(Received for publication, March 7, 1995; and in revised form, July 31, 1995)
From the
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.
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) .
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)
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.
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.
(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.
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.
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).
Figure 9:
AP-1/bZIP proteins and NF-B control
TF expression in vivo. Meth-A sarcoma (10
cells/animal) were implanted into C
H 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 monitored
in EMSA of tumor tissue (Fig. 10). Tumors derived from animals
transfected with vector DNA prior to TNF
had a stronger AP-1
binding activity than tumors derived from animals transfected with
mutated Jun (Fig. 10, top left). Consistently, tumors
from I-
B transfected animals demonstrated reduced NF-
B
binding activity at the TF derived NF-
B site (Fig. 10, top right) compared to vector controls. In addition, Northern
blot of mRNA, derived from whole tumors, showed decreased TF mRNA
levels, when the animals had been transfected with mutated Jun or
I-
B prior to TNF
application (Fig. 10, bottom). However, the suppression obtained was only partial,
since (i) members of the Jun and ATF family are less responsive to
inhibition by overexpression of mutated Jun than c-Fos, (ii) the in
vivo involvement of other transcription factors can not be
excluded, and (iii) transfection did not reach all cells.
Figure 10:
Efficiency of intravenous somatic gene
transfer. Transfection efficiency was monitored in EMSA (top)
and Northern blot (bottom). Top I, EMSA of tumor
nuclear extracts, derived from mice transfected with vector control (left) or mutated Jun (middle) before application of
TNF EMSA were performed with the proximal AP-1 site (A2) of the
human TF promotor. AP-1 binding was confirmed by suppressing the
observed shift in TNF
-induced vector controls by a 160-fold molar
excess of unlabeled AP-1 consensus competitor (right). Top
II, EMSA of tumor nuclear extracts, derived from mice transfected
with vector control (left) or I-
B (middle)
before application of TNF
. EMSA were performed with the TF-derived
NF-
B site. NF-
B binding was confirmed by suppressing the
observed shift in TNF
-stimulated vector controls by an 160-fold
molar excess of unlabeled NF-
B consensus competitor (right). Bottom I, Northern blot: total mRNA of
tumors from TNF
treated animals, transfected with vector control (left, 1) or mutated Jun (right, 3)
was hybridized against tissue factor (TF; top) or GAPDH (bottom) specific DNA probes. Bottom II, Northern
blot: total mRNA of tumors from TNF
-treated animals, transfected
with vector control (left, 1) or I-
B (right, 3) was hybridized against tissue factor (TF; top) or GAPDH (bottom) specific DNA
probes.
To give a
picture of the overall efficiency of transfection, microbeads were used
for measuring blood flow of the whole organ, avoiding potential
artifacts due to selection of a single area in histological studies.
When microbeads were injected into animals, a high number of beads was
present in tumors of animals not treated with TNF (Fig. 11). The number of beads reflecting tumor perfusion was
clearly decreased after TNF
injection with previous somatic gene
transfer with vector DNA (Fig. 11). This indicated that TNF
treatment resulted in loss of free blood flow, potentially due to
TF-mediated microvascular thrombosis. Therefore this method adds to the
histological study by providing data about the effect of I-
B and
mutated Jun on the whole organ. Gene transfer with I-
B or mutated
Jun partially reversed this effect of TNF
(Fig. 11). Hence
blocking TF on the transcriptional level reduced not only TF induction
by TNF
, but also reduced the fibrin/fibrinogen deposition and
restored free blood flow.
Figure 11:
Somatic gene transfer with mutated Jun or
I-B restores the free blood flow in tumors treated with TNF
.
10
Meth-A sarcoma cells were planted intracutaneously into
mice. Somatic gene transfer and TNF
application was performed as
described in Fig. 9. 3 h after intravenous injection of 5 µg
of TNF
, mice were anesthetized, microspheres were injected into
the left ventricle for 10-20 s (see ``Materials and
Methods''), and mice were sacrificed thereafter. Tumor tissue was
harvested and microbeads counted microscopically (see ``Materials
and Methods''). The free blood flow is shown, evidenced by the
number of latex particles per gram of tumor tissue. A, tumors
before TNF
application; B, tumors after TNF
,
pretreated with mutated Jun; C, tumors after TNF
,
pretreated with I-
B; D, tumors after TNF
, pretreated
with mutated Jun and I-
B; E, tumors after
TNF
.
Tissue factor (TF) is a potent initiator of the coagulation
cascade (1, 2, 4, 65, 66) and
normally is not expressed by quiescent endothelial
cells(1, 3, 6, 11, 12) . In vitro data showed induction of TF synthesis in endothelial
cells by inflammatory mediators such as endotoxin, phorbol esters,
oxygen-free radicals, or
cytokines(7, 8, 9, 10, 11, 67, 68, 69, 70) .
Recently members of the NF-B and the AP-1/bZIP family have been
reported to be involved in the lipopolysaccharide- and
cytokine-mediated TF induction in monocytes (15, 16, 17, 18, 62) and
porcine endothelial cells(11) . It has been more difficult to
show endothelial TF in vivo(3, 67) ; however,
recent studies demonstrate that in selected areas of the vascular bed
activators of the host response or TNF
lead to the synthesis and
expression of TF(12, 13, 14, 71) .
This study addresses the molecular mechanisms that underlie the
regulation of the human TF promotor in response to the proinflammatory
cytokine TNF
.
We used bovine aortic endothelial cells (BAEC),
which exhibit lower basal AP-1 and NF-B activity than porcine
(PAEC) or human (HUVEC) endothelial cells, and the human TF promotor. A
striking difference between the human and the porcine TF promotor is
seen at the proximal AP-1 site, which resembles a canonical high
affinity site in the human TF promotor(15, 16) , while
it is a low affinity non-canonical site in the porcine promotor, due to
a G
A switch at position 4 of the AP-1 heptamer(11) .
Thus, the proximal AP-1 site of the human promotor is more prominently
involved in the TNF
-mediated up-regulation of TF than it is in the
porcine promotor and, as a consequence, in addition to NF-
B
activation, AP-1 activation may be relevant for human disease.
In
transient transfection studies, the highest TNF inducibility was
only observed when the NF-
B(p65/c-Rel) site was present in the TF
promotor plasmids. Enhanced NF-
B(p65/c-Rel) binding to its
TF-derived motif was detectable within 5 min after TNF
stimulation
and rapidly down-regulated after 1 h (data not shown). This fast
activation of NF-
B(p65/c-Rel) reflects that TF mRNA can be rapidly
induced in the absence of protein synthesis; therefore, TF has been
classified as an immediate early gene(72) . The dependence of
TF induction by inflammatory mediators on NF-
B(p65/c-Rel)
activation may insure that this induction is transient, since it has
been recently reported that increased NF-
B levels lead to
increased expression of the inhibitor
I-
B(73, 74) , followed by NF-
B inactivation.
This might prompt the activated endothelial cells to return to a
quiescent state.
Therefore, the existence of increased TF mRNA
levels in HUVEC and BAEC 4-6 h after TNF stimulation (9, 10) cannot be explained solely on the basis of
NF-
B activation and demands the involvement of other inducible
transcription factors. Functional studies demonstrated that optimal TF
induction by TNF
was also mediated by both AP-1 sites in the human
TF promotor (Fig. 8). EMSA revealed that the TNF
-inducible
complex bound to the canonical proximal AP-1 site of the human TF
promotor consisted mainly of JunD/Fos (Fig. 7c);
however, it cannot be excluded that other members of the Jun family are
also involved. Highly vascularized organs (spleen, lung, intestine,
ovary, and brain) express high levels of JunD(30) . While the
expression of c-Jun and Jun B is rapidly up-regulated by various
stimuli, JunD is only modestly induced by growth factors and phorbol
esters(26, 30, 75, 76) .
Transactivation by JunD homodimers is significant lower than by c-Jun
homodimers(30) . In cooperation with c-Fos, however, JunD has
transactivation capacities similar to those of
c-Jun(29, 30) . The results displayed here demonstrate
that in cultured endothelial cells TNF
induces JunD/Fos
heterodimers that recognize the proximal AP-1 site of the human TF
promotor and thereby enhance TF transcription. In this respect the
human and the porcine system differ significantly. Binding of
JunD/Fos-containing complexes to the proximal AP-1 site is already
detected 30 min after TNF
stimulation. This rapid response
excludes newly synthesized JunD or Fos and indicates the rapid
activation of preexisting proteins. This availability of JunD/Fos
heterodimers therefore is a limiting factor. Consistently, the
canonical proximal AP-1 alone was not able to confer high
TNF
-mediated induction in transient transfection experiments and
required the presence of the NF-
B(p65/c-Rel)
site(11, 15, 16, 18) for optimal TF
expression. These data imply that the disposal of JunD/Fos heterodimers
is not sufficient for maximal induction by TNF
and need to recruit
NF-
B (p65/c-Rel) nuclear binding activity. Since
NF-
B(p65/c-Rel) translocation into the nucleus precedes JunD/Fos
activation only by 20 min, one might speculate that binding of one
transcription factor facilitates binding of the other.
The proximal
high affinity AP-1 site of the human TF promotor, which is missing in
the porcine TF promotor, is of particular importance with respect to
therapeutic interventions. A great variety of antioxidative agents has
been reported to suppress activation of NF-B in vitro and in vivo(77) and therefore might potentially be used
for reducing TF activity under certain pathophysiological conditions.
However, recent studies elucidated that changes in the cellular redox
system by radical scavengers suppress very fast NF-
B, but at the
same time induce time-dependent AP-1 activation
(Jun/Fos)(20, 21) . Antioxidative conditions strongly
induce c-Fos, which can form reactive heterodimers with preexisting Jun
homodimers (20, 21) . As pointed out before, tissues
with high endothelial portions contain constitutively high amounts of
JunD homodimers (30) and are therefore primed to generate large
amounts of JunD/Fos heterodimers under antioxidative therapy.
To
define the role of the non-canonical distal AP-1 site in human TF
regulation is more difficult. This site is a low affinity site for AP-1
binding and resembles the two non-canonical AP-1 sites of the porcine
TF promotor(11) . In accordance with these data, several
independent approaches demonstrated that Jun homodimers, but not
Jun/Fos heterodimers, bind to this site (Fig. 3, Fig. 4,
and Fig. 6). Differences in the structure of the DNA binding
domains for Jun homodimers and Jun/Fos heterodimers have been
described(78) ; therefore, it seems likely that a G A
switch at position 4 of this site facilitates Jun binding and excludes
significant Jun/Fos binding. Furthermore, specific properties of the
regions outside the defined AP-1 binding sites might be responsible for
preference in binding of the various homo- and heterodimer
complexes(79) . EMSA demonstrated (Fig. 6, a-c) that TNF
also induced protein complexes that
were different from Jun homodimers. These complexes have been
characterized to contain Jun and ATF family proteins (Fig. 6, a-c). In contrast, Moll et al.(11) recently reported constitutive binding of c-Jun,
JunD, and possibly Fra2 complexes to the porcine TF promotor-derived
non-canonical AP-1 sites. Since basal AP-1 binding activity is low in
BAEC compared to PAEC), this might explain why the study presented here
detected TNF
-inducible binding at the non-canonical distal AP-1
site of the human TF promotor. Consistent with our observations,
Donovan-Peluso and co-workers mentioned that in THP-1 cells large
differences between the distal and the proximal AP-1 site were detected
in EMSA, which indicate the involvement of different
heterodimers(17) . This finding differs from previous
observations in HUVEC, where Jun homodimer and Jun/Fos heterodimer
binding occurs at the distal and the proximal AP-1
site(47, 63) . This might be due to (i) a greater
availability of Jun homodimers, (ii) to a different composition of the
complexes induced, or (iii) to species differences in HUVEC versus BAEC. The low affinity distal AP-1 site of the human TF promotor
only marginally participates in TNF
-induced TF expression,
consistent with the data described for the two low affinity AP-1 sites
in the porcine TF promotor(11) . However, the non-canonical
AP-1 site significantly supports NF-
B-mediated TF induction, even
when the proximal AP-1 is deleted (Fig. 8). Since recently a
cooperative action of ATF proteins and NF-
B family members has
been demonstrated(80) , one might speculate that proteins bound
to the distal AP-1 site support and facilitate NF-
B activity.
Therefore a set of different transcription factors has to be activated
at the same time before endothelial TF is successfully induced.
The in vivo data presented (Fig. 9-11) support this
concept. Intravenous somatic gene transfer with plasmids overexpressing
I-B or mutated Jun reduce TF induction in vascular endothelial
cells of the tumor. They also decrease deposition of fibrin/fibrinogen.
The antibody used does not discriminate between fibrin and fibrinogen.
Therefore, the animals were perfused with 30-40 ml of PBS (see
``Materials and Methods'') prior to harvest of the organs to
remove non-clotted material. The reactive material represents at least
in part fibrin, since we observed striking differences in
fibrin/fibrinogen deposition between the different animal groups
corresponding to the perfusion studies with microbeads (the later ones
giving a better view of the overall efficiency of I-
B and mutated
Jun). However, in these experiments cells other than endothelial cells
may be affected. Nevertheless, the in situ hybridization and
immunohistochemical studies (Fig. 9) showed that endothelial
cell expression of TF is under control of NF-
B and AP-1 in the
animal model used. The incomplete suppression of TF and
fibrin/fibrinogen deposition can be explained (i) by the expected low
to moderate transfection efficiency, (ii) by local differences in
endothelial cells (capillaries still growing versus already
grown vessels, dividing vessels versus non-dividing
endothelial cells), (iii) by the involvement of other transcription
factors than AP-1 and NF-
B, and (iv) other EC genes influenced by
cytokines. Hence the TNF
-mediated activation of endothelial TF
transcription occurs in vitro and in vivo by members
of the NF-
B and AP-1/bZIP family.