(Received for publication, May 12, 1994; and in revised form, November 14, 1994)
From the
Tissue factor is up-regulated on endothelial cells and monocytes
in response to cytokines and endotoxin and is the main trigger of the
extrinsic pathway of the coagulation cascade. We have isolated the
porcine tissue factor gene and studied the regulation of the promoter,
which has not been investigated previously in endothelial cells.
Comparison of the promoter sequences with the respective human and
murine genes reveals short stretches of homology, which encompass
potential binding sites for AP-1, NFB, and Sp1 transcription
factors. Using DNase I footprinting, we detect binding of nuclear
factors to these promoter elements. Transfection experiments
demonstrate that a 300-base pair fragment containing the conserved
elements can mediate induced transcription and that the NF
B-like
element is essential. In accordance, electrophoretic mobility shift
assays show a strong increase in the binding of factors to the
NF
B-like site following induction. We further provide evidence
that RelA (p65), c-Rel, and possibly novel polypeptides bind to the
tissue factor NF
B element. In addition, we show constitutive
binding of members of the Fos/Jun and Sp1 families to the AP-1 and Sp1
sites, respectively. We propose a concerted action of AP-1-, NF
B-,
and Sp1-like factors in transcription from the tissue factor promoter
in endothelial cells.
The transmembrane glycoprotein tissue factor (TF) ()serves as the high affinity receptor and essential
cofactor for the plasma serine protease VII/VIIa and plays a central
role in the activation of the extrinsic pathway of the coagulation
cascade (for review, see (1) and (2) ). The relatively
high constitutive levels of TF in certain extravascular cells are
thought to represent a hemostatic barrier preventing excessive bleeding
following tissue injury. Endothelial cells (EC) normally function to
maintain an anti-coagulant environment and inhibit the formation of
thrombi(3, 4, 5) . Upon activation, EC change
the balance of anti- and procoagulation toward blood clotting through
the up-regulation of TF (1, 6, 7, 8) . In many situations of
inflammation, septic shock, various thromboembolic disorders, and
several forms of disseminated intravascular coagulation, aberrant TF
expression can result in life-threatening
diseases(2, 9) . In the special situation of vascular
rejection of xenotransplants, EC activation and consequent TF
expression has also been implicated as one of the key mechanisms
leading to blood clotting and organ death(10, 11) .
It was previously shown that TF is up-regulated in response to
endotoxin and inflammatory cytokines in EC and macrophages. Control of
expression is largely at the transcriptional and mRNA level. Exposure
of EC or monocytes to LPS or TNF- transiently induces TF gene
transcription(2, 12, 13, 14) ; in
addition, TF mRNA may be stabilized in response to
endotoxin(15, 16) . The determinants in the
5`-flanking region of the human gene that contribute to LPS-mediated
activation in a monocytic leukemia cell line have previously been
analyzed. A 56-bp region between -227 and -172 of the
promoter, which contains AP-1 and NF
B sites, is necessary for
transcriptional up-regulation(17, 18) . Recently, Oeth et al.(19) have described the binding of c-Rel/p65
heterodimers to the TF NF
B site and provided evidence that this
site confers LPS inducibility to a heterologous promoter in reporter
gene assays using monocytic cells. A central role for NF
B in the
up-regulation of genes in endothelial cells during inflammation has
been demonstrated by several
laboratories(20, 21, 22, 23) ,
including ours (24, 25) . In view of this, we have now
investigated a potential involvement of the TF promoter region
containing the NF
B and the two neighboring AP-1 sites in the
regulation of endothelial TF expression.
We report here for the
first time the importance of this region also for EC-specific TF
up-regulation. Using DNase I footprinting, we detect binding of nuclear
factors to the AP-1 and NFB as well as the Sp1-like promoter
elements. Reporter gene assays were used to show the functional
importance of this region and to demonstrate that the NF
B-like
site is essential for TF gene transcription in EC. By electrophoretic
mobility shift assays with extracts from unstimulated and induced EC,
we demonstrate inducible NF
B and constitutive AP-1 and Sp1
binding. In a biochemical analysis of the factors binding to the TF
promoter, we show binding of Rel-, Fos/Jun-, and Sp1-family members to
the respective sites.
Taq cycle sequencing was performed on an Applied Biosystem 373A DNA Sequencer according to the manuals provided by the manufacturer. The complete sequence of the promoter up to -1100 bp and of the transcribed region down to the beginning of the second intron was established on both strands. Sequence alignments were performed with the Pileup program of the Genetics Computer Group Sequence Analysis Software Package at default values(30) .
The
double-stranded synthetic oligonucleotides were radioactively labeled
by filling in the overhangs with Klenow enzyme in the presence of
[-
P]dATP and subsequently purified over a
7% polyacrylamide gel. The sequences of the probes used were as
follows: TF
B, 5`-AATTCTTGGAGTTTCCTACGGG-3`; TFm
B,
5`-AATTCTTGCAGTTTAGTACGGG-3`; hIg
B,
5`-AATTCAGAGGGGGATTTCCCAGAGG-3`; ECI6/BS2,
5`-AATTCGGCTTGGAAATTCCCCGAGCG-3`; Coll-AP-1,
5`-GCATAAAGCATGAGTCAGACACCTC-3`; TFAP-1,
5`-AATTGGGTTGAATCACGGGTGAATCAGCCCTTGCAGG-3`; mut1,
5`-AATTGGGTTCTAGAACGGGTGAATCAGCCCTTG-3`; mut2,
5`-AATTGGGTTGAATCACGGGTCTAGAAGCCCTTG-3`; mut1/2,
5`-AATTGGGTTCTAGAACGGGTCTAGAAGCCCTTGC-3`; TFSp1,
5`-AATTCGGGGGCGGGACCAGGGCGGGGCCTCG-3`; cons. Sp1,
5`-AATTCGGGGCGGGGCGATCGGGGCGGGGCG-3`; nonspecific oligo, 5`-
AATTCCGAATTCTTTG-3`.
Figure 1:
Induction of cellular procoagulant
activity. A, PAEC incubated with LPS for 1-10 h were
analyzed in an amidolytic thrombin assay. Cells were treated with 10
ng/ml (), 100 ng/ml (
), or 1000 ng/ml (
) of LPS.
Background values obtained with untreated samples were subtracted. B, cells were treated with increasing amounts of LPS,
IL-1
, or TNF-
for 6 h, and then the assay was performed. The
average value obtained with untreated samples is shown with an openbar (U). LPS was used in concentrations of 1,
10, 100, and 1000 ng/ml, IL-1
in 10, 100, 1000, and 10,000
units/ml; TNF-
was used in concentrations of 1, 10, 100, and 1000
units/ml, and the results are shown for increasing concentrations from left to right. The mean of four independent samples
and the standard deviations are given.
Figure 2:
Sequence of the porcine TF promoter and
alignment with the respective human and murine promoter sequences. The
sequence of the the porcine TF promoter (Por) from -366
to +122 including the 5`-untranslated region of the gene and a
sequence alignment with the corresponding regions of the human (Hum) and murine (Mur) TF gene is shown. Identical
nucleotides in the sequences of all three species are shown in capital
letters and as a consensus sequence (Con). Residues not
conserved in all three species(-) and gaps () in the
individual sequences introduced for optimal alignment to the sequences
of the other species are indicated. The two AP-1-like sites, the
NF
B-like site, 4 Sp1 elements, the EGR-1 site, and the TATA box
are underlined; the presumed transcription initiation site and
the translation initiation codon are shown in boldface. The
bases are numbered according to their relative position to the presumed
transcription initiation site on the porcine TF
promoter.
Figure 3: DNaseI footprint analysis of the porcine TF promoter. The XhoI-DraI fragment (position -329 to -27) from the TF promoter was end-labeled on the coding strand and incubated with nuclear extracts from either unstimulated (U) or LPS-treated (I) cells. For unstimulated cells, footprints obtained from two independent preparations of nuclear extracts are shown. Protected regions are outlined by bars, and the position on the TF promoter relative to the transcriptional start site is given on the left. G, Maxam/Gilbert G reaction sequencing ladder; -, no extract added.
Footprint 1 maps to a region of the
promoter that contains a potential Sp1 binding site. Regions 2 and 3
contain the distal and proximal AP-1 sites, implying that the TF AP-1
sites are bound by nuclear factors in endothelial cells. A similar
situation is indicated for the TF NFB element (region 4), which
also shows DNase I protection. The four potential Sp1 sites downstream
of the TF
B element are preferentially occupied in nuclear extracts
from induced cells (footprints 5-7). These results clearly
demonstrate that all of the conserved promoter elements located in the
footprint region are occupied at a time point when the TF gene is
actively transcribed, suggesting that these sites contribute in one or
the other way to transcription.
Initial transfection studies were carried out with luciferase reporter constructs containing the TF promoter from -330 to +34, approximately -4000 to +34 and -330 to +118. The average results obtained from at least two independent experiments with two independent isolates of PAEC each are shown in Fig. 4. Inducibility varied somewhat between individual experiments and PAEC isolates, but LPS stimulation of luciferase expression was reproducibly between 1.3-3-fold for all three constructs. This relative induction rate may not completely reflect the extent of up-regulation of the TF gene in quiescent EC since the relatively high basal levels of expression are consistent with the EC being already partially activated following the transfection procedure. Similar effects were observed for other endotoxin-induced promoters, and partial activation is indicated by increased levels of TF activity on transfected cells (data not shown). However, we assume that our transfection data faithfully reflect induced transcription levels, suggesting that the major regulatory elements mediating induced transcription rates in response to LPS in EC are contained within -330 and +34 of the TF promoter. Sequences upstream -330 and between +34 and +118 appeared to increase efficiency of transcription, but relative inducibility did not change when these regions were present.
Figure 4:
Functional analysis of the porcine TF
promoter in EC by transient transfection. Fragments of the porcine TF
promoter were fused to a luciferase gene as indicated and transfected
into primary aortic endothelial cells using adenovirus-polylysine
conjugates for DNA delivery. The cells were then cultured for 48 h
prior to the addition of 1 µg/ml LPS, and noninduced(-) and
LPS-treated (+) cells were harvested after 8 h. Luciferase
activity as determined in cell lysates from transfected cells was
normalized using expression from a cotransfected
cytomegalovirus-promoter--Gal expression vector as internal
control. Basal luciferase activity of pTF(-330/+118) was
arbitrarily set to 100% to emphasize the complete loss of
transcriptional activity from the TF
B mutant promoter
(pTF(-330/+118)mut
B).
Given the role of the NFB- and
AP-1-like elements in TF expression in a monocytic cell
line(18, 19) , we have tested the contributions of
these elements for LPS-inducible expression in EC. To this end, the
respective sites in the -330 to +118 luciferase promoter
construct were substituted with an XbaI linker sequence.
Strikingly, deletion of the NF
B element results in a complete
abrogation of transcription from the TF promoter, comparable with a
promoterless control construct (Fig. 4). Deletion of the two
AP-1 elements has a more moderate effect, which is somewhat variable
for different cell isolates. Nevertheless, LPS-inducibility of
transcription appears to be reduced.
Figure 5:
Binding of nuclear proteins to the TF
NFB site. A, binding on the TF NF
B site is
inducible. An oligonucleotide containing the NF
B site of the
porcine TF promoter was used for electrophoretic mobility shift assays
with extracts from untreated PAEC (lane1) or cells
induced for 2 h with 100 ng/ml LPS (lanes2-5)
or 100 units/ml TNF-
(lanes6-9). For
competition studies, a 50-fold excess of the TF
B oligonucleotide (TF
B, lanes3 and 7), a
mutated form thereof (mut
B, lanes5 and 9) or a
B oligonucleotide derived from the human
immunoglobulin
light chain enhancer (hIg
B, lanes4 and 8) were added to the binding
reactions. f.p., free probe. The triangles indicate the position of the two major protein-DNA complexes
formed. B, the complexes binding to the TF NF
B element
contain p65 and c-Rel. Electrophoretic mobility shift assays with the
TF NF
B site were performed with nuclear extracts isolated from
untreated PAEC (lane1) or cells treated with LPS (lanes2-7) for 2 h. The respective rabbit
polyclonal anti-human (p50, p52, p65) and
anti-mouse (c-Rel, RelB) peptide antibodies were
added to the binding reactions at a concentration of 0.2 µg/ml as
indicated (lanes3-7). Identical results were
obtained with TNF-
-treated cells.
Polyclonal
rabbit peptide antibodies against known subunits of NFB (p52, p50,
p65(RelA), c-Rel, and RelB; for review, see (42) ) were used to
probe the nuclear TF NF
B binding complexes for the presence of the
corresponding proteins (Fig. 5B). The addition of
anti-p65 antibodies leads to the formation of slower migrating
complexes and the intensity of both TF
B binding complexes is
strongly reduced. Interestingly, c-Rel antibodies interact specifically
with the upper complex. We conclude that p65 is contained within both
complexes, whereas c-Rel is a constituent only of the upper complex. In
contrast, there was no significant reactivity of p50 or p52 antibodies
with the TF NF
B complexes, whereas the p50 antibodies reacted well
with the proteins binding to the BS2-NF
B site of the porcine
I
B promoter(25) . RelB antibodies did not show any
reactivity with the TF NF
B complexes.
These findings are in
agreement with earlier observations (43, 44) that
substitution of the first G in the NFB consensus recognition site
to a C or T, as in the TF NF
B site, will prevent p50 from binding
to this element, although p65 and c-Rel still interact. When analyzing
the binding of in vitro translated NF
B subunits, p50,
p52, p65, as well as c-Rel are able to interact with the BS2-NF
B
element (Fig. 6, lanes18-21). In
contrast, only p65 and c-Rel bind to the wild-type TF NF
B site (lanes4 and 6). No binding of either p50 or
p52 could be detected to this element (lanes2 and 3), even in the presence of p65 or c-Rel (lanes8-11). Interestingly, substitution of the T in
position one of the TF NF
B element to a G creates a
B site
that interacts with p50, although still no binding of p52 (lanes13 and 14) is detected.
Figure 6:
DNA-binding of in vitro translated members of the NFB family. p50, p52, p65, and c-Rel were produced by in
vitro translation in a wheat germ extract, and the resulting
products were assayed in electrophoretic mobility shift assays
for binding to either the TF NF
B element (TF
B, left
panel, lanes 1-11), to a modified TF NF
B site (TF
B-G, middle panel, lanes 12-16) or to the
BS2-NF
B site from the porcine ECI-6 promoter (ECI-6/BS2, right
panel, lanes 17-21) as indicated. Antibodies against p65 (lane5) or against c-Rel (lane7)
were added as described in Fig. 5. WGE, control wheat
germ extract; f.p., free
probe.
To further
investigate the subunit composition of the nuclear TF NFB binding
complexes, photoreactive bromodeoxyuridine-substituted TF NF
B and
BS2-NF
B oligonucleotides were used for protein-DNA complex
formation and covalent cross-linking (Fig. 7). In agreement with
our earlier observations (25) two proteins of approximately 75
and 55-60 kDa, presumably p65 (RelA) and p50(45) ,
respectively, were found to interact with BS2-NF
B. Both proteins
appear to migrate as doublets, which may mean either that the subunits
are differentially modified or that the smaller forms represent
partially degraded proteins. On the TF NF
B site, a doublet at
approximately 75 kDa is seen, which comigrates with p65. In addition,
there is a weak but reproducible band at 84 kDa, which is the expected
molecular mass of cross-linked c-Rel(45) . Furthermore, two
additional polypeptides of approximately 63 and 55 kDa are found
associated with the TF
B oligo. Whereas no 63 kDa band could be
detected with the BS2-NF
B site, the smaller 55 kDa band migrates
similar to p50. Based on the binding data from the in vitro translated proteins (Fig. 6) and the antibody experiments (Fig. 5B), we nevertheless consider it unlikely that
this cross-linked protein actually represents p50.
Figure 7:
UV
cross-linking of nuclear proteins to the ECI-6/BS2 and the TF NFB
elements. Photoreactive bromodeoxyuridine-substituted ECI-6/BS2 and
TF
B oligonucleotides were synthesized by primer extension with
Klenow DNA polymerase in the presence of
[
P]dATP. The obtained oligonucleotides were
used for a preparative electrophoretic mobility shift assay. Following
separation of the NF
B/DNA complex on a native polyacrylamide gel
the DNA was cross-linked to the proteins by UV irradiation, and the
retarded material was loaded onto a 7.5% SDS-polyacrylamide gel
electrophoresis gel. The relative molecular masses of the cross-linking
adducts formed on either the TF
B (middle) or on the
BS2-NF
B (right) elements are given. The sizes of the
radioactive molecular mass marker proteins in kDa are given to the left.
Figure 8:
Binding of nuclear proteins to the TF AP-1
elements. A, binding to the TF AP-1 element is constitutive.
An oligonucleotide containing both of the AP-1 sites of the porcine TF
promoter was used for electrophoretic mobility shift assays
with nuclear extracts from untreated PAEC (nonind., lanes1-4) and cells induced with 100 ng/ml LPS (lanes5-8) or 100 units/ml of TNF- (lanes9-12) for 2 h. Competition was performed
with a 100-fold excess of the TF AP-1 oligonucleotide (TF AP-1,
lanes 2, 6, and 10), an oligonucleotide containing a
consensus AP-1 motif derived from the human collagenase promoter (CollAP-1, lanes3, 7, and 11), and with a control oligonucleotide (nonspec. comp., lanes4, 8, and 12). f.p., free probe. B,
contribution of individual AP-1 sites. Electrophoretic
mobility shift assays were performed with nuclear extracts from
LPS-treated EC and with either the dimeric TF AP-1 element (WT, lanes1, 7-14) or with
oligonucleotides where either the 5` (mut1, lanes2 and 3), the 3` (mut2, lanes4 and 5), or both AP-1 sites (mut1/2, lane 6) had been mutated. Competition
studies were carried out with an excess of non radioactively labeled
oligonucleotides as indicated (lanes3, 5, 7-14). Identical results were obtained with nuclear
extracts from unstimulated endothelial cells (data not shown). C, the TF AP-1 binding complex contains both Jun and Fos
family members. Electrophoretic mobility shift assays were
performed with the TF AP-1 element and nuclear extracts from
LPS-treated EC both in the absence (lane1) or
presence of 0.2 µg/ml peptide rabbit antibodies to mouse c-Jun,
JunB, JunD, human c-Fos, mouse FosB, human Fra1, and chicken Fra2 as
indicated (lanes2-8). In addition antibody
binding studies were carried out with two rabbit antisera broadly
reactive with Jun (lane9) and Fos (lane10) antigens, respectively. Identical results were
obtained with nuclear extracts from unstimulated endothelial cells
(data not
shown).
In order to determine whether binding to the two neighboring AP-1 sites was cooperative or would occur independently of each other, oligonucleotides were used in which either the distal (mut1), the proximal (mut2), or both (mut1/2) sites were individually substituted with an XbaI linker sequence. As is shown in Fig. 8B, substitution of either the distal (lane2) or the proximal (lane4) AP-1 site has little effect on complex formation, although binding is somewhat reduced. In contrast, substitution of both elements leads to a complete loss of binding (lane6). In addition, both single mutants efficiently compete for complex formation on the dimeric wild-type AP-1 element (lanes9-12). We conclude that binding to the individual AP-1 sites is not cooperative. There was no qualitative difference in the protein-DNA complexes formed with either a single or a double AP-1 element. This suggests that under the conditions used (oligonucleotide in excess over protein), only a single site is occupied on the oligo containing a dimeric AP-1 element and further supports the notion of independent binding to either AP-1 site in the TF promoter. In some experiments (see for example Fig. 8A), formation of a small amount of a second slower migrating complex was seen, which was only observed with the dimeric AP-1 element (data not shown). It is conceivable that this complex represents some double occupancy of the TF AP-1 element.
In
an initial analysis of the transcription factors constituting the TF
AP-1 binding complex, antibody supershift experiments were performed (Fig. 8C). Identical results were obtained with nuclear
extracts from both uninduced and LPS-treated cells, and only the
results for activated endothelial cells are shown. The addition of
rabbit antisera broadly reactive with either Jun or Fos antigens (lanes9 and 10) leads to an almost
complete prevention of complex formation or to the formation of slower
migrating complexes, suggesting that both Jun and Fos family members
are present in the TF AP-1 binding complex. Using peptide antibodies
against individual members of the Jun/Fos family of transcription
factors (lanes2-8), reactivity is seen both
with an anti-c-Jun and an anti-JunD antibody. None of the anti-Fos
peptide antibodies reacts strongly with the porcine TF AP-1 binding
complex, although some weak reactivity with an anti-Fra2 antibody is
observed.
Figure 9:
Binding of nuclear proteins to the Sp1
containing footprint region 5. An oligonucleotide spanning footprint
region 5 and containing two potential Sp1 binding sites was used for electrophoretic mobility shift assays with nuclear
extracts from noninduced PAEC (lanes1-6) and
cells induced with 100 ng/ml LPS (lane7) or 100
units/ml of TNF- (lane8) for 2 h. For
competition, a 100-fold excess of nonradioactive specific
oligonucleotide (lane2), of a consensus Sp1 (lane3), or a nonspecific oligonucleotide (lane4) was added. In lanes5 and 6, 0.2 µg/ml of a rabbit polyclonal peptide antibody
against Sp1 or a control IgG were added during the binding reaction,
respectively. f.p., free
probe.
TF synthesis is subject to tight control in EC and monocytes, presumably since any slight perturbation of the delicate balance between anti- and procoagulation can result in intravascular thrombosis (1, 2, 52) . On the cells surrounding the endothelial cell layer, constitutive expression has been documented, and the expression of TF may therefore be controlled by distinct mechanisms in these cells(3, 4) . Inappropriate expression of TF in the vasculature can be the cause of life-threatening diseases(9) . An understanding of the principles of TF gene control is therefore of great importance for the design of novel therapeutic approaches including gene therapy. In this context, the porcine system provides both a well established primary EC culture, where EC can be obtained in large quantities for in vitro studies, as well as an experimental animal, which might also serve as a donor of organs for xenotransplantation(10) .
For these
reasons we have isolated the porcine homologue of the TF gene and have
performed an analysis of the porcine TF promoter in primary aortic
endothelial cells. A triple alignment of the promoter with the
corresponding human and murine sequences reveals several well conserved
regions with homology to previously characterized transcription factor
binding motifs. The part of the promoter defined recently by Edgington
and co-workers (18) to function as an LPS-inducible element in
monocytic cells is among the most conserved. This element contains a
dimeric AP-1 site close to a single NFB site. Using DNaseI
footprint analysis of the TF promoter, these, in addition to several
Sp1 sites, are found to be the major occupied elements. In the porcine
TF promoter, the NF
B and both AP-1 sites differ from the main
consensus for the individual motifs by one nucleotide. Considering in
addition the conserved bases in the flanking regions of these sites, a
pattern of transcription factor binding unique to the TF NF
B and
AP-1 sites seems possible.
Using reporter gene constructs in which
up to 4 kilobase pairs of the porcine TF promoter were fused to the
luciferase gene, we observe an up to 3-fold induction of promoter
activity following treatment of the transiently transfected primary
endothelial cells with endotoxin. Since the transfection procedure
appears to partially induce the cells, we assume that the basal
transcription level in these experiments is higher than in normal
unstimulated cells, but the induced levels should faithfully reflect
the transcription rate in LPS-treated cells. LPS inducibility is
preserved in a construct that contains TF promoter sequences from
-330 to +34, suggesting that the LPS responsive promoter
elements required for transcriptional up-regulation of the TF gene in
EC are present in this part of the promoter. Mutation of the NFB
element demonstrates that this element plays an essential role in TF
gene transcription in EC. Deletion of the dimeric AP-1 element appears
to reduce the LPS inducibility, suggesting that this element is
involved in LPS responsiveness.
Previous analyses of the TF promoter
and its regulation in response to endotoxin have been restricted to a
monocytic cell line(18, 52) . These studies have
suggested the binding of nuclear factors to the TF NFB and AP-1
sites. Whereas in an earlier work binding to both sites was described
as inducible(18) , more recently a constitutive interaction of
transcription factors with the AP-1 elements was reported(52) .
Our analysis extends these studies to endothelial cells, demonstrates
that in our system the AP-1 binding is constitutive, and includes an
extended analysis of the nuclear factors binding to both the NF
B
and AP-1 sites.
The data obtained for the NFB complex with
nuclear extracts from primary PAEC demonstrate a strong induction of
binding following treatment of the cells with endotoxin or inflammatory
cytokines. We further observe that p65 (RelA) as well as c-Rel
participates in the binding complexes. In addition, UV cross-linking
experiments of the TF
B binding complexes reveal the presence of
two additional bands of 63 and 55 kDa, respectively, which could be two
differentially modified forms of a novel subunit or two separate
proteins. In a recent study, Oeth et al.(19) have
demonstrated the presence of p65 and c-Rel in the monocyte TF NF
B
complex. These authors have, however, not detected any other proteins
binding to this element. This could be accounted for because of a
differential availability of cross-linkable bromodeoxyuridine residues
in the specific oligos used. On the basis of the cross-linking data, we
cannot completely exclude the possibility that the 63- and 55-kDa
polypeptides we detect are degradation products of c-Rel or p65 that
retain DNA binding activity. Clearly more detailed studies will be
required to verify the nature of these proteins.
The binding of
proteins to the dimeric AP-1 site follows a different pattern from that
of the NFB site in endothelial cells. In this case we see
constitutive binding with extracts from unstimulated EC as well as with
extracts from cells induced with LPS or TNF-
. Although a slight
increase in the amount of complex formed is seen after induction, we
have not observed qualitative changes in the complexes formed on the
AP-1 elements. It is possible that the contribution of the AP-1 binding
factors to the regulation of the TF gene in EC may be exerted by
modifications of the proteins not grossly changing the binding
capabilities of the factors to DNA.
Recently, examples have been
published where a direct interaction of AP-1 or AP-1-related factors
with NFB proteins was demonstrated. In the case of the human
E-selectin promoter, ATF family members were found to cooperate with
NF
B in the cytokine-dependent activation of the gene. Furthermore,
a direct physical association between ATF family members and p50 or
p65, respectively, was shown in vitro(53) .
Similarly, physical interactions between c-Jun, c-Fos, and p65 and a
resulting functional synergism have also been suggested to occur in
vivo by reporter gene assays(54) .
In addition to
NFB and AP-1-like factors, the footprint analysis suggests a
potential importance of Sp1 binding factors for the transcription of
the TF gene. This seems even more significant given the almost complete
conservation of the Sp1 sites, even in their relative position to the
transcriptional start site in the TF promoter. Functional interactions
between inducible and constitutive cellular transcription factors like
Sp1 have been described. A well documented example is the human
immunodeficiency virus, type 1 enhancer, which is thought to be
regulated by a cooperative interplay of NF
B and Sp1 transcription
factors(55, 56) . It could very well be that a
comparable situation exists on the TF promoter. On the basis of our
results, we propose a concerted action of AP-1, NF
B, and Sp1
transcription factors in the regulation of the tissue factor promoter
in primary endothelial cells.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z46238[GenBank].