From the Cardiology Division, Beth Israel Deaconess
Medical Center, and Harvard Medical School, Boston, the
§ New England Baptist Bone and Joint Institute, Beth Israel
Deaconess Medical Center, and Harvard Medical School, Boston, and the
¶ Department of Medicine, Brigham and Women's Hospital,
Boston, Massachusetts 02115
Received for publication, July 21, 2000, and in revised form, October 17, 2000
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ABSTRACT |
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Inflammation is a hallmark of several vascular
diseases. The nuclear factor Inflammation is a prominent feature of several vascular diseases.
The most common vascular disease, atherosclerosis, begins when
lipoproteins, and in particular low density lipoprotein, enter the
subendothelium and become oxidized. Oxidized low density lipoprotein
stimulates the production of interleukin-1 and other inflammatory
cytokines. These cytokines activate adhesion molecules, including
VCAM-1, ICAM-1, and E-selectin, on the endothelial surface, which
promote the attachment of and transmigration of monocytes. The
expression of the inducible form of nitric-oxide synthase (NOS2)1 has also been shown
to be up-regulated by inflammatory cytokines and endotoxin in cultured
cells found in the atherosclerotic plaque including macrophages, smooth
muscle cells, and endothelial cells (1-3). Furthermore,
immunohistological studies have demonstrated the expression of NOS2 in
the atherosclerotic lesions in these cell types as well (1, 4). The
induction of the NOS2 is also associated with more acute forms of
vascular inflammation such as endotoxemia. The generation of the potent
vasodilator nitric oxide by NOS2 is at least in part responsible for
the hypotension seen in association with bacterial sepsis (5, 6). NOS2
expression is also induced in other types of vascular inflammation
including restenosis and in the accelerated atherosclerosis associated
with heart transplantation (7, 8).
Upon binding of cytokines or other inflammatory mediators to their
corresponding receptors, several classes of transcription factors are
involved in the induction of these stimuli. For example, within minutes
of interleukin-1 The propagation of inflammation is dependent on several other
transcription factors for the activation of multiple genes. The
Rel/NF- The ETS genes are a family of at least 30 members that function as
transcription factors (19). All ETS factors share a highly conserved
80-90-amino acid DNA binding domain, the Ets domain. ETS
factors play a central role in regulating genes involved in development, cellular differentiation, and proliferation. Many macrophage, B-, and T-cell-specific genes are regulated by ETS factors.
The role of ETS factors in the immune system has been substantiated by
experiments in mice where the genes encoding several ETS factors have
been disrupted by homologous recombination. The PU.1 knockout is
characterized by a lack of immune system development (20). T-cell
apoptosis and increased terminal B-cell differentiation are
characteristics of the Ets-1 knockout mice (21).
We recently identified a novel member of the ETS factor ESE-1 that is
the prototype member of a new subclass of ETS factors (22). ESE-1 has
several interesting features when compared with other ETS family
members. First, unlike other ETS factors that are either ubiquitously
expressed or primarily expressed in lymphoid cells, ESE-1 appears to
have an epithelial specific expression pattern under basal conditions.
Second, unlike all other ETS factors, ESE-1 has two DNA binding
domains, a classical ETS domain and, in addition, an A/T hook domain
also found in HMG proteins. In this report we demonstrate that ESE-1 is
inducible in vascular smooth muscle cells, endothelial cells, and cells
of the monocyte-macrophage lineage, in response to inflammatory
stimuli. This induction appears to be mediated via NF- Cell Culture--
Primary human umbilical vein endothelial cells
and primary human smooth muscle cells were obtained from Clonetics and
grown according to the manufacturer's recommendations. THP-1 and RAW 264.7 (monocytic cell lines) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Rat aortic smooth muscle cells (RASMCs) were harvested from male Harlan
Sprague-Dawley rats by enzymatic dissociation according to the method
of Gunther et al. (23).
RNA Isolation and Northern Blot Analysis--
Total RNA was
isolated using the RNAeasy kit (Qiagen). Northern blots were hybridized
with random prime-labeled ESE-1 and glyceraldehyde-3-phosphate
dehydrogenase cDNA in QuickHyb solution (Stratagene) according to
the manufacturer's recommendations and were washed at 50 °C with
0.2× SSC, 0.2% SDS.
RT-PCR Analysis--
cDNAs were generated from 1 µg of
mRNA isolated from different cells or tissues using
oligo(dT)12-18 priming (Life Technologies, Inc.) and
Moloney murine leukemia virus-reverse transcriptase (Life Technologies,
Inc.) in deoxyribonuclease I (Life Technologies, Inc.)-treated samples.
Each PCR used equivalent amounts of 0.1 ng of cDNA, 4 ng/µl of
each primer, 0.25 units of Taq polymerase (Promega, Madison,
WI), 150 µM of each dNTP, 3 mM of
MgCl2, reaction buffer, and water to a final volume of 25 µl and were covered with mineral oil.
The sequences of the ESE-1 primers are as follows: sense,
5'-CTGAGCAAAGAGTACTGGGACTGTC-3', and antisense,
5'-CCATAGTTGGGCCACAGCCTCGGAGC-3', with an expected
amplification product of 188 bp.
The sequences of the primers for glyceraldehyde-3-phosphate
dehydrogenase are as follows: sense, 5'-CAAAGTTGTCATGGATGACC-3', and
antisense, 5'-CCATGGAGAAGGCTGGGG-3', with an expected amplification product of 200 bp.
RT-PCR amplifications were carried out using a PerkinElmer Life
Sciences thermal cycler 480 as follows: 20-30 cycles of 1 min at
94 °C, 1 min at 56 °C, and 1 min at 72 °C followed by 15 min
at 72 °C. Lower numbers of cycles were used to verify linearity of
the amplification signal. 10 µl of the amplification product was
analyzed on a 2% agarose gel.
In Vitro Transcription/Translation--
Full-length cDNA
encoding the whole open reading frame of p50 and p65 were inserted
downstream of the T7 promoter into the pCRII TA cloning
vector (Invitrogen). Coupled in vitro
transcription/translation reactions were performed as described
previously (24).
Electrophoretic Mobility Shift Assays--
These were performed
as described previously (25). In brief, 2 µl of in vitro
translation product and 0.1-0.2 ng of 32P-labeled
double-stranded oligonucleotide probes (5,000-20,000 cpm) in the
presence or absence of competitor oligonucleotides (1 and 10 ng) were
run on 4% polyacrylamide gels containing as buffer 0.5× TGE as described.
Oligonucleotides used as probes and for competition studies are as
follows: 1) murine PSP promoter wild type oligonucleotide, 5'-TCGACGAACATCCAGGAAATAGGGCTC-3' and
3'-GCTTGTAGGTCCTTTATCCCGAGAGCT-5'; 2) NOS2 ETS site,
5'-GGCGACCAGGAAGAGATGG-3' and 3'-CCATCTCTTCCTGGTCGCC-5'.
Expression Vector and Luciferase Reporter Gene
Constructs--
A 1516- and 265-bp fragment corresponding to
nucleotides Site-directed Mutagenesis--
Site-directed mutagenesis of the
NOS2 promoter was performed using the QuickChange
Site-directed Mutagenesis Kit (Stratagene) according to the
manufacturer's recommendations. In brief, PCR primers encoding the
NOS2 promoter ETS site, DNA Transfection Assays--
Cotransfections of 2 × 105 RAW cells or RASMCs were carried out with 0.6 µg of
reporter gene construct DNA and 0.6 µg of expression vector DNA using
6 µl of LipofectAMINE (Life Technologies, Inc.) as described (27).
The cells were harvested 16 h after transfection and assayed for
luciferase activity. Transfections for every construct were performed
independently in duplicates and repeated 3 times with two different
plasmid preparations with similar results. Cotransfection of a second
plasmid for determination of transfection efficiency was omitted
because potential artifacts with this technique have been reported and
because many commonly used viral promoters contain potential binding
sites for ETS factors (28).
GST Pull-down Assay--
A series of GST-ESE-1 fusion proteins
were generated by PCR with specific primers to contain in frame
restriction enzyme sites and sequenced to confirm that there were no
mutations introduced by the PCR. GST-ESE-1 fusion proteins were
prepared as described previously (29).
[35S]Methionine-labeled and in vitro
translated full-length p50 and p65 were incubated with equal amounts of
GST-ESE-1 fusion proteins or GST on agarose beads in 200 µl of NETN
(0.5% Nonidet P-40, 1 mM EDTA, 20 mM Tris-HCl,
pH 8.0, 100 mM NaCl) for 3 h at 4 °C with gentle
shaking. Bound p50 or p65 proteins were then eluted after three
washings with NETN buffer and analyzed on a 12% SDS-polyacrylamide gel.
Rodent Model of Endotoxemia--
The model used is as described
previously (30). In brief, C57/B6 mice were injected with
Salmonella typhosa LPS (20 mg/kg intraperitoneally). Aortas
were harvested at 4 h after injection, fixed, and stained for
ESE-1 protein expression as described below.
Immunohistochemistry--
Using the models described above, the
mice were euthanized after 4 h of LPS treatment, and the blood
vessels were perfusion-fixed with 4% paraformaldehyde via the left
ventricle. Following fixation, the blood vessels were
paraffin-embedded. Deparaffined 5-µm sections were incubated with a
rabbit polyclonal anti-ESE-1 antibody. The primary antibody was applied
at a concentration of 1/200 for 1 h at room temperature and then
left overnight at 4 °C. After washing, sections were incubated with
a biotinylated goat anti-rabbit antibody (Vectastain ABC, Vector Labs,
Burlingame, CA) at 1.5 µg/ml in phosphate-buffered saline, 0.4%
Triton X-100 for 1 h at room temperature. After they were
incubated with avidin, the sections were developed with a peroxidase
3,3'-diaminobenzadine kit (Vector Laboratories). The ESE-1 antibody was
generated in our laboratory, and the NOS2 antibody was obtained from
Santa Cruz Biotechnology.
ESE-1 Is Inducible in Response to Inflammatory Stimuli--
We
have previously shown that under basal conditions ESE-1 expression is
restricted to cells of epithelial origin (22). To examine whether ESE-1
could be induced in nonepithelial cells, ESE-1 expression was evaluated
in human aortic smooth muscle cells, human umbilical endothelial cells,
and the human monocyte-macrophage cell line (THP-1). As shown in Fig.
1A, ESE-1 expression is
induced in response to the inflammatory cytokines interleukin-1 The ESE-1 Promoter Is Inducible in Response to Inflammatory
Cytokines--
We have previously isolated and characterized the human
ESE-1 promoter (31). As shown in Fig.
2A, the ESE-1
promoter contains a putative NF- ESE-1 Induction Is NF- NOS2 Is a Potential Target Gene for ESE-1--
In an attempt to
identify target genes for ESE-1, the ability of ESE-1 to transactivate
the promoters of several genes that are induced in response to
inflammatory stimuli were tested. As shown in Fig.
4A, ESE-1 significantly
transactivates the NOS2 promoter (5-fold) but not
E-selectin, CD44, or the interleukin-6 promoters.
Transactivation of the ICAM-2 promoter by ESE-1 led to a
3.5-fold induction. The promoter of another gene, Flt-1, which is known to be regulated by ETS factors, Flt-1, but is not induced in response to inflammatory cytokines was used as another control was mildly induced by ESE-1. We have also recently identified the COX-2 gene as another target for
ESE-1.2 The NOS2
gene promoter has two important regulatory regions, one that is
upstream ( ESE-1 Can Bind to an ETS Site within the NOS2 Promoter--
By
having identified putative targets for ESE-1, we were also interested
to determine whether ESE-1 could specifically bind to ETS sites within
the NOS2 promoter. An ETS-binding site was identified that
is highly homologous to the ETS consensus binding site for ESE-1 (33).
By using in vitro translated ESE-1 protein, EMSA was
performed comparing the potential binding of ESE-1 to the NOS2 ETS site
with an ETS site in the epithelial specific PSP gene
previously shown to bind to ESE-1 (33). As shown in Fig.
5, ESE-1 binds equally well to both ETS
sites.
Mutational Analysis of the NOS2 Promoter ETS Site--
To
determine the functional importance of the NOS2 promoter
ESE-1-binding site ( ESE-1 Synergizes with p50 and p65 to Transactivate the NOS2
Promoter--
Because p50 and p65 are also activated in response to
inflammatory stimuli, we were interested to examine whether the
interaction of the Rel domain proteins with ESE-1 has any functional
effect upon transactivation. Cotransfection experiments with ESE-1,
p50, and p65 were performed with the NOS2 promoter in
RAW264.7 and rat aortic smooth muscle cells. As shown in Fig.
7, although both ESE-1 and the
combination of p50 and p65 were able to transactivate the
NOS2 promoter, there was a marked synergistic response with the combination of p50, p65, and ESE-1 in both cell types tested. Thus,
ESE-1 substantially augments the NF- ESE-1 Binding to Rel Domain Proteins--
It has previously been
shown that both ETS factors and HMG proteins are capable of binding to
the Rel family members (35, 36). Interestingly, the binding regions
where these protein-protein interactions occur are within the DNA
binding domain of both types of transcription factors. For the HMG(I)Y
protein, this region of interaction has been precisely mapped within
one of the A/T hook domains (36). There is significant protein sequence
homology between the A/T hook domain of ESE-1 and the region
responsible for binding with p50 in the HMG(I)Y protein. Although the
ETS domain is known to interact with p50, the precise region within this domain has not been determined. To identify whether ESE-1 can bind
to either the p50 or p65 Rel domain proteins,
[35S]methionine-labeled p50 and p65 were generated by
in vitro translation (See Fig.
8A). Several GST-ESE-1 fusion
constructs and the GST protein alone were made. A diagram of the
constructs is shown in Fig. 8C. The GST fusion binding
studies demonstrate that not only does the full-length ESE-1 protein
bind to p50 but both the A/T hook and the ETS domain were capable of
interacting with p50 (Fig. 8B). In contrast, there was no
significant binding of any of the constructs to p65 (data not
shown).
ESE-1 Is Induced in Acute Vascular Inflammation--
To examine
the vascular expression of ESE-1 in response to an inflammatory
stimulus, ESE-1 expression was evaluated by immunohistochemical analysis in a rodent model of endotoxemia in which marked vascular inflammation occurs within hours of endotoxin administration. As shown
in Fig. 9, ESE-1 expression was markedly
induced 4 h after exposure to endotoxin. Intense expression of
ESE-1 is shown in the vascular endothelium (see arrows) and
first layer of the vascular smooth muscle cells. Some expression is
also evident in some of the other layers of vascular smooth muscle
cells. In contrast, minimal to no ESE-1 expression was observed in the
mouse aorta at base line. To determine whether ESE-1 has a similar
expression pattern as NOS2 in this model of inflammation, we examined
the same aortic specimens using a NOS2-specific antibody. As shown in
Fig. 9, NOS2 is expressed in the endothelium and first layer of
vascular smooth muscle cells, similar to ESE-1 expression. These
studies confirm the inducibility of ESE-1 in response to inflammatory
stimuli, in vivo as well as in vitro, and further support the role of ESE-1 in regulating NOS2 expression.
The ETS transcription factor family has been shown to regulate a
wide variety of normal cellular responses, including cellular growth
and differentiation. The promoters of several genes that are induced in
response to inflammatory cytokines have conserved binding sites for ETS
factors that are functionally important. The tumor necrosis factor- NOS2 is also induced in response to inflammatory cytokines in vascular
smooth muscle cells, endothelial cells, and monocytes. ETS factors have
not previously been shown to be important for the inducibility of the
NOS2 gene. However, it has recently been determined that the
expression of the NOS1, which is predominantly found in neurons and has
generally been thought to be constitutively expressed, can be
up-regulated with nerve growth factor. Analysis of the NOS1
promoter demonstrates multiple potential ETS-binding sites in the
regulatory region responsible for induction; however, no specific ETS
factors have been identified that regulate this gene (45).
Interestingly, we have also recently determined that ESE-1 is inducible
in other nonepithelial cells in response to inflammatory cytokines,
including glial cells.2 Although NOS2 is generally
considered an inducible enzyme, which is not constitutively expressed,
NOS2 is highly expressed in fetal and adult bronchial epithelium (46).
Interestingly, we also determined the highest level of ESE-1 expression
in bronchial epithelium (22). The functions of nitric oxide in the
mature airways include smooth muscle relaxation, neurotransmission,
bacteriostasis, and modulation of plasma exudation, mucin secretion,
and ciliary motility. Constitutive expression of NOS2 has also recently
been demonstrated at lower levels in both gastric and colonic
epithelium and is enhanced in association with infection or neoplasia
(47, 48).
The induction of the murine NOS2 by endotoxin, IL-1 Unlike any other ETS factors, ESE-1 is unique in that it has an A/T
hook DNA binding domain in addition to the classical ETS domain. A/T
hook domains are found in another family of transcription factors, the
high mobility group (HMG) proteins. These factors are nonhistone
chromosomal proteins that alter chromatin structure by binding to
AT-rich DNA sequences (54). DNA binding of HMG proteins is mediated by
A/T hook DNA binding domains that often exist in tandem clusters of two
or three domains that facilitate stronger binding to two or more
tandemly placed A/T-rich DNA sequences. HMG proteins have recently been
implicated in enhancing inflammatory responses (30). In addition to
affecting DNA structure, they also act to enhance transcription by
recruiting additional proteins such as NF- The ability of other ETS factors to bind to the p50 subunit of NF- In summary, our study demonstrates that ESE-1 is a novel ETS factor
that is induced in response to inflammatory stimuli and augments
NF-B (NF-
B) transcription factors are
dimeric proteins involved in the activation of a large number of genes
in response to inflammatory stimuli. We report the involvement of a
novel member of the ETS transcription factor, ESE-1, in mediating
vascular inflammation. ESE-1 is induced in response to inflammatory
cytokines and lipopolysaccharide in vascular smooth muscle cells,
endothelial cells, and cells of the monocyte-macrophage lineage. This
induction occurs within hours of stimulation and is mediated by NF-
B
transactivation of the ESE-1 promoter. We have
identified the inducible form of nitric-oxide synthase (NOS2) as a
putative target for ESE-1. ESE-1 can bind to the p50 subunit of
NF-
B, and cotransfection of ESE-1 with the p50 and p65 subunits of
NF-
B synergistically enhances transactivation of the
NOS2 promoter by ESE-1. An ESE-1-binding site within the
NOS2 promoter has been identified, the site-directed mutagenesis of which completely abolishes the ability of ESE-1 to
transactivate the NOS2 promoter. Finally, in a mouse model of endotoxemia, associated with acute vascular inflammation, ESE-1 is
strongly expressed in vascular endothelium and smooth muscle cells. In
summary, ESE-1 represents a novel mediator of vascular inflammation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(IL-1
) treatment, the expression of the
immediate early genes c-fos and c-jun are
induced. These transcription factors are the constituent proteins for
AP-1 (9, 10). One of the target genes of IL-1
, the collagenase gene, can be activated by AP-1 alone (11). Multiple signaling pathways have
been implicated in the activation of these immediate early genes by
IL-1
including the Janus kinases, mitogen-activated protein kinases,
and protein kinase A (12-16).
B transcription factor family is another set of genes, members of which are critical mediators of gene expression during inflammation. Activation of these factors does not require protein synthesis, as these factors are sequestered in the cytoplasm bound noncovalently to I
-B proteins, their endogenous inhibitors. Upon stimulation by inflammatory cytokines or endotoxin, these inhibitors are proteolytically degraded, allowing NF-
B to be translocated to
the nucleus where it binds to the regulatory regions of target genes as
a heterodimer. Although originally described as being important in
lymphoid cells and lymphoid-specific genes, NF-
B has clearly been
shown to play an important role in a whole host of other cell types and
target genes. The p50 and p65 subunits of NF-
B bind to other
transcription factors through protein interactions often resulting in
synergistic transactivation of the target genes of NF-
B (17, 18)
B. ESE-1 is
able to bind directly to the p50 subunit of NF-
B and can augment the
NF-
B-mediated activation of genes that are induced during
inflammation. Finally, we have identified the NOS2
gene as a target for ESE-1 and have demonstrated the induction of ESE-1
in the mouse aorta in vivo during acute inflammation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1485 to +31 and
234 to +31 of the murine
NOS2 gene promoter were subcloned into the PGL2 luciferase
reporter (Promega) (26).
190 to
180, and flanking
sequences, with TTAA substituted for GGAA were used. PCR
was performed with Turbo polymerase using the wild type NOS2 promoter luciferase reporter construct as a template. The PCR reaction
was digested with DpnI and the undigested plasmids were transformed into DH5
bacteria. Individual minipreps were sequenced to verify incorporation of the ETS site mutation.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
TNF-
and endotoxin. Interestingly, depending on the cell type, ESE-1 expression was induced as early as 1 h in the THP-1 cells to as late as 4 h in the human aortic smooth muscle cells. In all cells tested ESE-1 expression was almost completely absent by 24 h after stimulation, suggesting that ESE-1 expression in these cell types is
strongly linked to stimulation by inflammatory stimuli. To confirm the
results obtained by RT-PCR, we performed Northern blot analysis of
ESE-1 using total RNA derived from human aortic smooth muscle cells
stimulated with interleukin-1
. As shown in Fig. 1B, ESE-1
expression is absent prior to induction with IL-1
, and expression
peaks about 4 h after stimulation.
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Fig. 1.
ESE-1 induction to inflammatory stimuli.
A, RT-PCR analysis of ESE-1 expression in human aortic
smooth muscle cells (HASMCs), human umbilical vein
endothelial cells (HUVECs), and the THP-1 monocytic cell
line at different time points after stimulation with IL-1 , TNF-
,
or LPS (see "Experimental Procedures" for details of PCR).
B, Northern blot analysis of ESE-1 induction in primary
human aortic smooth muscle cells. 10 µg of total RNA were used per
lane. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (See
"Experimental Procedures" for details of Northern blot
analysis.)
B-binding site, in addition to sites
for other known transcription factors such as AP-1, ETS, and CRE. To
test whether the ESE-1 promoter was responsive to an
inflammatory stimulus, a luciferase reporter construct containing the
ESE-1 promoter was transfected into the mouse monocytic RAW
264.7 cell line. As shown in Fig. 2B, the ESE-1
promoter is inducible to LPS in the RAW cells, and a mutation in the
NF-
B-binding site significantly reduces the inducibility suggesting
that binding of NF-
B or related Rel proteins is required for
inducible ESE-1 expression (Fig. 2C). The same results were
obtained in the rat aortic smooth muscle cells (data not shown).
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Fig. 2.
ESE-1 promoter and induction by
LPS. A, schematic diagram of the human ESE-1
promoter demonstrating conserved transcription factor binding sites
(ETS, NF- B, Ap-1, and CRE) and the TATA box. B, induction
of the ESE-1 promoter in RAW 264.7 cells in response to LPS.
C, effect of mutation in NF-
B site (mESE-1
promoter) upon activation by LPS.
B-mediated--
To identify the specific
proteins binding to the ESE-1 NF-
B site, EMSAs were
performed using whole cell extracts derived from human aortic smooth
muscle cells stimulated with IL-1
. As shown in Fig.
3A, Il-1
stimulation is
associated with an inducible change in binding pattern to the
ESE-1 NF-
B-binding site. Interestingly, this binding
occurs as early as 1 h, despite the fact that ESE-1 expression was
only detected in Il-1
stimulated HASMCs at about 4 h. To
identify the proteins specifically binding to this site, EMSAs were
performed in the presence or absence of antisera to the different Rel
family members. As shown in Fig. 3B, the only two proteins
recognized in this complex are p50 and p65. This suggests that the
induction of ESE-1 is mediated by inducible binding of the dimeric
NF-
B proteins p50 and p65 to the ESE-1 promoter.
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Fig. 3.
Electrophoretic mobility shift assays with
ESE-1 promoter NF- B
site. A, EMSA using an oligonucleotide probe encoding
the ESE-1 promoter NF-
B site, with whole cell extracts
derived from human aortic smooth muscle cells at different time points
(0, 1, 2, 4, 6, and 24 h). B, EMSA using ESE-1 NF-
B
oligonucleotide probe and whole cell extracts derived from
IL-1
-stimulated cell at 0 h (control) or 1 h, in the
presence or absence of antibodies to the different Rel family members.
An antibody to the unrelated Bcl-3 protein is used as a negative
control.
1100 to
800) and one that is in the downstream region
proximal to the transcription start site (
234 to +31). To test
whether ESE-1 transactivation occurred principally through the upstream
region or through the region near the transcription start site,
cotransfections were performed with ESE-1 and reporter constructs
encoding either a long fragment of the NOS2 promoter containing both regions or one containing just the downstream promoter
region. As shown in Fig. 4B, transactivation by ESE-1 was
similar with both NOS2 promoter constructs tested,
suggesting that most of transactivation by ESE-1 occurs through the
promoter region near the transcription start site (
234 to +31).
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Fig. 4.
ESE-1 transactivation of the promoters of
potential target genes. A, cotransfection of the
mammalian expression plasmid with luciferase reporter constructs of
different potential target genes, including CD44,
interleukin-6, NOS2, E-selectin, and ICAM-2.
B, cotransfection of ESE-1 with the long ( 1485 to +31) and
short (
233 to +31) forms of the murine NOS2
promoter.
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Fig. 5.
Electrophoretic mobility shift assay of ESE-1
with NOS2 promoter ETS site. EMSA of in
vitro translated ESE-1 with an oligonucleotide probe encoding
either the PSP promoter ETS site (lane 2) or the
NOS2 promoter ETS site (lane 4) compared with
unprogrammed reticulocyte lysate control (lanes 1 and
3).
190 to
180), site-directed mutagenesis of this
site, substituting "TTAA" for the core "GGAA" sequence, was performed. Two independent constructs were verified by DNA sequencing. The effect of this mutation upon the ability of ESE-1 to transactivate the mutated NOS2 promoter was first examined. As shown in
Fig. 6A, this mutation
completely abolished the ability of ESE-1 to transactivate the
NOS2 promoter. The functional importance of this mutation
was next evaluated in the context of induction with an inflammatory
stimulus. As shown in Fig. 6B, the same mutation led to a
60% reduction in inducibility of the promoter with LPS. This reduction
is similar to that seen with mutation of the NF-
B or OCT sites
(34).
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Fig. 6.
Mutational analysis of NOS2 ETS-binding
site. A, cotransfection of wild type NOS2
promoter or the mutated NOS2 promoter with pCI or pCI-ESE-1
mammalian expression vectors. B, evaluation of effect of ETS
site mutation on LPS induction.
B-mediated transcriptional response to inflammatory stimuli.
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Fig. 7.
Synergistic effect of
NF- B with ESE-1 upon NOS2
transactivation. Cotransfection experiments of different
combinations of the pCI mammalian expression plasmid containing
cDNAs encoding either ESE-1, p50, or p65, were performed in RAW
264.7 (A) or RASMCs (B), with the NOS2
promoter luciferase reporter construct (short).
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Fig. 8.
ESE-1 interacts with p50. A,
[35S]methionine in vitro translated rabbit
reticulocyte lysates of p50, p65, and unprogrammed lysate (control)
separated by SDS-gel electrophoresis. Molecular weight markers are
shown on the left. B, binding of p50 to ESE-1,
another ETS factor NERF-2, and several deletion constructs, and the GST
fusion protein alone (control). C, schematic of
ESE-1 constructs used in GST fusion experiments. (See "Experimental
Procedures" for details of the GST-pull down experiment.)
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Fig. 9.
Expression of ESE-1 in the mouse aorta during
acute inflammation. Immunohistochemical evaluation of ESE-1
protein expression in the mouse aorta before and 4 h after
systemic administration of endotoxin (see "Experimental Procedures"
for details). Control represents immunohistochemical
evaluation with ESE-1 antibody at base line prior to administration of
endotoxin. NOS2 staining is performed at the 4-h time point.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF-
) gene promoter contains ETS-binding sites that are
functionally important for inducibility by phorbol esters (37).
Induction of JunB by interleukin-6 has been demonstrated to require
binding to a conserved ETS site (38). The induction of both the
macrophage inflammatory protein-1 (MIP-1
) and the chemokine
regulated on activation normal T-cell expressed by inflammatory cytokines has been shown to be at least in part mediated by ETS factors
(39, 40). The specific ETS factors responsible for the regulation of
these genes has not been identified. The effect of inflammatory
cytokines on the expression of selected ETS factors has been variable.
Macrophage gene expression of the ETS factor Fli-1 is reduced in
response to LPS, IL-1
, or phorbol 12-myristate 13-acetate (41). In
contrast, Ets-1 expression could be increased in response to phorbol
12-myristate 13-acetate and platelet-derived growth factor in rat
vascular smooth muscle cells (42). The proposed targets for Ets-1
include the matrix metalloproteinases collagenase and stromelysin,
which contain ETS-binding sites in their respective promoters. TNF-
has also been shown to induce Ets-1 in vascular smooth muscle cells
(43). Finally, Ets-1 has also been shown to be induced in fibroblasts
in response to stimulation with basic fibroblast growth factor,
epidermal growth factor, and platelet-derived growth factor (44).
Although Ets-1 expression can be increased in response to a variety of
stimuli in these cell types, Ets-1 is already expressed at base line in
these cells. In contrast, the expression pattern of ESE-1 is quite
different at base line in that it is predominantly expressed in cells
of epithelial origin and completely absent in vascular smooth muscle cells, endothelial cells, or monocytes. The rapid and transient induction of ESE-1 suggests that the target genes for ESE-1 in these
cell types are more likely to be directly associated with mediating the
inflammatory response than for the other ETS transcription factors that
are already expressed at base line.
, and interferon
requires binding of specific transcription factors to regulatory
regions within the NOS2 promoter. An NF-
B site (
85 to
76) is critical for activation of NOS2 by LPS and inflammatory cytokines in macrophages and vascular smooth muscle cells (49, 50). An
upstream promoter/enhancer region (
951 to
911) is responsible for
the synergistic activation of NOS2 by interferon-
and LPS. The
specific transcription factors binding to this region include
interferon regulatory factor-1, and Stat1
(51, 52). The
transactivation of the proximal promoter (
234 to +31) in response to
LPS or interleukin-1
can only partially be blocked by mutating the
NF-
B site (34). Inducible binding of additional factors to other
sites within this region has been demonstrated by in vivo
footprinting experiments. These sites include an NF-IL6 (
150 to
142) site and an octamer (
61 to
54) site (53). Mutational analysis of the OCT site has demonstrated the functional importance of
this site (34). Furthermore, the transcription factor HMG-I(Y) has been
shown to bind to the OCT site and cooperatively interact with NF-
B
to form a ternary complex and potentiate NOS2 transcription (34). Our
results are the first demonstration that ETS factors may be able to
regulate NOS2 expression.
B, ATF-2/c-Jun, and
interferon regulatory factor-1 (34). The binding of the p50 subunit of
NF-
B has been shown to be mediated via one of these A/T hook domains
(36). HMG-I(Y) expression is induced in vascular smooth muscle cells in
response to inflammatory stimuli including endotoxin and
interleukin-1
(30). The A/T hook domain within ESE-1 may facilitate
mediation of the inflammatory response either by recruiting other
proteins such as p50 or by enhancing binding of ESE-1 or other factors by altering DNA structure through binding to A/T-rich sequences.
B
has been shown for other Ets factors including Ets-1 and ELF-1 (32,
35). For both of these Ets factors the interaction occurs via the
conserved Ets DNA binding domain. It is therefore not surprising that
the Ets domain of ESE-1 is one of the domains capable of interacting
with p50. The degree of synergism associated with cotransfection of Ets
factors with the p50 and p65 subunits of NF-
B upon transactivation
of the regulatory regions of different genes is variable. For example,
when the enhancer regions of the HIV-1 and HIV-2
genes, which are known to bind to Ets factors, were tested, there was a
maximal 4-5-fold total transactivation when cotransfection experiments
were performed with the combination of Ets-1, p50, and p65 (35). When
the same type of experiment was performed with the GM-CSF
promoter, this led to over a 100-fold activation of this promoter,
which was ~10-fold greater than with either Ets-1 or NF-
B alone
(32). Thus, the functional role of these interactions may be highly
dependent upon the particular target genes. For expression of NOS2,
there was a marked synergistic effect with the combination ESE-1, p50,
and p65.
B-mediated induction of gene targets associated with inflammation. In particular, we have identified the inducible isoform
of nitric-oxide synthase (NOS2) as a target for ESE-1.
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ACKNOWLEDGEMENTS |
---|
We acknowledge fruitful discussions with Dr. Phil Auron, Dr. Ellen Gravallese, Dr. Mary Goldring, and Dr. Steven Goldring. We thank Alphie Tsay for his photographic assistance.
![]() |
FOOTNOTES |
---|
* This study was supported by National Institutes of Health Grants RO1/HL63008, KO8/CA71429 (to P. O.), RO1/CA76323 (to T. A. L.), and HL60788 and GM53249 (to M. A. P.).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: Harvard Institutes
of Medicine, 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-667-3390; Fax: 617-975-5299; E-mail: joettgen@caregroup.harvard.edu.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M006507200
2 F. Grall, X. Gu, L. Tan, T. Thamrongsak, B. Choy, C. Manning, Y. Akbarali, P. Stolt, S. Goldring, E. Gravallese, M. B. Goldring, P. Oettgen, and T. A. Libermann, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
NOS2, nitric-oxide
synthase;
LPS, lipopolysaccharide;
IL-1, interleukin-1
;
RASMCs, rat aortic smooth muscle cells;
PCR, polymerase chain reaction;
RT-PCR, reverse transcriptase-PCR;
GST, glutathione S-transferase;
HMG, high mobility group;
EMSA, electrophoretic mobility shift assays;
bp, base pairs;
TNF-
, tumor necrosis factor-
.
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