© 1995 American Heart Association, Inc.
Articles |
Transcriptional Regulation of Tissue Factor Expression in Human Endothelial Cells
From the Department of Immunology, The Scripps Research Institute, La Jolla, Calif.
Correspondence to Nigel Mackman, PhD, The Scripps Research Institute, 10666 N Torrey Pines Rd, IMM-17, La Jolla, CA 92037.
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Abstract |
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Abstract Tissue factor (TF) expression by endothelial cells is implicated in thrombotic episodes in patients with a variety of clinical disorders. In a baboon model of lethal sepsis, TF is expressed by endothelial cells in the splenic microvasculature. In vitro, endothelial cells are induced to express TF in response to tumor necrosis factor–
(TNF-), interleukin-1ß (IL-1ß), and
bacterial endotoxin (lipopolysaccharide [LPS]). Here, we identified
cis-acting regulatory elements that control TF gene
transcription in primary human endothelial cells. Functional
studies showed that the TF promoter contained a 56-bp enhancer
(-227 to -172 bp), which included two activator protein–1 (AP-1)
sites and a 
B-like site, that mediated induction by TNF-,
IL-1ß, and LPS. Electrophoretic mobility shift assays demonstrated
that endothelial cells contained constitutive AP-1 binding activity,
whereas the B-like site, 5'-CGGAGTTTCC-3', bound an inducible
nuclear complex composed of c-Rel–p65 heterodimers. Taken together,
our data suggest that induction of TF gene transcription in endothelial
cells is mediated by functional interactions between Fos-Jun and
c-Rel–p65 heterodimers.
Key Words: endothelium • tissue factor • gene expression
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Introduction |
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Tissue factor (TF) expression within the vasculature activates the coagulation protease cascades and promotes thrombotic episodes in patients with a variety of clinical disorders, including cancer, atherosclerosis, and septic shock (reviewed in References 1 and 21 2 ). Under normal physiological conditions, endothelial cells present an anticoagulant surface by expressing such proteins as thrombomodulin.3 However, in vitro, bacterial endotoxin (lipopolysaccharide [LPS]) and the inflammatory cytokines tumor necrosis factor–
(TNF-) and interleukin-1ß (IL-1ß) change
endothelial cells to a procoagulant state by inducing TF
expression.4 5 6 7 8 9 During endotoxemia, endothelial TF may be
induced directly by LPS or indirectly by TNF- and
IL-1ß.10 11 In experimental sepsis models, endothelial
TF may play a role in disseminated intravascular
coagulation.12 13 Indeed, TF expression is induced in
endothelial cells of the splenic microvasculature in a baboon model of
lethal Escherichia coli sepsis.14
Induction of TF gene expression in human endothelial cells exposed to
LPS, TNF-, phorbol 12-myristate 13-acetate (PMA), and oxidized LDL
is controlled at the level of transcription.4 9 15
However, posttranscriptional stabilization mechanisms also contribute
to TF mRNA accumulation in human umbilical vein endothelial cells
(HUVECs) exposed to LPS,9 although this mechanism was not
detected in LPS-stimulated human cardiac valve endothelial
cells.15 To date, cis-acting regulatory DNA
elements within the TF promoter that control TF gene expression in
endothelial cells have not been identified.
We have shown that a 56-bp enhancer (-227 to -172), which includes
two activator protein–1 (AP-1) sites and a B-like site, in the 5'
flanking region of the TF gene mediates LPS induction in human
monocytic cells.16 The AP-1 transcription factor family
includes fos-related antigens and jun proteins that form homodimers and
heterodimers, which specifically bind to sites matching the consensus
AP-1 recognition sequence, 5'-TGA(C/G)TCA-3'.17 Similarly,
members of the nuclear factor (NF)–B/Rel family specifically
recognize a decameric consensus sequence,
5'-GGGRNNYYCC-3',18 where R is A or G, Y is T or C, and N
is any nucleotide. The NF-B/Rel family includes p50 (NFKB1), p65
(RelA), and c-Rel, which also form various homodimers and
heterodimers.18 Our recent studies using nuclear extracts
from LPS-stimulated human monocytic cells demonstrate that the TF
promoter contains a B-like site, 5'-CGGAGTTTCC-3', that selectively
binds c-Rel–p65 heterodimers.19 In unstimulated
endothelial cells, NF-B/Rel family complexes are retained in the
cytoplasm by the binding of inhibitor proteins, including IB.
Cellular activation dissociates IB and allows translocation of
the transcription factors to the nucleus, where they bind to target
sites to regulate gene expression.20
In this study, we showed that induction of TF gene transcription in
primary cultures of human endothelial cells exposed to TNF-,
IL-1ß, and LPS was mediated by the 56-bp enhancer in the TF promoter.
The two AP-1 sites bound AP-1 proteins constitutively, whereas the
B-like site bound an inducible complex composed of c-Rel–p65
heterodimers, suggesting that binding of these two distinct families of
transcription factors regulates TF gene transcription.
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Methods |
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Reagents
LPS (E coli serotype O111:B4) was obtained from Calbiochem, human recombinant TNF-
and IL-1ß were obtained from
Collaborative Biomedical Products, and PMA was obtained from Sigma
Chemical Co.
Cell Culture
Primary cultures of human endothelial cells obtained from
collagenase-digested umbilical veins21 were cultured in
gelatin-coated flasks in medium 199 supplemented with 20% fetal bovine
serum (Gemini Bioproducts Inc), 90 µg/mL porcine intestinal heparin
(Sigma), 30 µg/mL endothelial cell growth supplement (H-Neurext,
Upstate Biotechnology Inc), 2 mmol/L L-glutamine, 100
U/mL penicillin, 100 µg/mL streptomycin (Irvine Scientific), and 25
mmol/L HEPES. Culture media contained <12.5 pg/mL endotoxin as
determined by Limulus amebocyte lysate assay (BioWhittaker).
All experiments used HUVECs between passages 3 and 5.
TF Activity
Cell pellets were solubilized with 15 mmol/L
octyl-ß-D-glucopyranoside at 37°C for 15 minutes, and
lysates were assayed for TF activity in a one-stage clotting
assay.22 Briefly, equal volumes of prewarmed human
citrated plasma and 20 mmol/L CaCl2 were added to the test
samples, and the clotting times were determined manually. Functional TF
was quantified by using a standard curve established with
phospholipid-reconstituted, affinity-purified TF from human
brain.22 A pool of neutralizing anti–human TF monoclonal
antibodies (TF8-5G9, TF8-6B4, and TF9-9C3) demonstrated that the
observed procoagulant activity was due to TF protein.
Analysis of TF mRNA
Total cellular RNA (8 µg) isolated from HUVECs was subjected
to denaturing electrophoresis in 1.2% agarose-formaldehyde
gels23 and transferred to a GeneScreen membrane
(DuPont–New England Nuclear). Membranes were hybridized with a TF cDNA
fragment labeled using [-32P]dCTP (>3000 Ci/mmol,
Amersham) as described.24 To control for variations in RNA
loadings, membranes were rehybridized with a radiolabeled cDNA fragment
from the human glucose-6-phosphate dehydrogenase gene. Membranes were
exposed to Kodak XAR film at -80°C.
Transfections
HUVECs were transfected using DEAE-dextran.25
Briefly, medium from subconfluent monolayers was replaced with 4 mL of
RPMI 1640 medium (BioWhittaker) containing 10% Nu-Serum I
(Collaborative Biomedical Products) and 2 mmol/L
L-glutamine before incubation for 4 hours with 200
µg/mL DEAE-dextran. Two micrograms and 10 µg of plasmid DNA were
used for six-well and 10-cm dishes, respectively. Cells were cultured
in complete medium for 20 hours before a 5-hour stimulation. Cell
lysates were assayed for luciferase activity as
described26 using a monolight 2010 luminometer (Analytical
luminescence Laboratory). To control for variation in transfection
efficiencies, cells were cotransfected with a control plasmid, pRSVCAT
(2 µg),27 which expresses the chloramphenicol
acetyltransferase reporter gene. Levels of chloramphenicol
acetyltransferase activity were determined as described28
and exhibited <20% variation between samples (data not shown).
Plasmids
Plasmids used in these studies have been described
previously.16 19 The cytomegalovirus (CMV)
promoter–containing plasmid soCMVIN was used for eukaryotic expression
of IB, p65, p50, and the chimeric proteins p50TA65 and c-RelTA65,
which contain the Rel homology domain of p50 and c-Rel, respectively,
fused to the transactivation domain of p65.29 30 These
plasmids were the generous gift of Drs C. Rosen, S. Ruben, and C.
Kunsch. pCMV–c-Rel contains the c-Rel cDNA cloned into the pRc/CMV
vector and was kindly provided by Dr N. Rice.
Electrophoretic Mobility Shift Assay (EMSA)
Nuclear and cytoplasmic extracts were prepared from
5x106 HUVECs as described.31 Protein
concentrations in nuclear extracts were 1 to 5 mg/mL, as determined by
BCA protein assay (Pierce). The following oligonucleotides were
obtained from Operon Technologies Inc: TF,
5'-GTCCCGGAGTTTCCTACCGGG-3'; TFm,
5'-GTCCCGGAGTTAGATACCGGG-3'; and Ig,
5'-CAGAGGGACTTTCCGAGA-3'. The B and B-like sites are
underlined, and the mutated TF (TFm) site contains a 3-bp
substitution. Oligonucleotides were radiolabeled using
[-32P]dCTP (>3000 Ci/mmol, Amersham) as
described.19 Analysis of NF-B/Rel family protein
binding was performed as follows. Nuclear extracts (1 to 2 µg) were
incubated with radiolabeled DNA probes (
10 ng;
1x106 cpm) for 20 minutes at room temperature in a
10-µL binding reaction containing 20 mmol/L HEPES, pH 7.9, 50 mmol/L
KCl, 0.5 mmol/L EDTA, 5% glycerol, 1 mmol/L DTT, 1 mg/mL bovine serum
albumin, 0.1% NP40, and 25 µg/mL poly(dI:dC). Protein-DNA complexes
were separated from free DNA probe by electrophoresis through 6%
nondenaturing acrylamide gels (Novex) in 0.5x Tris/borate/EDTA. AP-1
binding activity was analyzed with the Gel Shift System (Promega Corp)
using the following double-stranded oligonucleotides containing a
prototypic AP-1 site (underlined) or AP-1 sites from the TF promoter:
AP-1 consensus (AP-1C),
5'-CGCTTGATGAGTCAGCCGGAA-3'; distal TF AP-1 site
(AP-1D), 5'-CGCGGTTGAATCACTGGGG-3';
and proximal TF AP-1 site (AP-1P),
5'-CTGGGGTGAGTCATCCCTT-3'. For competition and antibody
supershift experiments, binding reactions were incubated with unlabeled
double-stranded oligonucleotides or monospecific antibodies for 20
minutes before the addition of the radiolabeled oligonucleotide.
Antibodies
Anti-p65, anti–c-Rel, anti–c-Fos, anti–Jun B, anti–Jun D,
and anti–Fos B rabbit polyclonal antibodies were purchased from Santa
Cruz Biotechnology. In addition, anti–c-Jun antisera were provided by
Dr P. Vogt, and anti-p50 and anti-IB antisera were kindly
provided by Dr. W. Greene.
Western Blot Analysis
Cytoplasmic extracts from HUVECs stimulated with IL-1ß (20
ng/mL) for various times were electrophoresed on 8% to 16%
SDS-polyacrylamide gels (Novex) and transferred to Hybond-ECL (Amersham
Corp). IB protein was detected according to the ECL protocol
(Amersham Corp) using a 1:2500 dilution of an IB
antiserum.32
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Results |
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Functional Analysis of the Human TF Promoter in HUVECs
Exposure of HUVECs to TNF-
(20 ng/mL), IL-1ß (20 ng/mL), LPS
(1 µg/mL), or PMA (50 ng/mL) rapidly induced TF mRNA and TF activity
(Fig 1
). These concentrations were found to maximally
induce TF expression (data not shown). Similar results were
obtained with LPS-stimulated human cardiac valve endothelial
cells and IL-1ß–stimulated human dermal microvascular endothelial
cells (data not shown). To localize cis-acting DNA elements
that regulate TF gene transcription, HUVECs were transfected with
plasmids containing sequential 5' truncations of the TF promoter
between -2106 and -21 bp cloned upstream of a luciferase reporter
gene (Fig 2A). After 20 hours, the cells were stimulated
for 5 hours to determine the fold induction of luciferase activity.
Plasmids containing at least -244 bp of the TF promoter, including the
56-bp enhancer (-227 to -172 bp), were all induced by TNF-,
whereas luciferase activity expressed by pTF(-153)LUC and
pTF(-111)LUC was not increased by TNF- stimulation (Fig 2B).
pTF(-21)LUC did not express any promoter activity. These results
suggest that the 56-bp enhancer may mediate TNF- induction of the TF
promoter in HUVECs. Similarly, luciferase activity expressed by
pTF(-278)LUC, which contained the enhancer, was induced by IL-1ß and
LPS, but these agonists failed to induce the enhancerless plasmid
pTF(-111)LUC (Fig 2C). PMA induction of pTF(-278)LUC was
significantly greater than the induction of pTF(-111)LUC, suggesting
that the 56-bp enhancer may mediate, at least in part, PMA induction of
the TF promoter. PMA induction of pTF(-111)LUC, although weak,
suggested the presence of a second PMA response element in the TF
promoter.
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To determine whether the two AP-1 sites and the B-like site within
the enhancer mediated TNF- induction, HUVECs were transfected with
plasmids containing mutations in these sites. Mutation of each of these
three sites independently abolished TNF- induction of the TF
promoter, suggesting that all three sites were required for inducible
expression (Fig 3A). To further examine the role of the
two AP-1 sites and the B-like site in TNF- induction, HUVECs were
transfected with a plasmid containing the 56-bp TF enhancer cloned
upstream of a heterologous promoter. The 56-bp enhancer present in
pTF(227/172)PAI-LUC conferred TNF inducibility to the minimal promoter
(Fig 3B). Plasmid pTF(227/189)PAI-LUC, which contained the two AP-1
sites alone, was not inducible by TNF-, whereas plasmid
pTF(192/172)PAI-LUC, which contained a single copy of the B-like
site alone, was induced by TNF-. Similar results were obtained with
IL-1ß, LPS, and PMA (data not shown). The function of the TF
B-like site also was examined by using plasmids containing four
tandem copies of either the TF B-like site or a mutated TF B-like
site cloned upstream of a minimal SV40 promoter expressing the
luciferase reporter gene. Luciferase activity expressed by
p(TF)4LUC, which contained four copies of the wild-type
site, was strongly induced by TNF-, IL-1ß, LPS, and PMA (Fig 3C).
In contrast, luciferase activity expressed by
p(TFmut)4LUC, which contained four copies of
the mutated TF B-like site, or the parental plasmid pSVLUC was not
induced by these agonists.
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AP-1 Binding Activity in HUVECs
To assess the binding of nuclear proteins to the two AP-1 sites in
the TF promoter, EMSAs were performed with oligonucleotides that
contained the distal (AP-1D) or proximal
(AP-1p) AP-1 sites from the TF promoter (see
"Methods"). Nuclear extracts from unstimulated HUVECs formed a
prominent protein-DNA complex with both the distal and proximal TF AP-1
sites, as well as with an oligonucleotide containing a prototypic AP-1
site (Fig 4A). Competition studies using unlabeled
oligonucleotides containing either an AP-1 site or an Sp1 site
demonstrated that the two AP-1 sites in the TF promoter and the
prototypic AP-1 site specifically bound AP-1 proteins (Fig 4A). To
determine whether stimulation of HUVECs increased AP-1 binding
activity, radiolabeled oligonucleotides were incubated with nuclear
extracts from HUVECs exposed to TNF-, IL-1ß, LPS, or PMA for 1
hour. The intensity of the protein-DNA complex formed with nuclear
extracts from unstimulated cells was similar to that observed with
nuclear extracts from stimulated cells (Fig 4B).
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Monospecific antibodies to various members of the AP-1 family
were used to determine the composition of the AP-1 complexes. The AP-1
complex formed between AP-1p and nuclear extracts from
unstimulated cells or IL-1ß–stimulated cells was not recognized by
antisera against Jun B, Jun D, Fos B, and p65 (Fig 4C
). In contrast,
this complex was supershifted with anti–c-Fos and anti–c-Jun antisera
(Fig 4C). Similar results were observed with AP-1D (data
not shown). Increasing the amount of c-Fos or c-Jun antisera or adding
both antisera simultaneously did not completely remove the residual
complex, suggesting that it did not represent binding of
Fos-Jun heterodimers or c-Jun homodimers. At present the identity
of this residual complex is unknown, but it may represent
binding of other bZIP proteins. These data suggest that in unstimulated
HUVECs the two AP-1 sites in the TF promoter constitutively bind
Fos-Jun heterodimers. Stimulation of the cells with various agonists
did not change the composition of the complex or increase binding of
the Fos-Jun heterodimers.
The TF B-like Site Binds c-Rel–p65 Heterodimers
EMSAs were performed to examine the binding of nuclear proteins to
an oligonucleotide containing the TF B-like site, 5'-CGGAGTTTCC-3'.
A constitutive complex was observed with nuclear extracts from both
unstimulated and IL-1ß–stimulated HUVECs, whereas an additional
inducible complex (TF complex) was observed with nuclear extracts from
IL-1ß–stimulated cells (Fig 5A, lanes 1 and 2). To
determine the specificity of these protein-DNA complexes, competition
studies were performed using unlabeled oligonucleotides containing
either the TF B-like site (TF) or a mutated TF B-like site
(TFm). Both complexes were competed with an oligonucleotide
containing the TF B-like site (Fig 5A, lane 4), whereas an
oligonucleotide containing the mutated TF B-like site only competed
with the faster-migrating complex (Fig 5A, lane 5). These data
indicated that the inducible TF complex represented
specific protein binding to the B-like site and the faster-migrating
complex represented nonspecific protein binding. The TF
complex was also observed with nuclear extracts from HUVECs stimulated
for 1 hour with TNF-, LPS, or PMA (Fig 5B, lanes 7 through 10). For
comparison, we examined the binding of NF-B (p50-p65) to a
prototypic B site from the mouse light-chain enhancer (Ig). The
low levels of p50-p65 heterodimers present in nuclear extracts from
unstimulated HUVECs were dramatically increased upon stimulation with
each of the four agonists (Fig 5B, lanes 1 through 5). Significantly,
the TF complex migrated more slowly than the Ig complex (Fig 5B),
suggesting that it represented binding of NF-B/Rel
proteins distinct from p50-p65 heterodimers.
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To assess the protein composition of the nuclear complex that bound to
the TF B-like site, the mobility of the TF complex was compared with
that of translated p50, p65, and c-Rel homodimers as well as p50-p65
and c-Rel–p65 heterodimers. The TF complex comigrated with c-Rel–p65
heterodimers and migrated independently of p65 and c-Rel homodimers
(Fig 5C, lanes 4 through 8). As expected, the Ig complex comigrated
with the p50-p65 heterodimers (Fig 5C, lanes 2 and 3). These data
suggest that the TF complex may represent binding of c-Rel–p65
heterodimers.
Monospecific antibodies to p50, p65, and c-Rel were used to
independently analyze the identity of the TF complex. Anti-p65 or
anti–c-Rel antibodies abolished formation of the TF complex, whereas
an anti-p50 antibody had no effect, indicating that the TF complex
represented binding of c-Rel–p65 heterodimers (Fig 5D).
These antibodies did not recognize the faster-migrating nonspecific
complex. The low levels of c-Rel–p65 heterodimers in HUVECs prohibited
analysis of this complex by UV cross-linking. c-Rel–p65
heterodimers also were activated in LPS-stimulated human cardiac valve
endothelial cells and IL-1ß–stimulated human dermal microvascular
endothelial cells (data not shown). Taken together, these data
demonstrated that stimulation of HUVECs with four diverse agonists
activated c-Rel–p65 heterodimers, which specifically bound to the
novel B-like site in the TF promoter.
Transactivation of the TF B-like Site in HUVECs by Expression of
NF-B/Rel Proteins
To investigate in vivo binding of NF-B/Rel family members to
the TF B-like site, HUVECs were cotransfected with
p(TF)4LUC and plasmids expressing p50, p65, or the chimeric
proteins p50TA65 and c-RelTA65, which contained the
transactivation domain of p65 fused to the DNA binding portions of p50
and c-Rel, respectively. To eliminate ambiguities created by
transactivation domains with different activities, all proteins
contained an identical transactivation domain. p50 does not contain a
transactivation domain and therefore was used as a negative control.
Transactivation by these NF-B/Rel proteins was measured by increased
expression of luciferase activity. p(TF)4LUC was
transactivated by p65 and c-RelTA65 but not by p50 or p50TA65 (Fig 6A). In contrast, p(Ig)4LUC was
transactivated by p65 and p50TA65 but not by p50 or c-RelTA65. These
NF-B/Rel proteins did not transactivate pSVLUC, which contained the
minimal SV40 promoter alone, or p(TFmut)4LUC,
which contained the mutated TF B-like site (data not shown).
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To examine transactivation by p65 or c-Rel homodimers as well as by
c-Rel–p65 or p50-p65 heterodimers, cells were cotransfected with
p(TF)4LUC in the presence of plasmids expressing p50, p65,
or c-Rel alone or in combination. c-Rel homodimers were weak
transactivators compared with p65 homodimers (Fig 6B), as reported
previously.33 Next, the cells were transfected with
plasmids expressing c-Rel and p65, or p50 and p65, at a 1:1 ratio to
generate c-Rel–p65 and p50-p65 heterodimers, with the assumption that
there is approximately equal expression from each plasmid. These
results indicated that c-Rel–p65 heterodimers transactivated the TF
B-like site, whereas formation of p50-p65 heterodimers abolished
transactivation, presumably by eliminating p65 homodimers (Fig 6B).
These results strongly suggested that c-Rel–p65 heterodimers regulated
the TF B-like site in human endothelial cells. To determine whether
c-Rel–p65 heterodimers were regulated by IB, HUVECs were
cotransfected with p(TF)4LUC and a plasmid that expresses
IB. TNF- induction of luciferase activity mediated by the
binding of c-Rel–p65 heterodimers to the TF B-like site was
abolished by expression of IB, indicating that binding of
exogenously expressed IB to endogenous c-Rel–p65 heterodimers
prevented nuclear translocation (Fig 6C).
Activation of c-Rel–p65 Heterodimers Precedes TF mRNA Induction in
HUVECs
TF mRNA was induced rapidly after TNF- or IL-1ß stimulation
of HUVECs (Fig 1). As shown in Fig 7 (upper panel),
stimulation of HUVECs resulted in a rapid and transient increase in TF
mRNA levels that were maximal at 60 minutes. To examine the kinetics of
activation of c-Rel–p65 heterodimers in HUVECs, nuclear extracts were
prepared from cells at various times after stimulation. c-Rel–p65
heterodimers were rapidly activated within 15 minutes and declined
after 30 minutes (Fig 7, middle panel). To determine whether
translocation of c-Rel–p65 to the nucleus was associated with loss of
the inhibitor protein IB, cytoplasmic extracts from stimulated
cells were analyzed by Western blotting and an anti-IB antibody.
IB protein was present in unstimulated HUVECs but was rapidly
and transiently reduced after stimulation (Fig 7, lower panel). Maximal
reduction of IB protein was observed at 15 minutes, which
corresponded to maximal levels of c-Rel–p65 heterodimers in the
nucleus. IB protein remained low at 30 minutes before increasing
after 1 hour. Taken together, these data indicated that activation of
c-Rel–p65 heterodimers was regulated by IB. The rapid activation
and nuclear translocation of c-Rel–p65 heterodimers within 15 minutes
preceded increases in TF mRNA levels at 40 minutes, consistent with a
role for this transcription factor in the induction of TF gene
transcription.
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Discussion |
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This study elucidated a transcriptional regulatory mechanism that coordinated induction of TF expression in primary cultures of human endothelial cells exposed to TNF-
, IL-1ß, and LPS. Functional
analysis of the human TF promoter demonstrated that a 56-bp
enhancer (-227 to -172), containing two AP-1 sites and a B-like
site, mediated induction in response to these agonists. Mutational
analysis indicated that all three sites were required for induction
of the homologous TF promoter. EMSAs revealed that the two AP-1 sites
constitutively bound Fos-Jun heterodimers, whereas the B-like site
bound an inducible nuclear complex, which was composed of c-Rel–p65
heterodimers. Nuclear extracts from LPS-stimulated human cardiac valve
endothelial cells and IL-1ß–stimulated human dermal microvascular
endothelial cells also contained c-Rel–p65 heterodimers (data not
shown). Nuclear translocation of c-Rel–p65 heterodimers preceded
induction of TF mRNA, consistent with a positive regulatory role for
this transcription factor in TF gene transcription. Taken together,
these data suggest that Fos-Jun and c-Rel–p65 heterodimers regulate TF
gene expression in human endothelial cells exposed to TNF-, IL-1ß,
and LPS.
Comparable levels of TF activity and TF mRNA were induced in HUVECs
exposed to optimal doses of TNF-, IL-1ß, and LPS, whereas PMA was
a more potent agonist (Fig 1). The apparent discrepancy between
increases in the steady-state levels of TF mRNA and the magnitude of
induction of the TF promoter expressing the luciferase reporter gene
suggested that posttranscriptional control mechanisms contributed to TF
mRNA accumulation in these cells, as reported previously.9
Nevertheless, studies of TF gene regulation in human endothelial cells
have consistently reported that LPS, TNF-, and PMA increase the rate
of TF gene transcription,4 9 15 indicating that TF
expression is controlled, in part, at the level of transcription. Our
studies with transfected HUVECs were consistent with the changes in the
rate of transcription of the endogenous TF gene in LPS-stimulated
HUVECs.4 9 However, we cannot discount the possibility
that DNA elements not present between -2106 and +121 bp also
regulate TF gene expression in HUVECs, which may explain why PMA did
not strongly induce the cloned TF promoter despite a dramatic induction
of TF mRNA. Our studies indicated the presence of two PMA response
elements in the cloned TF promoter: the TF enhancer (-227 to -172 bp)
and a region (-111 to +121 bp) that contains Sp1 and EGR-1 binding
sites.34 TNF- induction of the TF promoter in HUVECs
was mediated by the 56-bp enhancer alone because it was abolished by
deletion of the enhancer and by individual mutations of the two AP-1
sites and the B-like site. Previously, we showed similar results in
LPS-stimulated monocytic THP-1 cells,16 suggesting that a
common mechanism regulates TF gene expression in both human monocytes
and endothelial cells.
A recurring theme in the regulation of gene expression in eukaryotic
cells is the binding of multiple transcription factors.35
Here, we showed that in stimulated endothelial cells, Fos-Jun
heterodimers bound to the two AP-1 sites and c-Rel–p65 heterodimers
bound to an adjacent B-like site in the 56-bp TF enhancer. The
binding of Fos-Jun heterodimers was unaffected by stimulation of the
cells, and the composition of the complex was not changed upon
stimulation. NF-B/Rel family proteins cooperate with AP-1 proteins
as well as with NF-IL6, SRF, and Sp1 to activate both cellular and
viral genes.36 37 38 39 40 Recent studies by Stein and
colleagues40 demonstrated that the bZIP regions of Fos and
Jun proteins from the AP-1 family directly interacted with the Rel
homology domain of p65 from the NF-B/Rel family to form a
functionally active transcription factor complex. Thus, in a similar
manner, binding of Fos-Jun heterodimers to the TF enhancer may
cooperate with c-Rel–p65 heterodimers to facilitate activation of TF
gene transcription.
Endothelial cell activation induces the rapid expression of many genes
involved in inflammation, which contain B and B-like sites in
their promoters.20 Here, we have shown that the TF
B-like site plays a central role in regulating TF gene transcription
in endothelial cells. The TF B site, 5'-CGGAGTTTCC-3', specifically
bound c-Rel–p65 heterodimers. The nonconsensus C at position 1 is
conserved in the mouse TF promoter and is a T in the porcine TF
promoter.41 42 Other genes that are inducibly expressed in
endothelial cells, including IL-8 and intercellular adhesion
molecule–1,43 44 45 contain B-like sites with a T at
position 1 and bind c-Rel–p65 heterodimers,46 suggesting
that a distinct subset of genes are regulated by c-Rel–p65
heterodimers.
LPS, TNF-, IL-1ß, and PMA all activate c-Rel–p65 heterodimers,
which activation represents a common mechanism for induction of
TF expression in HUVECs. This conclusion is supported by a recent
study demonstrating that both activation of NF-B/Rel family proteins
and induction of TF mRNA expression in HUVECs exposed to LPS are
dependent on the presence of soluble CD14 receptors.31 LPS
stimulation of cells induces the rapid phosphorylation of IB and
subsequent translocation of NF-B/Rel complexes to the
nucleus.47 TNF- induction mediated by the TF B-like
site was abolished by overexpression of IB, indicating that
c-Rel–p65 heterodimers are regulated by binding of IB. Thus,
despite the binding of LPS, TNF-, and IL-1ß to distinct
cell-surface receptors, intracellular signaling pathways converge to
activate c-Rel–p65 heterodimers, which interact with Fos-Jun
heterodimers to coordinate induction of TF expression in endothelial
cells.
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Acknowledgments |
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This work was supported by a Research Fellowship (Dr Parry) from the American Heart Association, California Affiliate, San Diego County Chapter, and by National Institutes of Health grants HL-16411 and HL-48872 (Dr Mackman). This is publication IMM-8547 from The Scripps Research Institute. We wish to thank P. Nantermet, H. Larson, and P. Oeth for expert technical assistance and D. Nielsen for the preparation of the manuscript. We thank P. Vogt for anti–c-Jun antibodies; W. Greene for the anti-p50 and anti-I
B antibodies; N. Rice for the
c-Rel expression vector; C. Rosen, C. Kunsch, and S. Ruben for the
soCMVIN expression vectors; D. Loskutoff and K. Roegner for endothelial
cells; T. Drake for human cardiac valve endothelial cells; and S.
Cordle and M. Read for advice on EMSA. We thank L. Curtiss and C. Banka
for critical review of this manuscript. Received October 6, 1994; accepted February 17, 1995.
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Bach RR. Initiation of coagulation by tissue factor. Crit Rev Biochem. 1988;23:339-368. [Medline] [Order article via Infotrieve]
2. Edgington TS, Mackman N, Brand K, Ruf W. The structural biology of expression and function of tissue factor. Thromb Haemost. 1991;66:67-79. [Medline] [Order article via Infotrieve]
3.
Esmon CT. The regulation of natural
anticoagulant pathways. Science. 1987;235:1348-1352.
4.
Scarpati EM, Sadler JE. Regulation of
endothelial cell coagulant properties. J Biol Chem. 1989;264:20705-20713.
5.
Bevilacqua MP, Pober JS, Majeau GR, Fiers W, Cotran
RS, Gimbrone MA. Recombinant tumor necrosis factor induces
procoagulant activity in cultured human vascular endothelium:
characterization and comparison with the actions of interleukin
1. Proc Natl Acad Sci U S A. 1986;83:4533-4537.
6.
Bevilacqua MP, Pober JS, Majeau GR, Cotran RS,
Gimbrone MA. Interleukin 1 (IL-1) induces biosynthesis and cell
surface expression of procoagulant activity in human vascular
endothelial cells. J Exp Med. 1984;160:618-623.
7. Moore KL, Andreoli SP, Esmon NL, Esmon CT, Bang NU. Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro. J Clin Invest. 1987;79:124-130.
8. Colucci M, Balconi G, Lorenzet R, Pietra A, Locati D, Donati MB, Semeraro N. Cultured human endothelial cells generate tissue factor in response to endotoxin. J Clin Invest. 1983;71:1893-1896.
9.
Crossman DC, Carr DP, Tuddenham EGD, Pearson JD, McVey
JH. The regulation of tissue factor mRNA in human endothelial
cells in response to endotoxin or phorbol ester. J Biol
Chem. 1990;265:9782-9787.
10. Michie HR, Manogue KR, Spriggs DR, Revhaug A, O'Dwyer S, Dinarello CA, Cerami A, Wolff SM, Wilmore DW. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med. 1988;318:1481-1486. [Abstract]
11. van der Poll T, Büller HR, ten Cate H, Wortel CH, Bauer KA, van Deventer SJH, Hack CE, Sauerwein HP, Rosenberg RD, ten Cate JW. Activation of coagulation after administration of tumor necrosis factor to normal subjects. N Engl J Med. 1990;322:1622-1627. [Abstract]
12.
Warr TA, Rao LVM, Rapaport SI. Disseminated
intravascular coagulation in rabbits induced by administration of
endotoxin or tissue factor: effect of anti-tissue factor antibodies and
measurement of plasma extrinsic pathway inhibitor activity.
Blood. 1990;75:1481-1489.
13. Taylor FB Jr, Chang A, Ruf W, Morrissey JH, Hinshaw L, Catlett R, Blick K, Edgington TS. Lethal E coli septic shock is prevented by blocking tissue factor with monoclonal antibody. Circ Shock. 1991;33:127-134. [Medline] [Order article via Infotrieve]
14. Drake TA, Cheng J, Chang A, Taylor FB Jr. Expression of tissue factor, thrombomodulin, and E-selectin in baboons with lethal Escherichia coli sepsis. Am J Pathol. 1993;142:1-13.
15.
Fei H, Berliner JA, Parhami F, Drake TA.
Regulation of endothelial cell tissue factor expression by
minimally oxidized low density lipoprotein and
lipopolysaccharide. Arterioscler Thromb. 1993;13:1711-1717.
16.
Mackman N, Brand K, Edgington TS.
Lipopolysaccharide-mediated transcriptional activation of the
human tissue factor gene in THP-1 monocytic cells requires both
activator protein 1 and nuclear factor B binding sites.
J Exp Med. 1991;174:1517-1526.
17. Abate C, Curran T. Encounters with Fos and Jun on the road to AP-1. Semin Cancer Biol. 1990;1:19-26. [Medline] [Order article via Infotrieve]
18. Grilli M, Chiu JJ-S, Lenardo MJ. NF-kB and Rel: participants in a multiform transcriptional regulatory system. In: Jeon KW, Friedlander M, Jarvik J, eds. International Review of Cytology. San Diego, Calif: Academic Press Inc; 1993:1-62.
19.
Oeth PA, Parry GCN, Kunsch C, Nantermet P, Rosen CA,
Mackman N. LPS induction of tissue factor gene expression in
monocytic cells is mediated by binding of c-Rel–p65 heterodimers to a
kB-like site. Mol Cell Biol. 1994;14:3772-3781.
20.
Read MA, Whitley MZ, Williams AJ, Collins T.
NF-kappaB and IkappaB: an inducible regulatory system in
endothelial activation. J Exp Med. 1994;179:503-512.
21.
Van Mourik JA, Lawrence DA, Loskutoff DJ.
Purification of an inhibitor of plasminogen activator
(antiactivator) synthesized by endothelial cells. J Biol
Chem. 1984;259:14914-14921.
22. Morrissey JH, Fair DS, Edgington TS. Monoclonal antibody analysis of purified and cell-associated tissue factor. Thromb Res. 1988;52:247-261. [Medline] [Order article via Infotrieve]
23. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1982.
24.
Brand K, Fowler BJ, Edgington TS, Mackman N.
Tissue factor mRNA in THP-1 monocytic cells is regulated at both
transcriptional and posttranscriptional levels in response to
lipopolysaccharide. Mol Cell Biol. 1991;11:4732-4738.
25. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Current Protocols in Molecular Biology. New York, NY: Green Publishing Association & Wiley Interscience; 1987.
26.
De Wet JR, Wood KV, Deluca M, Helinski DR, Subramani S.
Firefly luciferase gene: structure and expression in mammalian
cells. Mol Cell Biol. 1987;7:725-737.
27.
Gorman CM, Merlino GT, Willingham MC, Pastan I, Howard
BH. The rous sarcoma virus long terminal repeat is a strong
promoter when introduced into a variety of eukaryotic cells by
DNA-mediated transfection. Proc Natl Acad Sci
U S A. 1982;79:6777-6781.
28. Neumann JR, Morency CA, Russian KO. A novel rapid assay for chloramphenicol acetyltransferase gene expression. BioTechniques. 1987;5:444-447.
29.
Beg AA, Ruben SM, Scheinman RI, Haskill S, Rosen CA,
Baldwin AS Jr. IB interacts with the nuclear localization
sequences of the subunits of NF-B: a mechanism for cytoplasmic
retention. Genes Dev. 1992;6:1899-1913.
30.
Ruben SM, Narayanan R, Klement JF, Chen C-H, Rosen CA.
Functional characterization of the NF-B p65 transcriptional
activator and an alternatively spliced derivative. Mol
Cell Biol. 1992;12:444-454.
31.
Read MA, Cordle SR, Veach RA, Carlisle CD, Hawiger J.
Cell-free pool of CD14 mediates activation of transcription
factor NF-B by lipopolysaccharide in human endothelial
cells. Proc Natl Acad Sci U S A. 1993;90:9887-9891.
32.
Sun S-C, Ganchi PA, Ballard DW, Greene WC. NF-kB
controls expression of inhibitor IB: evidence for an inducible
autoregulatory pathway. Science. 1993;259:1912-1915.
33.
Hansen SK, Baeuerle PA, Blasi F. Purification,
reconstitution, and IB association of the c-Rel-p65 (RelA) complex,
a strong activator of transcription. Mol Cell Biol. 1994;14:2593-2603.
34.
Cui M-Z, Parry GCN, Edgington TS, Mackman N.
Regulation of tissue factor gene expression in epithelial cells:
induction by serum and phorbol 12-myristate 13-acetate.
Arterioscler Thromb. 1994;14:807-814.
35.
Mitchell PJ, Tjian R. Transcriptional regulation
in mammalian cells by sequence-specific DNA binding proteins.
Science. 1989;245:371-378.
36.
Stein B, Baldwin ASJ. Distinct mechanisms for
regulation of the interleukin-8 gene involve synergism and
cooperativity between C/EBP and NF-B. Mol Cell
Biol. 1993;13:7191-7198.
37.
Matsusaka T, Fujikawa K, Nishio Y, Mukaida N,
Matsushima K, Kishimoto T, Akira S. Transcription factors NF-IL6
and NF-B synergistically activate transcription of the inflammatory
cytokines, interleukin 6 and interleukin 8. Proc Natl
Acad Sci U S A. 1993;90:10193-10197.
38.
Kuang AA, Novak KD, Kang S-M, Bruhn K, Lenardo MJ.
Interaction between NF-B- and serum response factor-binding
elements activates an interleukin-2 receptor -chain enhancer
specifically in T lymphocytes. Mol Cell Biol. 1993;13:2536-2545.
39.
Desai-Yajnik V, Samuels HH. The NF-B and Sp1
motifs of the human immunodeficiency virus type 1 long terminal repeat
function as novel thyroid hormone response elements. Mol
Cell Biol. 1993;13:5057-5069.
40.
Stein B, Baldwin AS Jr, Ballard DW, Greene WC, Angel P,
Herrlich P. Cross-coupling of the NF-B p65 and Fos/Jun
tran-scription factors produces potentiated biological
function. EMBO J. 1993;12:3879-3891. [Medline]
[Order article via Infotrieve]
41.
Mackman N, Imes S, Maske WH, Taylor B, Lusis AJ, Drake
TA. Structure of the murine tissue factor gene: chromosome
location and conservation of regulatory elements in the
promoter. Arterioscler Thromb. 1992;12:474-483.
42. Hofer E, Moll T, Czyz M, Holzmüller H, Bach FH. Induction of the tissue factor gene in porcine aortic endothelial cells. J Cell Biochem. 1994;(suppl 18A):323. Abstract.
43.
Degitz K, Lian-Jie L, Wright Caughman S. Cloning
and characterization of the 5'-transcriptional regulatory region of the
human intercellular adhesion molecule 1 gene. J Biol
Chem. 1991;266:14024-14030.
44.
van de Stolpe A, Caldenhoven E, Stade BG,
Koenderman L, Raaijmakers JAM, Johnson JP, van der Saag PT.
12-O-tetradecanoylphorbol-13-acetate- and tumor
necrosis factor -mediated induction of intercellular adhesion
molecule-1 is inhibited by dexamethasone. J Biol
Chem. 1994;269:6185-6192.
45. Mukaida N, Shiroo M, Matsushima K. Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8. J Immunol. 1989;143:1366-1371. [Abstract]
46.
Parry GCN, Mackman N. A set of inducible genes
expressed by activated human monocytic and endothelial cells contain
B-like sites that specifically bind c-Rel-p65 heterodimers.
J Biol Chem. 1994;269:20823-20825.
47.
Cordle SR, Donald R, Read MA, Hawiger JJ.
Lipopolysaccharide induces phosphorylation of MAD3 and
activation of c-Rel and related NF-kB proteins in human monocytic THP-1
cells. J Biol Chem. 1993;268:11803-11810.
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|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
 S. IMOKAWA, A. SATO, H. HAYAKAWA, M. KOTANI, T. URANO, and A. TAKADA Tissue Factor Expression and Fibrin Deposition in the Lungs of Patients with Idiopathic Pulmonary Fibrosis and Systemic Sclerosis Am. J. Respir. Crit. Care Med., July 1, 1997; 156(2): 631 - 636. [Abstract] [Full Text]
|
|
|
|
|
|
P. Oeth, G. C.N. Parry, and N. Mackman Regulation of the Tissue Factor Gene in Human Monocytic Cells: Role of AP-1, NF-{kappa}B/Rel, and Sp1 Proteins in Uninduced and Lipopolysaccharide-Induced Expression Arterioscler. Thromb. Vasc. Biol., February 1, 1997; 17(2): 365 - 374. [Abstract] [Full Text]
|
|
|
|
 |
|
 E. M. Boyle Jr, E. D. Verrier, and B. D. Spiess Endothelial Cell Injury in Cardiovascular Surgery: The Procoagulant Response Ann. Thorac. Surg., November 1, 1996; 62(5): 1549 - 1557. [Abstract] [Full Text]
|
|
|
|
|
|
V. Ollivier, G. C.N. Parry, R. R. Cobb, D. d. Prost, and N. Mackman Elevated Cyclic AMP Inhibits NF-kappa B-mediated Transcription in Human Monocytic Cells and Endothelial Cells J. Biol. Chem., August 23, 1996; 271(34): 20828 - 20835. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Groupp and M. Donovan-Peluso Lipopolysaccharide Induction of THP-1 Cells Activates Binding of c-Jun, Ets, and Egr-1 to the Tissue Factor Promoter J. Biol. Chem., May 24, 1996; 271(21): 12423 - 12430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-M. Kiely, M. I. Cybulsky, F. W. Luscinskas, and M. A. Gimbrone Jr Immunoselective Targeting of an Anti-Thrombin Agent to the Surface of Cytokine-Activated Vascular Endothelial Cells Arterioscler. Thromb. Vasc. Biol., August 1, 1995; 15(8): 1211 - 1218. [Abstract] [Full Text]
|
|
|
|
|
|
S. Blum, K. Issbruker, A. Willuweit, S. Hehlgans, M. Lucerna, D. Mechtcheriakova, K. Walsh, D. von der Ahe, E. Hofer, and M. Clauss An Inhibitory Role of the Phosphatidylinositol 3-Kinase-signaling Pathway in Vascular Endothelial Growth Factor-induced Tissue Factor Expression J. Biol. Chem., August 31, 2001; 276(36): 33428 - 33434. [Abstract] [Full Text] [PDF]
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