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B Site of the Promoter Mediates Endothelial ICAM-1 Expression and Neutrophil Adhesion1
Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL 60612
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B site (-533 bases from translation start site) had no
effect on thrombin responsiveness, whereas mutation/deletion of
downstream NF-B site (-223 bases from the translation start site)
prevented the activation of ICAM-1 promoter, indicating that the
downstream NF-B site is critical for thrombin inducibility.
NF-B-directed luciferase activity increased 
3-fold when cells
transfected with the plasmid pNF-BLUC containing five copies of
consensus NF-B site linked to a minimal adenovirus E1B
promoter-luciferase gene were exposed to thrombin, indicating that
activation of NF-B was essential for thrombin response. Gel
supershift assays demonstrated that thrombin induced binding of
NF-Bp65 (Rel A) to downstream NF-B site of the ICAM-1 promoter.
Thrombin receptor activation peptide, a 14-amino-acid peptide
representing the new NH2 terminus of proteolytically
activated receptor-1, mimicked thrombin’s action in inducing ICAM-1
expression. These data indicate that thrombin activates endothelial
ICAM-1 expression and polymorphonuclear leukocyte adhesion by
NF-Bp65 binding to the downstream NF-B site of ICAM-1 promoter
after proteolytically activated receptor-1
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies have shown that thrombin is an important regulator of
polymorphonuclear leukocyte (PMN) adhesion to endothelial cells (9, 10). The basis of increased endothelial adhesivity may involve type I
activation of adhesive proteins such as ICAM-1 (CD54) on the
endothelial cell surface. However, there is no evidence indicating that
thrombin mediates type II activation of ICAM-1 expression in
endothelial cells. The interaction of ICAM-1 with its counter-receptors
on the surface of leukocytes, CD11a/CD18 and CD11b/CD18
ß2 integrins, is a critical requirement for PMN adhesion
and transendothelial PMN migration (19, 20). ICAM-1 is constitutively
present in low levels, but its expression can be transcriptionally
up-regulated by cytokines via the activation of NF-B (21, 22, 23).
NF-B/Rel transcription factors are composed of five distinct
DNA-binding subunits, called p50, p52, p65 (RelA), c-Rel, and Rel-B
(24). The different family members can associate in various homo- or
heterodimers through a highly conserved NH2-terminal
sequence, NRD (NF-B/rel/Dorsal) (25) or Rel homology domain.
Inactive NF-B is sequestered in the cytoplasm by the inhibitory
protein I-B (IB) and released after phosphorylation of IB
either on serine residues 32 and 36 of IB
and serines 19 and 23
of IBß by IB kinases and ß, respectively (26, 27), that
regulates their ubiquitin-dependent degradation through the 26S
proteasome (28, 29, 30, 31), or on tyrosine residues of IB that does not
involve its degradation (32). The activated NF-B dimer then
translocates to the nucleus and regulates transcription of genes such
as ICAM-1 involved in inflammatory responses (33, 34, 35).
Sequence analysis of ICAM-1 promoter has revealed the presence of two
NF-B sites (22, 23, 36): the upstream NF-B
(5'-CGGGAGGATTCCTGGGCC-3', element underlined, within -542
to -524 bases from the translation start site) and the downstream
NF-B (5'-AGCTTGGAAATTCCGGAGCTG-3', element underlined,
within -231 to -211 bases from the translation start site) (Fig. 1
). In the present study, we demonstrate
that thrombin-induced expression of ICAM-1 is regulated at the level of
transcription, and that this expression is mediated by binding of
NF-Bp65 to the downstream NF-B site of ICAM-1 promoter. These
data provide evidence linking the activation of the procoagulant,
thrombin, to induction of the inflammatory response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HUVEC were obtained from Clonetics (La Jolla, CA) and grown on
gelatin-coated flasks or plates in endothelial cell growth medium (EGM)
containing 10% FCS, and 3 mg/ml of endothelial-derived growth factor
from bovine brain extract protein. Human thrombin with an activity of
3170 NIH U/mg protein was purchased from Enzyme Research Laboratories
(South Bend, IN). All experiments, except where indicated, were made
using cells under eighth passage. Eahy926 cells, a hybrid cell line of
HUVEC and A549 cell line (derived from human lung epithelial type II
cells), were provided by Dr. C. J. Edgell (University of North
Carolina, Chapel Hill) and cultured as described (38). Eahy926 cells
retain endothelial morphology and express endothelial cell-specific
marker human factor VIII-related Ag (38), and upon stimulation with
TNF- these cells also express the endothelial cell-specific adhesion
molecule E-selectin (39). Confluent HUVEC or Eahy926 cells were starved
for 2 h in EGM containing 1–2% FCS or in RPMI containing 0.5%
FCS, respectively, and were then incubated in the same medium with
thrombin or TRAP for the times and at concentrations indicated in each
experiment.
Northern blot analysis
Total RNA was isolated from HUVEC with RNeasy kit (Qiagen,
Chatsworth, CA), according to manufacturer’s recommendations.
Quantification and purity of RNA were assessed by
A260/A280 absorption, and
an aliquot of RNA (20 µg) from samples with ratio above 1.6 was
fractionated using a 1% agarose formaldehyde gel. The RNA was
transferred to Duralose-UV nitrocellulose membrane (Stratagene, La
Jolla, CA) and covalently linked by UV irradiation using a Stratalinker
UV cross-linker (Stratagene). Human ICAM-1 (0.96-kb SalI to
PstI fragment) (40) and rat GAPDH (1.1-kb PstI
fragment) were labeled with [-32P]dCTP using the
random primer kit (Stratagene), and hybridization was conducted as
described (41). Briefly, the blots were prehybridized for 30 min at
68°C in QuikHyb solution (Stratagene) and hybridized for 2 h at
68°C with random primed 32P-labeled probes. After
hybridization, the blots were washed twice for 30 min at room
temperature in 2x SSC with 0.1% SDS, followed by two washes for 15
min each at 60°C in 0.1x SSC with 0.1% SDS. Autoradiography was
performed with an intensifying screen at -70°C for 12–24 h. The
signal intensities were quantified by scanning the autoradiograms with
a laser densitometer (Howtek, Hudson, NH) linked to a computer analysis
system (PDI Imageware Systems, Huntington Station, NY). The
nitrocellulose membrane was soaked for stripping the probe with boiled
water containing 0.1x SSC with 0.1% SDS.
Reporter gene constructs, endothelial cell transfection, and luciferase assay
The ICAM-1LUC reporter plasmid and its 5' deletion derivatives
have been described (36). The constructs containing 600 bp of ICAM-1
promoter with wild-type (pGL2-WT) and mutated versions of Sp-1
(pGL2-Sp-1-MU, C/EBP (pGL2-C/EBP-MU), and downstream NF-B site
(pGL2-NF-B-MU) (22) were provided by Dr. Z. Cao (Tularik, San
Francisco, CA). The plasmid pNF-BLUC containing five copies of
consensus NF-B site linked to a minimal E1B promoter-luciferase
reporter gene was purchased from Stratagene. HUVEC under the fifth
passage or Eahy926 cells were plated into six-well Primaria culture
dishes 18–24 h before transfection. Transfections of HUVEC (except for
the experiment shown in Fig. 8A) were performed using
superfect (Qiagen) according to manufacturer’s recommendations.
Briefly, reporter DNA (1 µg) was mixed with 5–7.5 µl of superfect
in 100 µl serum-free EGM (Clonetics, La Jolla, CA). We used 0.2 µg
pTKRLUC plasmid (Promega, Madison, WI) containing Renilla
luciferase gene driven by a constitutively active thymidine kinase
promoter to normalize transfection efficiencies. Since we did not
observe any significant difference in transfection efficiencies in the
initial experiments, we did not cotransfect the pTKRLUC construct in
the later experiments. After a 5–10-min incubation at room
temperature, 0.6 ml EGM containing 10% FCS was added, and the mixture
was applied onto the cells that had been washed once with PBS. Two to
three hours later, the medium was changed to EGM containing 10% FCS
and the cells were grown to confluency.
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Nuclear extract preparation
After appropriate treatments, cells were washed twice with ice-cold Tris-buffered saline (TBS) and resuspended in 400 µl of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF). After 15 min, Nonidet P-40 was added to a final concentration of 0.6%. Nuclei were pelleted and resuspended in 50 µl of buffer C (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). After 30 min at 4°C, the lysates were centrifuged and supernatants containing the nuclear proteins were transferred to new vials. The protein concentration of the extract was measured using a Bio-Rad protein determination kit (Bio-Rad, Hercules, CA).
Electrophoretic mobility shift assays (EMSA)
EMSA were performed as described (42). Briefly, 10 µg of
nuclear extract was incubated with 1 µg of poly(dI-dC) in a binding
buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM DTT, and 10%
glycerol (20 µl final volume)) for 15 min at room temperature. Then
end-labeled double-stranded oligonucleotides containing the downstream
NF-B site of ICAM-1 promoter (30,000 cpm each) were added in the
absence or presence of 25- or 100-fold molar excess cold competitor,
and the reaction mixtures were incubated for 15 min at room
temperature. In Ab supershift experiments, nuclear extracts were
incubated for 15 min at room temperature with polyclonal rabbit Ab to
human NF-B proteins (p65 [RelA], p50, p52, c-Rel, and RelB)
(obtained from Santa Cruz Biotechnology, Santa Cruz, CA) at a
concentration of 2 µg/20 µl before incubation with the labeled
probe for another 15 min at room temperature. The DNA-protein complexes
were resolved in 5% native polyacrylamide gel electrophoresis in low
ionic strength buffer (0.25x Tris-borate-EDTA). Oligonucleotides used
for the gel shift analysis were as follows: ICAM-1 NF-B,
5'-AGCTTGGAAATTCCGGAGCTG-3'; mut-ICAM-1 NF-B,
5'-AGCTTccAAATTCCGGAGCTG-3'.
The oligonucleotide designated as ICAM-1NF-B represents a 21-bp
sequence of ICAM-1 promoter encompassing the downstream NF-B binding
site located 223 bp upstream of translation initiation site (22, 23).
The oligonucleotide mut-ICAM-1NF-B is the same as ICAM-1NF-B,
except that it has 2-bp mutations in NF-B site. Sequence motifs
within the oligonucleotides are underlined and the mutations are shown
in lower case.
Flow cytometry analysis
Flow cytometry analysis was perfomed as described (42). Briefly, HUVEC monolayers in six-well Primaria tissue culture dishes were stimulated with thrombin for various time points. After completion of incubation period, cells were washed twice with cold PBS, removed by careful trypsinization, and washed again with Ca2+/Mg2+-free PBS before incubating with 20% horse serum for 30 min. Following two washes, cells were incubated with a mouse mAb directed against human ICAM-1, BIRR0001 (kindly provided by Dr. Robert Rothlein, Boeringer Ingleheim, Ridgefield, CT) (43), in Ca2+/Mg2+-free PBS containing 3% horse serum for 30 min at 4°C. Cells were then washed twice with PBS/horse serum and incubated for 30 min at 4°C with a goat anti-mouse IgG FITC-conjugated secondary Ab. Cells were then fixed with 2% paraformaldehyde, and analyzed by flow cytometry in a FACScan cytofluorometer (Becton Dickinson, Mountain View, CA), and the results were gated for mean fluorescence intensity above the fluorescence produced by the secondary Ab alone.
PMN adhesion assay
PMN adhesion assay was performed as described (9). Briefly, confluent HUVEC monolayers in 24-well plates were incubated with thrombin for 2 or 6 h. At the end of each incubation period, HUVEC were fixed with 1% paraformaldehyde/PBS at room temperature for 15 min, and washed three times with DMEM without serum. The 51Cr-labeled human PMN (2 x 106 cells/ml DMEM) were distributed at 1 ml/well over the HUVEC and coincubated for 1 h at 37°C in 5% CO2 and 98% humidity.
To determine the contribution of ICAM-1 in thrombin-induced PMN adherence, HUVEC were treated with RR1/1 (mAb to ICAM-1) 15 min before 51Cr-labeled PMN administration at a concentration of 10 µg/ml. HUVEC monolayers were then gently washed three times with DMEM without serum to remove the nonadherent PMN. Endothelial monolayers were kept overnight in 1 ml of 1 N NaOH at 4°C. The cell lysates were scraped, collected in polypropylene tubes, and counted for radioactivity in a Tm analytical gamma counter. Phase-contrast microscopy confirmed HUVEC integrity and PMN adherence to HUVEC.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously showed that thrombin increased endothelial
adhesivity toward PMN within 0.5 h and that this response was
associated with type I activation of ICAM-1 expression, that is, this
early response was protein synthesis inhibitor cycloheximide (CHX)
insensitive and did not require increased ICAM-1 mRNA expression (9).
To examine whether thrombin-induced ICAM-1 expression is initially
independent of de novo ICAM-1 protein synthesis, but later becomes
protein synthesis dependent, we determined the time course of
thrombin-induced ICAM-1 expression on endothelial cell surface as
assessed by flow cytometry (Fig. 2).
ICAM-1 expression increased within 6 h of thrombin challenge and
continued to increase further at 12 and 24 h (Fig. 2). TNF-,
used as a positive control, also induced cell surface expression of
ICAM-1 (Fig. 2).
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). Approximately 50% of the increased
PMN adherence to HUVEC at 6 h was CHX sensitive (Fig. 3),
consistent with the finding that 50% of ICAM-1 expression at 6 h
was CHX sensitive (data not shown). The CHX sensitivity of PMN
adhesion in the 6-h thrombin-stimulated cells (Fig. 3) was in
contrast to the thrombin response at 2 h, which was insensitive to
CHX (9). Fig. 3 shows that pretreatment of HUVEC with mAb RR1/1 reduced
PMN adherence at 6 h by 80%, indicating a role of ICAM-1 in
mediating thrombin-induced PMN adhesion.
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Thrombin induces ICAM-1 mRNA expression
To determine whether protein synthesis-dependent expression of
ICAM-1 is preceded by an increase in ICAM-1 mRNA expression, Northern
blot analysis was performed to measure the abundance of ICAM-1
transcript, up to 24 h, after addition of thrombin (2.5 U/ml) to
the medium. Thrombin induced ICAM-1 mRNA expression in a time-dependent
manner, with maximum induction occurring between 2 and 4 h after
thrombin challenge, followed by a 55% decrease at 8 h, and
returned to the basal level by 24 h (Fig. 4, A and B).
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We evaluated the effects of TRAP, a 14-amino-acid peptide
representing the new NH2 terminus of PAR-1 generated after
thrombin cleavage, to determine whether the induction of ICAM-1 mRNA
expression requires activation of PAR-1. Both thrombin and TRAP induced
the expression of ICAM-1 transcript in a dose-dependent manner (Fig. 5, A and B). The
3.3-kb transcript increased slightly with 1 U/ml thrombin or 12.5 µM
TRAP; the maximum induction occurred with 5 U/ml or 50 µM TRAP (Fig. 5, A and B), indicating that thrombin-induced
ICAM-1 transcription occurs secondary to the cleavage of PAR-1.
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We used CHX to determine whether thrombin induction of ICAM-1
transcript was a direct effect of thrombin treatment or it required the
synthesis of additional proteins. The presence of CHX in the medium
during thrombin treatment of HUVEC did not prevent the thrombin-induced
ICAM-1 mRNA expression (Fig. 6),
suggesting that proteins necessary to mediate thrombin response were
already present in cells. CHX alone caused a slight induction of ICAM-1
transcript (Fig. 6), a characteristic of NF-B-dependent genes, since
CHX is known to activate nuclear transport of NF-B, presumably by
inhibiting synthesis of relatively labile IB proteins (24).
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B activation prevents thrombin-induced ICAM-1
mRNA expression
To further assess the role of NF-B in mediating ICAM-1
expression by thrombin, we used PDTC, an antioxidant that prevents
NF-B activation, and thereby its translocation to the nucleus
through its ability to chelate metal ions and deliver thiol groups to
cells (46). Confluent HUVEC monolayers were treated with PDTC for
0.5 h before stimulation with thrombin for 3 h. PDTC
prevented thrombin-induced ICAM-1 mRNA expression in a dose-dependent
manner (Fig. 7). These data suggest that
reactive oxygen species, produced during inflammatory response,
function as second messengers in activating NF-B and may mediate
ICAM-1 transcription in thrombin-stimulated endothelial cells.
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We assessed the effects of thrombin on transcriptional
activity of the ICAM-1 promoter to demonstrate that thrombin was
capable of activating ICAM-1 gene transcription. HUVEC were transfected
with a full-length wild-type construct containing 1393 bp of the ICAM-1
promoter linked to the firefly luciferase gene (ICAM-1 LUC). Thrombin
increased ICAM-1 promoter activity (Fig. 8
A). A similar but more
pronounced activation by thrombin or TRAP was observed in Eahy926 cells
(Fig. 8B). TNF- or PMA, used as positive controls, also
activated the promoter in these cells (Fig. 8, A and
B). Since transfection efficiency and the consequent
luciferase activity were 2- to 3-fold higher in Eahy926 cells, we used
these cells to localize the thrombin-responsive region within the
ICAM-1 promoter (as described below).
Localization of thrombin-responsive region within ICAM-1 promoter
The ICAM-1 promoter contains a number of cis-acting
elements of potential importance in mediating the activation of ICAM-1
gene (Fig. 1). We used a set of 5' deletion mutation constructs
containing different lengths of the ICAM-1 promoter linked to the
firefly luciferase reporter gene to localize the thrombin-responsive
elements. Deletion of ICAM-1 promoter sequences 393 bp upstream of the
ATG start codon decreased basal promoter activity (Fig. 9). AP-1, AP-1/Ets repeats, NF-B, and
AP-3 are the apparent elements in this region, indicating that these
sites are important for basal expression of construct 393 ICAM-1 LUC.
However, the expression of this construct was still strongly activated
by thrombin and TRAP, indicating that AP-1, AP-1/Ets, AP-3, and the
upstream NF-B site (533 bp upstream of translation start site) are
not essential for thrombin responsiveness (Fig. 9). Thrombin
responsiveness was lost upon deletion from position 393 to 176 bp,
suggesting that cis-regulatory elements responsible for
thrombin response reside within this region (Fig. 9). Potential binding
sites for AP-1 (tetradecanoylphorbol-13-acetate responsive element
(TRE)), SP1, C/EBP, and NF-B transcription factors are located
within this region.
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,
TRE-directed promoter activity did not increase significantly when the
transfected cells were exposed to thrombin or TRAP.
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B site prevents thrombin-induced
ICAM-1 promoter activation
To ascertain the role of Sp-1, C/EBP, and downstream NF-B sites
in mediating ICAM-1 promoter activation by thrombin, we transfected
HUVEC with pGL2-WT and -MU vectors containing wild-type and mutant
versions of Sp-1 (pGL2-Sp-1-MU), C/EBP (pGL2-C/EBP-MU), and downstream
NF-B (pGL2-NF-B-MU) site. Thrombin induced a 4-fold increase
in ICAM-1 promoter activity when pGL2-WT was used, whereas thrombin
failed to increase ICAM-1 promoter activity in cells transfected with
pGL2-NF-B-MU, which is the same as pGL2-NF-B-WT, except that it
has 2-bp mutations in downstream NF-B site (Fig. 10). However, Sp-1 and C/EBP sites do
not appear to be important, as the mutations in these sites failed to
prevent thrombin-induced luciferase activity in cells transfected with
pGL2-Sp-1-MU and pGL2-C/EBP-MU vectors. These results indicate that the
downstream NF-B site is critical in mediating thrombin-induced
activation of ICAM-1 promoter.
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B activation is essential to mediate thrombin response
To determine whether activation of NF-B site is necessary
to confer thrombin inducibility of ICAM-1 gene, we transfected HUVEC
with a plasmid pNF-BLUC containing five copies of consensus NF-B
sequence from Ig gene linked to a minimal adenovirus E1B
promoter-luciferase reporter gene. As shown in Fig. 11, NF-B-directed promoter activity
increased 3-fold when the transfected cells were exposed to
thrombin. These data indicate that NF-B sequence alone is capable of
mediating thrombin response.
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B site of
ICAM-1 promoter
To determine whether the downstream NF-B site of ICAM-1
promoter is capable of binding thrombin-activated DNA-binding proteins,
we synthesized an oligonucleotide containing this sequence, prepared
nuclear extracts from thrombin-stimulated HUVEC, and determined the
binding activity by EMSA. Fig. 12 shows
that thrombin-induced DNA-binding activities, when assayed on
nondenaturing gel electrophoresis, resolved into three closely
migrating bands. We performed competition experiments to determine the
specificity of these binding activities. These DNA-binding activities
were competed by specific oligonucleotide probe, ICAM-1NF-B
(lane 4), yet remained intact when challenged with an
oligonucleotide (mut-ICAM-1NF-B) bearing 2-bp substitution in the
downstream NF-B site of ICAM-1 promoter (lane 5).
The fast migrating activities (bands 2 and 3) are nonspecific, as
evidenced by the appearance of these bands when the same nuclear
extract was tested for its ability to bind to the mutant version of
downstream NF-B site (lane 6).
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B-binding complex. Incubation with Ab to p65
abolished the slowest migrating complex (band 1), with a concomitant
supershift, but did not affect the fast migrating protein:DNA complexes
(bands 2 and 3) (Fig. 13A).
Abs to p50, p52, c-Rel, or Rel B had no effect on any of these bands
(Fig. 13A). As a positive control, we used TNF-, which is
known to induce NF-Bp65 binding to the downstream NF-B site of
ICAM-1 promoter (23). As in the case of thrombin, Ab to p65 also
disrupted TNF--induced band 1, with a concomitant supershift,
whereas Abs to p50 or p52 did not have an effect (Fig. 13B).
To demonstrate that the failure of p50 or other Abs to elicit a
supershifted complex is the result of the absence of these proteins in
the NF-B complexes interacting at the ICAM-1 NF-B site rather
than the inability of these Abs to supershift under the conditions
used, we performed another control experiment in which anti-p50
indeed caused a supershift when mixed with purified p50 protein (data
not shown). Thus, these data indicate that thrombin exposure of HUVEC
results in a binding complex (as evidenced by band 1) containing the
p65 homodimer.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-dependent pathway. Thrombin stimulation of HUVEC
resulted in increased ICAM-1 mRNA and cell surface expression, and
consequently ICAM-1-dependent PMN adhesion to endothelial cells. The
expression of ICAM-1 message did not require novel protein synthesis,
as it was insensitive to CHX. The mRNA expression peaked between 2–4 h
after thrombin challenge, and returned to the basal level by 24 h.
EMSA, mutation/deletion construct reporter gene transfection assays,
and NF-B inhibition experiments indicated that binding of NF-B
p65 to the downstream NF-B site of ICAM-1 promoter mediated
thrombin-induced ICAM-1 transcription.
NF-B complexes containing the p65 subunit are known to play an
important role in the generation of an inflammatory response. Several
lines of evidence suggest that activation of downstream NF-B site is
necessary for thrombin induction of the ICAM-1 gene. The inability of
CHX, as well as the ability of PDTC to prevent the thrombin-induced
increase in ICAM-1 mRNA expression, is consistent with a
NF-B-dependent signaling pathway. Moreover, the slight induction of
ICAM-1 mRNA by CHX alone may be due to inhibition of IB synthesis by
CHX and is characteristic of NF-B-dependent genes (24). EMSA showed
that thrombin induced nuclear NF-B translocation and DNA-binding
activity. The binding complex consisted of NF-B p65 homodimer, as
shown by the supershift experiments. Inhibition of NF-B, either by
blocking its activation and thereby its nuclear translocation or by
mutating the downstream NF-B site, and thereby preventing binding of
activated NF-B to the ICAM-1 promoter, abolished thrombin-induced
increase in ICAM-1 mRNA expression and ICAM-1 promoter activity,
respectively.
Studies have shown that NF-B p65 mediates a number of
thrombin-induced cellular responses such as proliferation and cytokine
production in vascular smooth muscle cells (37, 48). Activation of
NF-B requires the phosphorylation either on serine or tyrosine
residues of IB, which sequesters NF-B in the cytosol. Many, if
not all, activators of NF-B induce serine phosphorylation of IB
that targets it for rapid polyubiquitination, followed by degradation
through 26S proteasome (28, 29, 30, 31). However, in certain settings such as
reoxygenation of hypoxic cells, tyrosine phosphorylation of IB
leads to NF-B activation without proteolytic degradation of IB
(32). We do not exclude any of these possible mechanisms for NF-B
activation following thrombin stimulation of endothelial cells.
The fact that TNF- or IL-1ß activates ICAM-1 expression via
NF-B, coupled with the observation that TNF-- or IL-ß-induced
ICAM-1 expression is rapid, with mRNA being detectable as early as
0.5 h, peaking at steady state level by 2 h (49) as opposed
to thrombin-induced ICAM-1 mRNA expression, which peaks between 2–4 h,
raises the possibility that the thrombin response may be secondary to
TNF- or IL-1ß expression. However, such a possibility is ruled out
by the findings that thrombin response was not prevented by the protein
synthesis inhibitor CHX (Fig. 6). In addition, Kaplanski et al. (12)
have recently shown that receptor antagonist, Abs, or antisense
oligonucleotides to IL-1ß, TNF-, or IL-1 failed to inhibit
thrombin-induced E-selectin and IL-8 gene expression in HUVEC.
Furthermore, IL-1, IL-1ß, and TNF- were not detected in the
supernatants of thrombin-activated HUVEC (12).
Analysis of ICAM-1 promoter showed that deletion of upstream NF-B
site (-533 bases from translation start site) did not prevent the
thrombin response. Thus, we did not examine whether this site can bind
p65 homodimer in response to thrombin or TRAP. We found that the
thrombin-responsive region (spanning from -393 to -176 bases from the
start codon) contained AP-1/TRE, SP1, C/EBP, and downstream NF-B
binding sites. Further analysis of this region revealed that TRE by
itself is not functionally important for thrombin activation of ICAM-1
gene; however, we do not exclude the possibility that it may cooperate
with other cis-acting elements such as NF-B to mediate
thrombin response. Interestingly, the close arrangement of C/EBP and
NF-B binding sites is reminiscent of the IL-8 gene promoter (50).
These sites have also been shown to be critically involved in the
mediation of TNF- signal to the ICAM-1 promoter (22). However, the
mutation of C/EBP or Sp-1 site failed to prevent thrombin-induced
activation of ICAM-1 promoter, suggesting that these sites are not
necessary to confer thrombin inducibility of ICAM-1 gene. Mutation of
downstream NF-B site, on the other hand, abrogated the response,
indicating that the downstream NF-B site is critical for
thrombin-mediated ICAM-1 gene transcription in endothelial cells.
Furthermore, the ability of thrombin to induce NF-B-mediated
activation of a minimal heterologous promoter established that NF-B
is essential for ICAM-1 expression in endothelial cells (Fig. 11).
The downstream NF-B site is also necessary for hypoxia-induced
cyclooxygenase-2 (COX-2) expression in HUVEC. Human COX-2 promoter is
characterized by the presence of two NF-B sites: the upstream
NF-B site (5'-GGGGATTCCC-3', -445 bp relative to transcriptional
start site) and the downstream NF-B site (5'-GGGGACTACC-3', -223 bp
relative to transcriptional start site). Schmedtj et al. (51) have
reported recently that the downstream NF-B site of COX-2 promoter is
necessary for hypoxia-mediated COX-2 gene transcription in endothelial
cells, whereas the upstream NF-B site has no effect.
PMN sequestration into the extravascular space is mediated by not only the action of chemotactic agents, but also by PMN adhesion to endothelial cells (52). We and others have shown that thrombin is a potent activator of PMN adhesion to endothelial cells through P-selectin (CD62P) and ICAM-1 expression. However, this phenomenon is rapid (occurring within 0.5 h) and does not involve induction of mRNAs for ICAM-1 and P-selectin, and occurred in the presence of CHX (9). In the present study, we demonstrate that the delayed thrombin response (occurring after 2 h) resulted in further increase in PMN adhesion and required de novo protein synthesis of ICAM-1. Thrombin also induces both IL-8 and E-selectin (CD62E) gene expression in endothelial cells, with similar kinetics as that of ICAM-1 (12). It should be noted that in the sequence of adhesion events, E-selectin mediates PMN rolling, whereas ICAM-1 contributes to firm arrest and IL-8 promotes transmigration of PMN across endothelial cells; hence, the ability of thrombin to induce these genes correlates with the findings that thrombin infusion produced PMN sequestration in pulmonary microvessels secondary to the attachment of PMN to vascular endothelial cells (53, 54). The resultant vascular injury and tissue inflammation were critically dependent on ICAM-1-mediated PMN adhesion to microvessel endothelial cells (55).
In summary, we demonstrate that in addition to its role in
intravascular coagulation, thrombin serves as a critical mediator of
the inflammatory process through its ability to induce activation of
NF-Bp65 and the expression of ICAM-1 and ICAM-1-dependent
endothelial adhesivity toward PMN. Thus, thrombin-induced ICAM-1
expression is an important linkage between the coagulation cascade and
inflammatory response, which may be important in the mechanism of
vascular endothelial adhesivity, PMN adhesion, and PMN migration across
the endothelial barrier.
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Acknowledgments |
|---|
B, CAAT
enhancer-binding protein, or promoter selective-1 site in intercellular
adhesion molecule-1 promoter. We also thank Drs. Michael Karin and
Elizabeth Nabel for their useful suggestions. We are grateful to Robert
Rothlein for anti-intercellular adhesion molecule-1 antibodies
(RR1/1 and BIRR0001). |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Arshad Rahman, Department of Pharmacology, College of Medicine, University of Illinois, 835 South Wolcott Avenue, Chicago, IL 60612-7343. E-mail address:
3 Abbreviations used in this paper: PAR-1, protease-activated receptor-1; C/EBP, CAAT enhancer-binding protein; CHX, cycloheximide; COX-2, cyclooxygenase-2; EGM, endothelial growth medium; IB, inhibitory protein-B; LUC, luciferase; PDTC, pyrrolidinedithio-carbamate; PMN, polymorphonuclear leukocyte; RLU, relative light unit; Sp-1, promoter selective-1; TRAP, thrombin receptor activation peptide; TRE, tetradecanoylphorbol-13-acetate responsive element.
Received for publication July 31, 1998. Accepted for publication February 9, 1999.
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