.5 µg of anti-ATF-1
antibodies; lane 7, 1 nM TNF + 2.5 µg
anti-ATF2 antibodies; lane 8, 0.3 µg of recombinant c-Jun; lane 9, 10 µl of c-Jun/Fos programmed lysate. Antibodies
were added directly before addition of the P-labeled
distal AP-1 oligonucleotides. Complexes I and II and the nonspecific
complex III are indicated by arrows. b, binding of
recombinant Jun, recombinant ATF-2, recombinant Jun/ATF-2 heterodimers,
and Jun/Fos programmed lysate to the distal non-canonical AP-1 site
compared to binding of TNF-induced nuclear proteins (10
µg/reaction). The experiment was performed three times with
identical results. One typical experiment is shown: lanes 1 and 2 represent cellular extract from control (lane
1) or TNF (1 nM, 2 h) treated cells (lane
2). Lanes 3-6 represent EMSA of the distal AP-1
site (A1) incubated with various recombinant members of the AP-1/bZIP
family. Lane 1, control; lane 2, 1 nM TNF (1 nM, 2 h); lane 3, 0.5 µg of Jun
homodimers; lane 4, 0.5 µg of ATF-2 homodimers; lane
5, 0.25 µg of Jun and 0.25 µg of ATF-2, coincubated for 30
min at room temperature before addition to the binding reaction; lane 6, 8 µl of Jun/Fos programmed lysate; lane
7, 1 nM TNF + 500-fold molar excess of
unlabeled AP-1 consensus oligonucleotides. The nuclear extract-derived
complexes I, II, and III are indicated by arrows (left). The complexes formed by recombinant Jun
homodimers, ATF-2 homodimers, and Jun/ATF-2 heterodimers are marked
with filled triangles (right). c, time
course of the TNF-inducible complex II, binding to the distal AP-1
site (A1). Nuclear extracts were prepared from BAEC induced for various
times (0-6 h) with TNF (1 nM) (lanes
1-8). To demonstrate that the observed bands were not due to
the oligonucleotide preparation used, a reaction without nuclear
extract was included (lane 9). 10 µg of nuclear extract
were used in each binding reaction. DNA-protein complexes were analyzed
on native 4% polyacrylamide gels. The very weak binding of complex I,
the TNF-inducible complex II, and the nonspecific complex III are
indicated by arrows.
Since complex III (Fig. 5b) was not at all competed by unlabeled consensus AP-1 oligonucleotides (see below) and also present in unprogrammed rabbit reticulocyte lysate (Fig. 3b), it is regarded as nonspecific as depicted in Fig. 3b.
To analyze the complexes I and II that were observed at the distal non-canonical AP site (Fig. 5b), characterization with supershifting antibodies was performed (Fig. 6a). The upper gel shift band (complex I; Fig. 6a, lane 1) was reduced in the presence of pan-Jun antibodies (Fig. 6a, lane 2) and anti-JunD antibodies (Fig. 6a, lane 4) and suppressed in the presence of anti-c-Jun (Fig. 6a, lane 3) and anti-ATF-2 antibodies (Fig. 6a, lane 7). In addition, anti-c-Fos (Fig. 6a, lane 5) and anti-ATF-1 antibodies (Fig. 6a, lane 6) slightly decreased the shift. Complex II was suppressed when anti-ATF-2 antibodies were included in the reaction (Fig. 6a, lane 7) and reduced in the presence of anti-pan-Jun and anti-c-Jun antibodies (Fig. 6a, lanes 2 and 3), while anti-JunD, anti-c-Fos, and anti-ATF-1 antibodies did not affect binding (Fig. 6a, lanes 4-6). Thus complex I and II also consist of different members of the AP-1/bZIP family. Intensity of the lower band (complex III), previously characterized as nonspecific (Fig. 5b), was not affected by any of the antibodies.
To further confirm this
hypothesis, recombinant Jun and recombinant ATF-2 (0.5 µg each),
produced as inclusion bodies in Escherichia coli and able to
heterodimerize, were used in binding reactions and their migration was
compared to the complexes seen in extracts of control and TNF
stimulated cells (Fig. 6b). Consistent with the above
data Jun homodimers bound to the distal non-canonical AP site (Fig. 6, panel a, lane 8, and panel
b, lane 3) forming complexes that migrated in the gel at
the same position as complex I (Fig. 6, panel a, lane 1, and panel b, lane 2). The faster
migrating ATF-2 homodimers demonstrated stronger binding to the distal
non-canonical AP-1 site (Fig. 6b, lane 4) than
Jun homodimers (Fig. 6b, lane 3). When
equimolar amounts of recombinant ATF-2 and recombinant c-Jun were
coincubated in the binding reaction, a slightly faster migrating
complex was observed (Fig. 6b, lane 5). No
significant binding was observed, when programmed Jun/Fos lysate was
included in the binding reaction (Fig. 6b, lane
6). The observed binding, seen in lane 6, is also present
in unprogrammed lysate (Fig. 3b and Fig. 4b) and therefore regarded as nonspecific. To
further characterize the TNF-inducible complexes, a 500-fold molar
excess of unlabeled AP-1 oligonucleotides (instead of 160-fold; Fig. 5b, lane 5) was included in the binding
reaction with TNF stimulated nuclear extract (Fig. 6b, lane 7). This unusual high excess of
AP-1 competitor abolished binding of complexes I and II, but not of
complex III. Since unlabeled oligonucleotides did not compete binding
of complex III that was also detected in unprogrammed lysate (Fig. 3b and 4b), we defined complex III as
nonspecific. The data shown in Fig. 6(a and b) indicate that the distal non-canonical AP-1 site forms
complexes with members of the Jun and ATF family. However, more
detailed studies have to be performed to elucidate the nature and the
functional significance of the proteins involved in complex I and II.
Complex II was the major specific binding observed after TNF
stimulation. Therefore the time course of complex II induction by
TNF was studied (Fig. 6c). TNF-mediated
binding of complex II to the distal AP-1 site was biphasic, with a fast
initial response at approximately 5 min and a slower response, maximal
between 2 and 6 h (Fig. 6c). No signal was observed in
the absence of nuclear extracts (Fig. 6c, lane
9).
At the proximal canonical AP-1 site TNF induced
time-dependent (Fig. 7a) and dose-dependent (Fig. 7b) induction of proteins, which reached a
maximum between 30 min and 2 h. These proteins were characterized as
AP-1 by (i) competing the binding with an excess of cold AP-1 consensus
oligonucleotides (Fig. 7, panel a, lane 8 and panel b, lane 7) and (ii) by reducing binding
activity by overexpression of mutated Jun (Fig. 5a, lane 4). For the characterization using polyclonal antibodies,
extract of TNF-stimulated cells (Fig. 7c, lane
1) was incubated with the antibodies (Fig. 7c, lanes 2-7). Migration was compared to the shift observed
with recombinant Jun homodimers (Fig. 7c, lane
8) and Jun/Fos programmed lysate (Fig. 7c, lane 9). Pretreatment with anti-pan-Jun (Fig. 7c, lane 2) or anti-JunD antibodies (Fig. 7c, lane 4) resulted in supershifted
bands; pretreatment with anti-c-Fos antibodies abolished the observed
binding (Fig. 7c, lane 5). No reaction was
observed with antibodies directed against members of the ATF family (Fig. 7c, lanes 6 and 7). Consistent
with the above results (Fig. 4a), no binding was
observed with 0.3 µg of Jun homodimers (Fig. 7c, lane 8). Therefore, the TNF-induced binding to the
proximal canonical AP-1 site comprises JunD/Fos heterodimers. Although
c-Jun-specific antibodies did not result in supershifted or reduced
binding, c-Jun might contribute to the observed complexes, since it
might be possible that the anti-c-Jun antibody fails to recognize c-Jun
in Jun/Fos heterodimers.
Figure 7:
TNF induces time- and dose-dependent
binding of AP-1 to the proximal AP-1 site (A2) of the human TF
promotor. a, time course of AP-1 binding to the proximal AP-1
site (A2) of the human TF promotor. Nuclear extracts were prepared from
BAEC induced for various times (0-3 h) with TNF (1
nM) (lanes 1-7). 10 µg of nuclear extract
were included in each binding reaction. DNA-protein complexes were
analyzed on 4% native polyacrylamide gels. EMSA detected AP-1 binding
to the proximal AP-1 site (Table 2) derived from the human TF
promotor. The TNF-inducible AP-1 complex is indicated with an arrow. Specificity of binding was ascertained by competing
with 160-fold molar excess of cold AP-1 consensus oligonucleotides (Table 2) included in the binding reaction (lane 8). b, dose response of AP-1 binding to the proximal AP-1 site
(A2). BAEC were stimulated with various doses of TNF (0 pM to 1000 pM) for 30 min (lanes 1-6).
Nuclear extracts were prepared, and 10 µg of this extract were
included in each binding reaction and analyzed as above. The
TNF-inducible AP-1 complex (JunD/Fos) is indicated with an arrow. Specificity of binding was ascertained by competing
with 160-fold molar excess of cold AP-1 consensus oligonucleotides (Table 2) included in the binding reaction (lane 7). c, characterization of the complex bound to the proximal AP-1
site (A2) after TNF induction. Characterization was performed two
times, using two different nuclear extract preparations, with identical
results. One typical experiment is shown. 10 µg of nuclear extract
were included in each binding reaction: lane 1, 1 nM TNF (1 nM, 1 h); lane 2, 1 nM TNF + 2.5 µg of anti-pan-Jun antibodies; lane
3, 1 nM TNF + 2.5 µg of anti-c-Jun
antibodies; lane 4, 1 nM TNF + 2.5 µg
of anti-JunD antibodies; lane 5, 1 nM TNF +
2.5 µg of anti-c-Fos antibodies; lane 6, 1 nM TNF + 2.5 µg of anti-ATF-1 antibodies; lane
7, 1 nM TNF + 2.5 µg of anti-ATF2
antibodies; lane 8, 0.3 µg of recombinant c-Jun; lane
9, 10 µl of c-Jun/Fos programmed lysate. Antibodies were added
directly before addition of the P-labeled proximal AP-1
oligonucleotide. The TNF-inducible complex is indicated with an arrow.
-induced Binding of p65/c-Rel to the TF-derived
NF-B Site resulted in time- and
dose-dependent binding of two distinct protein DNA-complexes to the TF
derived NF-B site, which were identified as NF-B (data not
shown). Consistent with previous studies, binding of the slower
migrating complex was characterized as NF-B(p65/c-Rel)
complex(11, 18, 62, 63) , while in
the faster migrating band only NF-B(p65) seemed to participate in
the binding complex (11) (data not shown). Binding of
NF-B(p65/c-Rel) was already detected after 5 min, reached its
maximum after 10 min, and decreased to basal levels after 1 h (data not
shown). The p65-containing faster migrating complex was induced between
5 min and 3 h (data not shown). Taken together, these data indicate
that activation of the human TF promotor follows a kinetic that
includes first activation of NF-B(p65/c-Rel), followed by
activation of JunD/Fos heterodimers that bind to the proximal AP-1
site. While NF-B(p65/c-Rel) activation declines, the distal AP-1
site exhibits maximal binding of AP-1/bZIP proteins, characterized to
contain Jun and ATF proteins. This might explain, why TF transcription
is still enhanced after 4-6 h ( (9) and (10) and
data not shown), even TNF-induced NF-B(p65/c-Rel) activation
has already dropped to base-line level.
Mediates Induction of Endothelial TF by a
Concerted Action of the AP-1 and NF-B SitesB family functionally
act on the human TF promotor and contribute to TF induction by
TNF, transient transfections of BAEC with TF promotor mutants (Fig. 8) were performed. When BAEC were stimulated with
TNF, promotor activity compared to basal expression was highest,
when the proximal AP-1 (A2) and the NF-B site were present (Fig. 8, a and b). Loss of the proximal AP-1
site or the NF-B site resulted in significantly decreased TF
induction (Fig. 8, a and b). Loss of the
non-canonical distal AP-1 site had a less prominent effect, as expected (Fig. 8, a and b). Promotor mutants with only
the proximal AP-1 site (A2) cloned in front of the minimal promotor
were still inducible by TNF, while the distal AP-1 site (A1) alone
was unable to confer TF induction (Fig. 8, a and b). The proximal AP-1 significantly contributed to TF basal
expression (Fig. 8a). Therefore mutants that contained
the NF-B, but not the proximal AP-1 site, were more inducible by
TNF than mutants comprising the proximal AP-1 site (Fig. 8b); however, the overall TF expression of
mutants without proximal AP-1 was lower (Fig. 8a).
Highest TF expression was observed only when both AP-1 sites and the
NF-B site were intact, consistent with participation of all three
sites in regulation of TF transcription (Fig. 8a). The
TNF-induced activity of the construct A1-A2-N, spanning the
complete promotor, is higher (396 Luc units) than the added activity of
the constructs containing each element alone (A1 = 50, A2
= 125, N = 106 Luc units; total 281 Luc units). Thus,
these elements act in concert on the human TF promotor in mediating
TNF-induced TF transcription.
Figure 8:
TNF induces tissue factor expression
by a concerted action of AP-1/bZIP- and NF-B-like proteins.
Functional analysis of TF expression in unstimulated and
TNF-induced BAEC. BAEC were transfected with various TF promotor
plasmids (Table 1; for detail see ``Materials and
Methods'') for 36 h before TNF (1 nM) was added for
6 h, where indicated. After harvest luciferase activity was determined
in the cell lysates and normalized for transfection efficiency to the
amount of -galactosidase activity expressed by the control plasmid
pSV--Gal (Promega). Corrected values were expressed as relative
luciferase units. The results represent the mean of at least three
independent experiments ± S.D. that were performed in
triplicate. a, functional analysis of TF expression in
unstimulated BAEC compared with TF expression in TNF stimulated
BAEC; the mean of six independent experiments ± S.D. performed
in triplicate is shown. b, the inducibility by TNF
relating to basal expression is shown. The level of basal expression is
indicated with B. c, to directly demonstrate the role
of NF-B(p65) in TNF-mediated TF induction, various TF
promotor plasmids (Table 1) were cotransfected with plasmids
overexpressing CAT (= mock) or the NF-B(p65) specific
inhibitor I-B and cultivated for 36 h, before TNF (1
nM) was added to the cells for 6 h. After harvest luciferase
activity and transfection efficiency were determined as above. The data
represent the mean of three different experiments performed in
triplicate. d, to directly demonstrate the role of JunD/Fos
(AP-1) in TNF-mediated TF induction, various TF promotor plasmids (Table 1) were cotransfected with plasmids overexpressing CAT
(= mock) or mutated Jun and cultivated for 36 h before TNF
(1 nM) was added to the cells for 6 h. Data were obtained from
three different experiments ± S.D. performed in
triplicate.
The concept that both AP-1 sites
and the NF-B site act in concert was further supported by studies
overexpressing specific inhibitors of NF-B(p65)(I-B) or
AP-1/bZIP (mutated Jun). Overexpression of I-B reduced
TNF-mediated TF induction as long as the NF-B site was
present (Fig. 8c). Overexpression of mutated Jun
reduced TF induction by TNF as long as the proximal AP-1 site was
present (Fig. 8d).
-mediated TF Induction Is Dependent on AP-1/bZIP
and NF-B Proteins in VivoB-mediated TF induction in
vivo. Since in the human and the mouse TF promotor the same
sequences are found for the distal and the proximal AP-1, as well as
for the NF-B recognition motif, we used a mouse model to study the
dependence of TNF-mediated TF expression of endothelial cells on
Jun and NF-B in vivo. In this model, TNF induces the
expression of TF and fibrin formation on endothelial cells of the tumor
vasculature(14, 64) . 3 h after intravenous injection
of TNF TF expression was induced, based on in situ hybridization (Fig. 9a) and immunohistochemistry (Fig. 9b). TF was expressed by subendothelial
structures including tumor cells and also by endothelial cells. With
respect to the topic of this study, we focused on endothelial cells.
More than 150 vessels were evaluated in this part of the study.
Figure 9:
AP-1/bZIP proteins and NF-B control
TF expression in vivo. Meth-A sarcoma (10 cells/animal) were implanted into CH mice. After the
tumors reached an average size of 0.5 cm, intravenous somatic gene
transfer was performed with plasmids overexpressing a vector control,
mutated Jun, or I-B. 12 days after planting the tumors, mice
received PBS (control) or 5 µg of TNF/animal for 3 h. Mice
were sacrificed and perfused with 30-40 ml of PBS by intracardiac
injection of PBS into the left ventricle. Tumors were harvested and TF
transcription (a, in situ hybridization), TF antigen (b, immunohistochemistry), and fibrin/fibrinogen deposition (c; immunofluorescence) was evidenced in the tissue. a, in situ hybridization with a mou
se TF-specific
riboprobe (see ``Materials and Methods'') in control (top) and TNF (bottom) treated animals,
transfected with vector control (left), mutated Jun (middle), or I-B (right). Magnification,
160. b, immunohistochemistry using an anti-mouse TF antibody
(see ``Materials and Methods'') in control (top) and
TNF (bottom) treated animals, transfected with vector
control (left), mutated Jun (middle) or I-B (right). Magnification, 160. c,
immunofluorescence of fibrin/fibrinogen deposition in control (top) and TNF (bottom) treated animals,
transfected with vector control (left), mutated Jun (middle), or I-B (right). Magnification,
40.
When
animals were treated by intravenous somatic gene transfer with mutated
Jun 24 h prior to TNF injection, a decrease in the endothelial
response to TNF was observed by in situ hybridization (Fig. 9a) and immunohistology (Fig. 9b)
compared to vector-transfected animals. Thus, by blocking the
interaction of Jun with other members of the bZIP family by somatic
gene transfer with a plasmid overexpressing mutated Jun, endothelial TF
induction could be partially reduced (Fig. 9, a and b). Similar data were obtained when a plasmid overexpressing
I-B was used (Fig. 9, a and b). Mutated
Jun and I-B both reduced the inducibility of TF in endothelial
cells in this tumor model in vivo. The tumor model was further
used to examine the functional effect of TF; when the fibrin/fibrinogen
deposition in response to TNF was studied in animals perfused with
30-40 ml of PBS (see ``Materials and Methods''), a
reduction by mutated Jun and I-B was demonstrated in some, but not
all vessels (Fig. 9c). Thus TF expression in vivo is under the control of AP-1/bZIP and NF-B-like proteins.
Successful transfection with mutated Jun or I-B was moni
