From the Departments of Medicine and Biochemistry & Molecular Biology, W. K. Warren Medical Research Institute, University of Oklahoma Health Sciences Center, and Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104
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ABSTRACT |
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Tumor necrosis factor- (TNF-
) or
lipopolysaccharide (LPS) increases expression of the P-selectin gene in
murine, but not in human, endothelial cells. These mediators augment
expression of a reporter gene driven by the murine, but not the human,
P-selectin promoter in transfected endothelial cells. The regions from
593 to
474 and from
229 to
13 in the murine P-selectin promoter are required for TNF-
or LPS to stimulate reporter gene expression. Within these regions, we identified two tandem
B elements, a reverse-oriented
B site and a variant activating transcription factor/cAMP response element (ATF/CRE), that participate in TNF-
- or
LPS-induced expression. The tandem
B elements bound to NF-
B heterodimers and p65 homodimers, the reverse-oriented
B site bound
to p65 homodimers, and the variant ATF/CRE bound to nuclear proteins
that included activating transcription factor-2. Mutations in each
individual element eliminated binding to nuclear proteins and decreased
by 20-60% the TNF-
- or LPS-induced expression of a reporter gene
driven by the murine P-selectin promoter in transfected endothelial
cells. Simultaneous mutations of all elements further decreased, but
did not abolish, induced expression. Co-overexpression of p50 and p65
enhanced murine P-selectin promoter activity in a
B
site-dependent manner. These data indicate that the
B
sites and the variant ATF/CRE are required for TNF-
or LPS to
optimally induce expression of the murine P-selectin gene. The presence of these elements in the murine, but not the human, P-selectin gene may
explain in part why TNF-
or LPS stimulates transcription of
P-selectin in a species-specific manner.
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INTRODUCTION |
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Tumor necrosis factor-
(TNF-
)1 and LPS are
mediators that increase expression of many proteins in a variety of
cells. Either agent stimulates endothelial cells to synthesize diverse
proteins that participate in inflammation or coagulation (1, 2). These
include the adhesion molecules E-selectin, vascular adhesion molecule-1, intercellular adhesion molecule-1, and mucosal addressin cell adhesion molecule-1; the cytokines IL-1, IL-6, and IL-8; and the
coagulation protein tissue factor (3-10). TNF-
and LPS induce
expression through both transcriptional and post-transcriptional mechanisms (2, 6, 7, 11). Transcriptional activation requires specific
combinations of basally expressed and signal-regulated transcription
factors (2, 12). The best characterized transcription factors regulated
by signaling through TNF-
and LPS are NF-
B/Rel proteins and
proteins activated by MAP kinases.
In mammals, the NF-B/Rel family includes NF-
B1 (p50), NF-
B2
(p52), RelA (p65), c-Rel, and RelB (13). The DNA-binding forms of
NF-
B/Rel proteins are homodimers or heterodimers that recognize
decameric
B elements. Most
B elements have incomplete dyad
symmetry and have a characteristic 5' to 3' orientation relative to the
transcriptional start site. The prototypical NF-
B heterodimer (p50/p65) recognizes
B elements with the consensus 5'-GGGRNNYYCC-3'. However, some variant
B sites are recognized only by specific homodimeric or heterodimeric combinations of NF-
B/Rel proteins (14-17). The p65-containing dimers are retained in the cytoplasm through complex formation with I
B
and related proteins. Upon cellular stimulation by TNF-
or LPS, I
B-
is degraded. Dimers containing p65 then migrate into the nucleus and activate many genes
with
B elements, including the gene encoding I
B-
(18, 19). The
newly synthesized I
B-
terminates the activity of p65-containing
dimers, resulting in post-inductional repression of transcription (20,
21).
TNF- or LPS signals the activation of the JNK and p38 MAP kinases
(22, 23), which translocate to the nucleus and phosphorylate substrates
that include ATF-2 and c-Jun (24-26). ATF-2 is a member of the
ATF/cAMP response element binding protein family of transcription factors; it functions as homodimers or heterodimers that recognize an
8-bp variant ATF/CRE site (5'-TGACATCA-3') (27, 28). c-Jun is a member
of the AP1 family of transcription factors; it functions as homodimers
or heterodimers that bind to a 7-bp AP1 site (5'-TGANTCA-3') (29).
Phosphorylation of ATF-2 and c-Jun proteins enhances the abilities of
dimeric complexes, notably ATF-2/c-Jun and c-Fos/c-Jun heterodimers, to
activate transcription (24, 26).
The pathways for activation of NF-B/Rel proteins and JNK/p38 MAP
kinases are evolutionarily conserved from insects to mammals (30, 31).
TNF-
and LPS also generally use conserved pathways to activate a
specific gene. For example, the LPS response element of the tissue
factor promoter contains two AP1 sites and a
B site that are
conserved in at least four different mammals (11). TNF-
-induced
expression of the E-selectin gene is mediated by an enhanceosome that
contains three
B sites, a variant ATF/CRE, and four A/T-rich
sequences that bind to the architectural HMG I(Y) proteins (32, 33).
All these elements are required for TNF-
or LPS to maximally induce
transcription, and they are conserved in both the human and murine
E-selectin genes (34).
By contrast, TNF- or LPS increases expression of the P-selectin gene
in murine endothelial cells but not in human endothelial cells
(35-37). In the preceding paper (38), we demonstrated that these
mediators augment expression of a reporter gene driven by the
murine, but not the human, P-selectin promoter in transfected endothelial cells. Furthermore, the sequences from
593 to
474 and
from
229 to
13 in the murine P-selectin promoter are required for
TNF-
or LPS to stimulate expression of the reporter gene (38). To
dissect the molecular basis for this unusual species-specific gene
activation event, we employed pharmacologic agents, DNA-binding experiments, transfection studies, and mutational analysis to characterize the regulatory elements and their cognate proteins that
contribute to TNF-
- or LPS-induced expression of the murine P-selectin gene. We identified two tandem
B sites, a
reverse-oriented
B site and a variant ATF/CRE, that participate in
TNF-
- or LPS-induced expression. These elements are not present in
the corresponding regions of the human P-selectin gene, which may
account in part for the species-specific response of the P-selectin
gene to TNF-
or LPS.
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MATERIALS AND METHODS |
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Reagents and Antibodies--
Recombinant human TNF- and ALLN
(calpain inhibitor I) were purchased from Boehringer Mannheim. PDTC,
cycloheximide, anisomycin, actinomycin D, and LPS from Salmonella
typhosa were obtained from Sigma. A stock solution of ALLN was
made in dimethyl sulfoxide (American Type Culture Collection) at a
concentration of 50 mM. Stock solutions of cycloheximide,
anisomycin, and actinomycin D were made in ethyl alcohol (Quantum
Chemical Co., Tuscola, IL) at a concentration of 5 mg/ml. Antibodies
against p50, p52, p65, c-Rel, RelB, ATF-2, c-Jun, and c-Fos were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Expression
plasmids encoding p50 or p65 were a generous gift from Dr. Craig Rosen
(39).
Gel Mobility Shift Assay--
Nuclear extracts from murine
bEnd.3 endothelioma cells or BAEC were prepared as described (40). Gel
mobility shift assays were performed as described (15). The sequences
of the oligonucleotides used in gel mobility shift assays are shown in
the figures. The oligonucleotides were 5' end-labeled by
[-32P]ATP using T4 polynucleotide kinase.
Construction of Chimeric Luciferase Expression
Vectors--
Plasmids mp1379LUC, p1379IBI, and p0LUC were described in
the preceding paper (38). Plasmids mpMutB, mpMutR
B, mpMutATF, mpMutDouble, and mpMutTriple, which carry mutations in
B and/or variant ATF/CRE as indicated in the text, were constructed in the
following two steps. 1) The KpnI-PstI or
PstI-SacI fragment in p1379IBI was replaced with
respective PCR products generated according to an overlap extension
protocol (41, 42). 2) The KpnI-HindIII fragment
(from
1379 to
13) that carried each mutation was excised and
inserted between the KpnI and HindIII sites of p0LUC. All constructs were confirmed by restriction mapping, and the
fidelity of the PCR-generated cassettes was verified by sequencing.
Cell Culture, Transfection, and Stimulation--
Murine bEnd.3
cells and BAEC were cultured as described (37, 42). Preparation of
plasmids, transfections and co-transfections, and luciferase assays
were described previously (15). The total amount of DNA for each
co-transfection was held constant at 8 µg/dish of cells by adding an
appropriate amount of a plasmid with a lacZ insert driven by
a cytomegalovirus promoter. For cell stimulation, culture medium
containing the indicated concentration of recombinant human TNF-,
LPS, ALLN, PDTC, cycloheximide, anisomycin, or actinomycin was added to
cells for the indicated time.
Northern Blot Analysis-- Total RNA was prepared from bEnd.3 cells by acid guanidinium thiocyanate/phenol/chloroform extraction (43). Northern blot analysis was performed as described (43), using previously characterized probes (37).
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RESULTS |
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TNF--induced Expression of P-selectin in Murine Endothelial
Cells Is Prevented by the Proteasome Inhibitor ALLN or the Antioxidant
PDTC but Superinduced by the Translation Inhibitor Cycloheximide or
Anisomycin--
In the preceding paper (38), we showed that TNF-
or
LPS requires the sequences from
593 to
474 and from
229 to
13
in the 5'-flanking region of the murine P-selectin gene to induce expression of a reporter gene in transfected endothelial cells. Inspection of these regions revealed several putative
B elements and
a variant ATF/CRE site, suggesting that NF-
B/Rel proteins and
ATF-2/c-Jun heterodimers may participate in inducible expression. As an
initial test of this hypothesis, we used the proteasome inhibitor ALLN
or the antioxidant PDTC that blocks activation of NF-
B, and the
translation inhibitor cycloheximide or anisomycin that prevents
resynthesis of I
B-
and activates JNK/p38 MAP kinases (44-46). We
incubated murine bEnd.3 endothelioma cells with TNF-
or LPS in the
absence or presence of a pharmacologic agent for various times. Levels
of P-selectin mRNA from each group of cells were then measured by
Northern blot analysis.
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Characterization of Two Tandem B Sites and a Reverse-oriented
B Site in the Murine P-selectin Promoter--
To identify sites for
binding to NF-
B/Rel proteins, we synthesized double-stranded
oligonucleotide probes encompassing each putative
B site from
593
to
13 in the 5'-flanking region of the murine P-selectin gene (Fig.
2A). Each labeled probe was
assessed for binding to nuclear proteins from endothelial cells
incubated with or without LPS or TNF-
.
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Characterization of a Variant ATF/CRE in the Murine P-selectin
Promoter--
A putative ATF/CRE in the murine P-selectin promoter is
identical in sequence to the ATF/CRE in the E-selectin gene and
deviates one nucleotide from the CRE consensus sequence (27, 28, 49). To test whether the P-selectin ATF/CRE competes with the other two
elements for binding to common nuclear proteins, we synthesized oligonucleotide probes encompassing the murine P-selectin ATF/CRE, the
human E-selectin ATF/CRE, or a CRE consensus sequence (Fig. 5A). A labeled probe
encompassing the P-selectin ATF/CRE formed a DNA-protein complex when
incubated with extracts from unstimulated bEnd.3 cells (Fig.
5B) or TNF--stimulated bEnd.3 cells (data not shown).
Relatively little complex formation was detected, consistent with a
possibly lower affinity of the variant ATF/CRE for nuclear proteins.
Complex formation was sequence-specific, as it was prevented by
addition of a 100-fold molar excess of the unlabeled probe but not of
an unrelated GATA element. Complex formation also required the ATF/CRE
sequence, because it was prevented by addition of a 100-fold excess of
an unlabeled probe encoding the E-selectin ATF/CRE or a CRE consensus
sequence. The labeled CRE consensus sequence probe formed two
complexes, A and B, when incubated with nuclear extracts from bEnd.3
cells (Fig. 5C). Formation of each complex was
sequence-specific, as it was prevented by addition of a 50-200-fold
excess of the unlabeled probe. Formation of complex A but not B was
significantly diminished by addition of a 50-200-fold excess of an
unlabeled probe encoding the P-selectin or E-selectin ATF/CRE. An
unlabeled probe containing mutations in the murine P-selectin ATF/CRE
or a probe containing the corresponding sequence from the human
P-selectin gene did not inhibit complex formation (Fig. 5C).
These data suggest that the murine P-selectin ATF/CRE, the E-selectin
ATF/CRE, and a CRE consensus sequence bind to common nuclear factors in
complex A.
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The Tandem B Sites, the Reverse-oriented
B Site, and the
Variant ATF/CRE Are Required for TNF-
or LPS to Maximally Induce
Expression of a Reporter Gene Driven by the Murine P-selectin Promoter
in Transfected Endothelial Cells--
To determine whether the tandem
B sites, the reverse-oriented
B site, and the variant ATF/CRE
allowed TNF-
or LPS to induce expression of murine P-selectin, we
mutated these elements, individually or in combination, in a reporter
construct driven by the murine P-selectin promoter. The mutations were
the same as those made in the mutant probes that eliminated binding to
nuclear proteins. Following transfection of the wild-type construct or
each mutant construct into BAEC, the cells were incubated in the
absence or presence of TNF-
or LPS for 4.5 h and then harvested
for assay of luciferase activity. Mutations in each individual element
decreased TNF-
- or LPS-induced expression by 20-60% relative to
that of the wild-type construct (Fig. 6).
Combined mutations in the reverse-oriented
B site and the variant
ATF/CRE or in all three elements further decreased, but did not
abolish, TNF-
- or LPS-induced expression. These data demonstrate
that the tandem
B sites, the reverse-oriented
B site, the variant
ATF/CRE, and still uncharacterized elements are required for TNF-
or
LPS to maximally induce expression of a murine P-selectin reporter gene
in transfected endothelial cells.
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A Reporter Gene Driven by the Murine P-selectin 5'-Flanking Region
Recapitulates Post-inductional Repression--
Since translocation of
p65-containing NF-B/Rel proteins into the nucleus is transient
because of feedback inhibition by newly synthesized I
B proteins (20,
21), NF-
B-dependent transcription usually declines after
its initial induction. We measured the activity of a luciferase
reporter gene driven by the murine P-selectin promoter in transfected
BAEC that were stimulated with TNF-
for various times. Because
luciferase protein and mRNA levels turn over rapidly, the kinetics
of luciferase activity accurately reflect the kinetics of
transcriptional activity of the reporter gene (51). Luciferase activity
increased at 3 h, reached a maximum at 4.5 h, then declined
rapidly and returned to a basal level by 13 h (Fig.
7). These data are consistent with
post-inductional repression of transcription of the murine P-selectin
gene.
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Co-overexpression of p50 and p65 Augments
B-dependent Expression of a Reporter Gene Driven by the
Murine P-selectin Promoter--
To test directly the role of
NF-
B/Rel proteins in inducing expression of murine P-selectin, we
co-transfected BAEC with plasmids encoding the NF-
B/Rel protein p50
or p65 with a reporter gene driven by the wild-type P-selectin promoter
or the promoter with mutations in the
B and ATF/CRE. Co-expression
of p50 alone slightly decreased promoter activity of the wild-type or
mutant construct. However, co-expression of increasing amounts of p65
with a fixed concentration of p50 markedly increased promoter activity
of the wild-type construct but not of the mutant construct (Fig.
8). Co-expression of p65 alone also
increased expression of the wild-type reporter gene, but to a lesser
extent than that elicited by co-expression of p65 with p50 (data not
shown). These data demonstrate that p65-containing NF-
B/Rel proteins
regulate
B-dependent expression of the murine P-selectin
gene.
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DISCUSSION |
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We identified and characterized two tandem B sites, a
reverse-oriented
B site and a variant ATF/CRE site, that are
required for TNF-
or LPS to optimally induce expression of the
murine P-selectin gene. The presence of these elements in the murine, but not human, P-selectin gene may help explain why TNF-
or LPS increases expression of P-selectin in murine, but not in human, endothelial cells.
In contrast to the unique B site in the human P-selectin gene that
binds p50 or p52 homodimers (15, 42), the three
B sites in the
murine gene bind inducible p65-containing NF-
B/Rel proteins. The
unique
B site in the human gene is required for optimal constitutive
expression of a reporter gene in transfected endothelial cells (15). In
contrast, the three
B sites in the murine gene are important for
TNF-
- or LPS-inducible gene expression but not for constitutive
expression. The interactions of p50 or p52 homodimers with the
proto-oncoprotein, Bcl-3, regulate the activity of the human
B site
(15), whereas p65-containing NF-
B/Rel proteins regulate the activity
of the three murine
B sites. These findings indicate that distinct
members of the NF-
B/Rel family proteins regulate transcription of
the murine and human P-selectin genes. The binding of newly synthesized
I
B
to p65-containing NF-
B/Rel proteins may explain the
post-inductional repression of reporter gene expression driven by the
murine P-selectin promoter. The stabilization of I
B
by the
proteasome inhibitor ALLN or the antioxidant PDTC may prevent TNF-
-
or LPS-induced expression of P-selectin mRNA in murine endothelial
cells. In contrast, the translational inhibitors cycloheximide or
anisomycin may superinduce expression by preventing the resynthesis of
I
B
.
The two tandem B elements in the murine P-selectin gene bind avidly
to NF-
B heterodimers and p65 homodimers, although they lack the
typical consensus sequence for binding to NF-
B/Rel proteins (13,
16). However, the tandem
B elements have striking sequence similarity to the two tandem
B sites in the human or murine
E-selectin genes (32-34, 52). The sequences flanking or overlapping
the
B elements in the E-selectin gene and the murine P-selectin gene have at least three A/T-rich sequences for binding to the architectural HMG I(Y) proteins (53). In the E-selectin gene, binding of HMG I(Y)
proteins to the A/T-rich sequences enhances binding of NF-
B heterodimers to the
B sites and facilitates interaction of NF-
B heterodimers with ATF-2/c-Jun proteins that bind to an adjacent ATF/CRE. These interactions contribute to formation of a highly organized enhanceosome (2, 32, 33). By analogy, the binding of HMG I(Y)
proteins to A/T-rich sequences in the murine P-selectin gene may
enhance binding of NF-
B to the tandem P-selectin
B sites and may
facilitate binding of NF-
B to the weak
B element located 13 base
pairs 3' to the tandem
B sites. Proteins binding to the P-selectin
tandem
B sites and the A/T-rich sequences may also cooperate with
other transcription factors to form an enhanceosome.
The reverse-oriented B site in the murine P-selectin gene binds
preferentially to p65 homodimers and is required for optimal TNF-
-
or LPS-induced expression of a reporter gene driven by the murine
P-selectin promoter. To our knowledge, this is the first reported
example of an asymmetric
B element with an opposite orientation
relative to the transcriptional start site. This element may function
because it binds to symmetrical p65 homodimers that do not require a
specifically oriented
B sequence to transactivate gene expression.
The identification of this unusual
B site further supports the
notion that p65 homodimers and NF-
B heterodimers have distinct
biological functions (54-56).
Mutation of the variant ATF/CRE in the murine P-selectin gene
eliminated binding to ATF-2 and other nuclear proteins, and it also
decreased the TNF-- or LPS-induced expression of a reporter gene
driven by the murine P-selectin promoter. This suggests that activation
of the JNK/p38 MAP kinases, which phosphorylate ATF-2, is required for
TNF-
or LPS to optimally induce expression of P-selectin. Combined
mutations of the
B sites and the variant ATF/CRE further decreased,
but did not abolish, the TNF-
- or LPS-inducible expression,
indicating that other regulatory elements also participate. Candidate
elements include three putative AP1 sites identified in the preceding
paper (38) and the low affinity
B site identified in this study.
Deletional analysis did not reveal a role for these elements in
TNF-
- or LPS-inducible expression of the murine P-selectin reporter
gene. However, deletions may alter other regulatory elements that
modulate the function of the deleted element.
TNF- and LPS use strikingly similar mechanisms to induce expression
of the murine P-selectin gene (this study) and the human and murine
E-selectin genes (2). Both genes use
B sites and a variant ATF/CRE
to transmit signals received from extracellular stimuli. The use of two
or more signal-regulated elements may allow optimal adjustment of gene
expression in response to a variety of challenges (12). The similar
pathways by which TNF-
and LPS regulate the E-selectin gene and the
murine P-selectin gene are consistent with the origin of the selectin
gene family by gene duplication (57). This may partially explain the
overlapping functions of P-selectin and E-selectin in the mouse
(58-60). It also predicts that agents such as ultraviolet light and
IL-1
, which activate NF-
B or JNK/p38 kinases (2), will stimulate expression of the murine, but not the human, P-selectin gene.
Our studies have the inherent limitation that cultured endothelial
cells may not contain the same transcription factors and signaling
proteins found in microvascular endothelial cells in vivo.
However, TNF- or LPS clearly augments expression of both P- and
E-selectin in murine endothelial cells in vivo (35, 36, 61,
62). By contrast, intradermal injection of LPS increases expression of
E-selectin, but not P-selectin, in venules of non-human primates (63).
Furthermore, E-selectin mRNA levels increase in the atria of
patients after cardiopulmonary bypass, whereas P-selectin mRNA
levels decline (64). These in vivo studies further support a
species-specific response of the P-selectin gene to TNF-
, LPS, or
related mediators. Thus, the mechanisms for regulating the inducible
transcription of the P-selectin gene appear to have evolved from mice
to humans. This species-specific fine tuning of gene expression may
extend to other inflammatory mediators and adhesion molecules. For
example, either IL-4 or oncostatin M induces expression of P-selectin
in murine and human endothelial cells. But induced expression in human
cells is delayed and requires new protein synthesis, whereas induced
expression in murine cells is more rapid and does not require new
protein synthesis (37).2 IL-4
suppresses the TNF-
-inducible expression of E-selectin in human
endothelial cells (65). IL-4 prevents E-selectin expression by
activating Stat6, which binds to a DNA element that overlaps one of the
B sites in the human E-selectin gene, thereby competitively inhibiting binding of NF-
B (66). The Stat6 element, however, is not
conserved in the murine E-selectin gene (34), suggesting that IL-4 may
not suppress TNF-
-inducible expression of E-selectin in mice.
Therefore, distinct mechanisms may be used to regulate expression of
selectin genes in different species.
Our results also suggest that the function assigned to a selectin in a
particular animal model may not necessarily apply to humans. In many
rodent models of inflammation, tissue injury or other insults generate
thrombin, histamine, oxygen-derived radicals, or other mediators that
stimulate endothelial cells to redistribute P-selectin from
Weibel-Palade bodies to the cell surface (67-69). Depending on the
specific challenge, TNF-, IL-
, or LPS may also be elaborated.
These agents cause murine endothelial cells to increase synthesis of
P-selectin, which may travel directly to the cell surface if the
machinery that sorts proteins into Weibel-Palade bodies is saturated.
Some models may induce expression of other cytokines such as IL-4 and
oncostatin M, which can also increase synthesis of P-selectin. The
relative contributions of these distinct pathways of inducible
expression may be difficult to distinguish in vivo. Our data
suggest that mediators that activate p65-containing NF-
B dimers or
ATF-2-containing dimers may increase transcription of the P-selectin
gene in rodents, but not in humans. Close scrutiny of the mechanisms
for inducing P-selectin expression in animal models may be necessary to
interpret the relevance of the findings for human biology.
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ACKNOWLEDGEMENTS |
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We thank Ginger Hampton for technical assistance and Dr. Craig Rosen for valuable reagents. We are grateful to Dr. James Morrissey for critical reading of the manuscript. We also thank Dr. Kenneth Jackson (Molecular Biology Resource Facility, University of Oklahoma Health Sciences Center) for synthesis of oligonucleotides.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: W. K. Warren
Medical Research Institute, University of Oklahoma Health Sciences Center, 825 N.E. 13th St., Oklahoma City, OK 73104. Tel.: 405-271-6480; Fax: 405-271-3137; E-mail: rodger-mcever{at}ouhsc.edu.
1
The abbreviations used are: TNF-, tumor
necrosis factor-
; ALLN,
N-acetyl-leucinyl-leucinyl-norleucinal-H; ATF, activating transcription factor; BAEC, bovine aortic endothelial cells; CRE, cAMP
response element; IL, interleukin; JNK, c-Jun N-terminal kinase; LPS,
lipopolysaccharide; MAP, mitogen-activated protein; PDTC, pyrrolidine
dithiocarbamate.
2 L. Yao and R. P. McEver, unpublished observations.
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REFERENCES |
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