The Ets transcription factor ESE-1 mediates induction
of the COX-2 gene by LPS in monocytes
Franck T. Grall, Wolf C. Prall, Wanjiang Wei, Xuesong Gu, Je-Yoel Cho, Bob K. Choy,
Luiz F. Zerbini, Mehmet S. Inan, Steven R. Goldring, Ellen M. Gravallese, Mary B. Goldring,
Peter Oettgen and Towia A. Libermann
New England Baptist Bone and Joint Institute and BIDMC Genomics Center, Beth Israel Deaconess Medical Center and Harvard Medical
School, Boston, USA
Cyclooxygenase (COX) is an enzyme that converts
arachidonic acid into the prostaglandin H2. This prod-
uct is the critical point of the synthetic pathway of
numerous members of the prostaglandin family. COX
exists as two major isoforms derived from two separate
genes: COX-1 and COX-2. COX-1 is constitutively
expressed, whereas COX-2 expression is inducible.
Pro-inflammatory substances are some of the major
activators of COX-2. Examples include interleukin
(IL)-1 [1], tumor necrosis factor (TNF)-a [2], and bac-
terial lipopolysaccharide (LPS) [3]. A third isoform
COX-3 has also been reported [4].
The mechanisms leading to COX-2 expression
involve various combinations of different transcription
factors, depending on the cell type and stimulus. The
members of the C⁄ EBP family have been identified as
Keywords
COX-2; ESE-1; Ets; gene expression; LPS
Correspondence
T. A. Libermann, New England Baptist Bone
& Joint Institute, Department of Medicine,
Beth Israel Deaconess Medical Center,
Harvard Institutes of Medicine, 4 Blackfan
Circle, Boston, MA 02115, USA

Ad, Adenovirus; ChIP, chromatin immunoprecipitation; CMV, cytomegalovirus; COX, cyclooxygenase; CRE, cAMP responsive element;
ESE, epithelium specific Ets factor; Ets, E26 transformation specific; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; HRP,
horseradish peroxidase; ICAM, intercellular adhesion molecule; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide;
MMP, matrix metalloproteinase; NFAT, nuclear factor of activated T cells; NF-jB, nuclear factor-jB; TNF, tumor necrosis factor.
1676 FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS
important regulators of COX-2 expression in osteo-
blasts [5], T lymphocytes [6], amnion epithelial cell
WISH [7], macrophages [8,9], and chondrocytes [10].
The nuclear factors of activated T cells (NFAT) are
essential for COX-2 activation in T lymphocytes [6].
The role of nuclear factor-jB (NF-jB) appears to be
more cell-specific. According to Allport et al. [7], the
mutation of the major NF-jB site of the COX-2 pro-
moter abolishes IL-1-induced COX-2 expression in the
human amnion epithelial cell line WISH. Crofford
et al. [11] successfully employed p65 antisense oligo-
nucleotides to inhibit IL-1-mediated induction of the
COX-2 promoter in synoviocytes. Furthermore, a frag-
ment of the COX-2 promoter starting downstream of
the NF-jB sites could not be activated by IL-1 in
chondrocytes [10]. However, in macrophages, Wad-
leigh et al. [9] mutated the NF-jB site of the murine
COX-2 promoter without loss of LPS mediated
COX-2 activation. The cAMP responsive element
(CRE) site overlapping an E-box element is another
important site for transcription factors as the mutation
of the CRE site within the COX-2 promoter or the
expression of a dominant negative mutant of CREB
reduces inducibility of the COX-2 promoter [9,12–14].
The transcription factors binding this site include

we show the capacity of a dominant-negative form
of ESE-1 to diminish COX-2 promoter induction in
response to LPS or IL-1 exposure.
Results
COX-2 induction by pro-inflammatory stimuli
correlates with ESE-1 induction
Our previous data had indicated that ESE-1 expression
is rapidly and transiently induced by pro-inflammatory
cytokines in a variety of vascular and connective tis-
sue cell types [20,21]. We also demonstrated that the
iNOS gene, a target for pro-inflammatory cytokines, is
a downstream target for ESE-1. To further our under-
standing of ESE-1 function during inflammatory
processes, we have now explored the involvement of
ESE-1 in the regulation of another inflammation-related
gene, COX-2, in monocytic cells and chondrocytes. We
previously demonstrated that LPS stimulation of
human monocytic THP1 cells leads to an induction of
ESE-1 mRNA expression within 1 h of exposure,
reaching a peak at 4 h and leveling off after 24 h [12].
To investigate the level of ESE-1 protein following
LPS exposure, we performed western blot analysis in
murine monocytic RAW 264.7 cells. The intensity of
the ECL signal was determined using the alphaease
fc sofware and divided by the protein concentration
of the sample. ESE-1 protein was detected 4 h after
LPS stimulation and increased levels were observed
until 10 h. Analysis of COX-2 protein expression in
response to LPS in RAW 264.7 cells by western blot
revealed that the temporal pattern of COX-2 pro-

promoter was not restricted to RAW 264.7 cells,
since transfection of pCI ⁄ ESE-1 also stimulated tran-
scription of the )170 bp COX-2 promoter in the
human chondrocyte cell line T ⁄ C28a2 (Fig. 2C), a
cell type shown to express ESE-1 in response to
IL-1 [21].
As another Ets factor, PEA3, has previously been
shown to activate the COX-2 promoter, we compared
the relative activities of ESE-1 and PEA3, cloned
downstream of the cytomegalovirus (CMV) promoter,
in a dose–response curve. Different amounts of ESE-1
and PEA3 expression vector DNA were cotransfected
with the COX-2 promoter luciferase construct, main-
taining the total amount of transfected DNA con-
stant by adding the parental pCI vector. As
illustrated in Fig. 2D, ESE-1 at all concentrations
was more effective than PEA3 in transactivating the
COX-2 promoter. This result does not appear to be
due to a higher production of the ESE-1 protein.
Western blot analysis of ESE-1 and PEA3 expression
after transfection of equal amounts of expression vec-
tor into 293ft cells, shows that ESE-1 protein expres-
sion is lower than PEA3 (Fig. 2E). Indeed we have
observed that generally ESE-1 protein expression after
transfection is significantly lower than most other Ets
factors.
ESE-1 binds to the human COX-2 promoter
To investigate whether this induction could be due to
a direct effect of ESE-1 on the COX-2 promoter, we
assessed the ability of ESE-1 to bind to the COX-2

COX-2 promoter in vivo.
A
B
Fig. 1. Induction of ESE-1 and COX-2 expression in monocytic
cells. RAW cells were grown in the absence or presence of LPS
(100 ngÆmL
)1
) for 0, 2, 4, 10 or 12 h. ESE-1 and COX-2 protein lev-
els were measured by western blotting using ESE-1 and COX-2
specific antibodies. (A) ECL signal on a photographic film. (B) Lev-
els of ESE-1 and COX-2 shown as integrated intensity divided by
the protein load for each lane.
ESE-1 activates COX-2 expression F. T. Grall et al.
1678 FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS
Mutation of multiple ESE-1 binding sites
drastically reduces activation of the COX-2
promoter by ESE-1 and by LPS
To examine whether the Ets sites in the COX-2 promo-
ter are responsive to ESE-1 and to determine whether
LPS induction of the COX-2 promoter is mediated via
ESE-1, we introduced mutations into individual or
multiple Ets sites of the COX-2 promoter. Wild type
or mutant constructs of pXP2 ⁄ COX-2–170 were
cotransfected into RAW 264.7 cells in the absence or
presence of pCI ⁄ ESE-1. These experiments indicated
that individual mutations of the Ets binding sites 2, 3
and 4 led to more than 50% reduction of ESE-1-medi-
ated COX-2 promoter activation (Fig. 4A). Simulta-
neous mutation of site 3 along with sites 1, 2 or 4
almost completely eliminated inducibility by ESE-1

was left when sites 1, 2, 3 and 5 were mutated in
combination.
LPS response was also significantly affected when
sites 3, 4 or 5 were mutated individually (Fig. 4B).
Combined mutation of the Ets sites 1, 2, 3, and 5,
leaving the NFAT element in site number 4 intact, led
to a drastic inhibition of promoter activation in
response to LPS (Fig. 4B). This experiment demon-
strates that LPS activation of the COX-2 promoter is
at least partially mediated via ESE-1 or a related Ets
factor.
The mutation of the C ⁄ EBPb site that inhibited the
activity of PEA3 [16] led to only a diminution of the
activity of ESE-1.
ESE-1 and NFAT act synergistically on the COX-2
promoter
As the NFAT factors have been reported as activators
of COX-2 [6], we evaluated whether ESE-1 and NFAT
could cooperate in the context of the COX-2 promoter.
The )170 COX-2 promoter luciferase construct was
cotransfected into RAW cells together with pCI ⁄ ESE-1
or a constitutively active form of one member of the
NFAT family, NFAT3, cloned into pRK5, or a combi-
nation thereof, and either empty pRK5 or pCI, respect-
ively (Fig. 5A). ESE-1 enhanced COX-2 promoter
activity 10-fold compared to only 2.5-fold activation by
NFAT3. Combined expression of ESE-1 and NFAT3
synergistically enhanced COX-2 promoter activity more
than 20-fold. These results indicate that ESE-1 and
NFAT3 most likely act via different sites or different

Fig. 4. ESE-1 and LPS transactivate the COX-2 promoter through
multiple Ets binding sites. (A) Mutation of multiple Ets binding sites
within the COX-2 promoter inhibits induction by ESE-1. RAW cells
were cotransfected with the pCI ⁄ ESE-1 expression vector and the
COX-2 promoter luciferase constructs containing either wild-type
(WT) or multiple mutants of potential binding sites (mut) alone or in
combination. Luciferase activity in the lysates was determined 16 h
later, as described elsewhere [23]. Data shown are means of dupli-
cate measurements from one representative transfection. The
experiment was repeated four times with different plasmid prepara-
tions with comparable results. Error bars represent SD of the two
replicates. (B) Mutation of the Ets binding sites reduces LPS-
induced transactivation of the COX-2 promoter. RAW cells were
transfected with the wild-type or Ets mutant COX-2 promoter luci-
ferase constructs and then stimulated with LPS. Luciferase activity
in the lysates was determined 16 h later. Error bars represent the
SD of the two replicates.
A
B
C
Fig. 5. ESE-1 cooperates with NFAT and NF-jB in transactivating
the COX-2 promoter. (A) RAW cells were cotransfected with the
pCI ⁄ ESE-1 expression vector or the pRK5 ⁄ NFAT3 expression vec-
tors or a combination thereof and the )170 COX-2 promoter luci-
ferase construct. Luciferase activity in the lysates was determined
16 h later, as described elsewhere [23]. Data shown are means of
duplicate measurements from one representative transfection. The
experiment was repeated twice with different plasmid preparations
with comparable results. Error bars represent the standard devi-
ation of the two replicates. (B and C) RAW cells were cotransfected

dominant-negative mutants of ESE-1 as tools to block
endogenous COX-2 gene expression. We constructed
two dominant-negative forms of ESE-1. One of these
two constructs, Dominant Negative 1 (DN1), encom-
passes the carboxy-terminal Ets DNA binding domain
of ESE-1 and competes with intact endogenous ESE-1
for binding to target gene promoters. The second
dominant-negative mutant, Dominant Negative 2
(DN2), encompasses the amino-terminal transactiva-
tion domain and Pointed domain fused to a nuclear
localization signal and presumably acts as a dominant
negative ESE-1 due to its ability to interact with coac-
tivators and other cofactors needed for transactivation
of ESE-1, thereby depriving intact ESE-1 of its factors
needed for transactivation. DN1 and DN2 were cloned
into adenovirus vectors and the expression plasmid
pCI.
We tested the effect of dominant-negative ESE-1 on
inducible COX-2 gene expression in the human chond-
rocyte cell line T ⁄ C28a2. Very little COX-2 mRNA
expression was detected in unstimulated cells, but a
strong induction was observed upon exposure to IL-1
(Fig. 6A). Infection with AdE1-DN1 or AdE1-DN2
inhibited IL-1-induced expression of COX-2 mRNA
by 50–70% compared to Ad-bGal infection (Fig. 6A).
As RAW monocytic cells are difficult to infect with
adenoviruses and also do not transfect with high effi-
ciency, they are not suitable for assessing the effects of
the dominant negative ESE-1 on endogenous COX-2
mRNA levels. Therefore, we evaluated the effects of

)1
) for 16 h. Luciferase
activity in the lysates was determined 16 h after addition of LPS.
Data shown are means of duplicate measurements from one rep-
resentative transfection. Error bars represent the SD of the two
replicates.
ESE-1 activates COX-2 expression F. T. Grall et al.
1682 FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS
the mechanisms by which the promoter of COX-2 is
activated. Several transcription factor families have
been shown to be involved in this process such as
C ⁄ EBP [5,9,10,26], NF-jB [7,10,11], NFAT [6,10] and
Ets [16,18,27].
We now report the involvement of another Ets tran-
scription factor ESE-1 in the regulation of COX-2
expression in monocytes ⁄ macrophages and chondro-
cytes. ESE-1 (also named ELF3, Jen, ERT, ESX) is an
Ets family transcription factor, recently discovered by
us and others [22,23,28,29], whose expression under
normal physiological conditions is restricted to epi-
thelial cells. However, we uncovered an unexpected
function for ESE-1 in the vascular system and in con-
nective tissue cells where its expression is induced fol-
lowing exposure to pro-inflammatory stimuli such as
IL-1, TNF-a, and LPS [20,21].
We show here that LPS-mediated induction of COX-2
gene expression is, at least partially, dependant upon
ESE-1 upregulation. ESE-1 binds to the promoter of
COX-2 on several sites and activates its expression.
The integrity of these sites is required for full COX-2

activator of the COX-2 promoter than PEA3, further
supporting the notion that ESE-1 may be relevant for
COX-2 regulation. However, in contrast to ESE-1,
PEA3 does not appear to be regulated by pro-inflam-
matory stimuli. Surprisingly, the effect of PEA3 seems
to be mediated via the C ⁄ EBPb site. Our data show
that mutation of the C ⁄ EBPb site also affects ESE-1
mediated transactivation, but only partially. This sug-
gests that ESE-1 may also cooperate with C ⁄ EBPb or
another factor binding to this site in this process but
that does not account for the entire activation activity
as the mutation of Ets sites leads to a more drastic
abolition of this induction.
It has also been shown that the pattern of expression
of PEA3 in breast cancer samples correlates with the
patterns of expression of HER-2 ⁄ neu and COX-2 [17]
suggesting that the levels of COX-2 may results from
an HER-2 ⁄ neu stimulation of PEA3. Interestingly, a
similar correlation between the expression of HER-2 ⁄
neu and ESE-1 has also been observed [28,30,31] and
HER-2 ⁄ neu could itself activate the expression of
ESE-1 [31].
The role of Ets factors in inflammation and in the
regulation of cytokine-responsive genes has not been
studied in detail. However, several genes, including
urokinase-type plasminogen activator, matrix metallo-
proteinase (MMP)-1, MMP-3, TNF-a, scavenger
receptor, intercellular adhesion molecule (ICAM)-1,
ICAM-2, and IL-12 have been shown to depend on
Ets factors for their inducibility by cytokines such as

It can activate some important genes such as iNOS and
COX-2. Our studies also suggest that, by modulating
the activity of ESE-1, we could decrease the inflamma-
tory reaction in response to LPS exposure.
Experimental procedures
Cell culture and patient samples
THP-1 (human monocytic) and RAW 264.7 (murine mono-
cytic) cells (ATCC, Manassas, VA, USA) were cultured in
DMEM with 10% serum (Hyclone, Logan, UT, USA) with
undetectable levels of endotoxin. Immortalized human
chondrocytes T ⁄ C28a2 were grown and treated with cyto-
kines as described [37,38]. Lipopolysaccharide was obtained
from Sigma (St Louis, MO, USA) (catalogue number L8274).
RT ⁄ PCR analysis
Total RNA was harvested using QIAshreder (Qiagen,
Valencia, CA, USA) and RNeasy
Ò
Mini Kits (Qiagen). The
cDNAs were generated from 1 lg total RNA using Ready-
To-Go
TM
You-prime First-Strand Beads (Amersham Phar-
macia Biotech Inc., Piscataway, NJ, USA).
SYBR Green I-based real-time PCR was carried out on
the Opticon Monitor (MJ Research, Inc., Waltham, MA,
USA). All PCR mixtures contained: PCR buffer (final con-
centration 10 mm Tris ⁄ HCl pH 9.0, 50 m m KCl, 2 mm
MgCl
2
, 0.1% TritonX-100), 250 lm deoxy-NTP (Roche

aryotic expression vector downstream of the T7 and CMV
promoter as described [39]. The dominant negative form of
ESE-1, DN1, encodes the ESE-1 peptidic sequence deleted
of the amino acid residues 76–198, and DN2 encodes the
amino acid residues 1–231 fused in frame to a nuclear local-
ization signal motif repeated three times and a sequence
coding for EYFP in the pEYFP-NLS vector from Clon-
tech. Full length ESE-1 was fused in-frame with the Flag
peptide at the amino terminus in the pcDNA3-1 vector.
The human COX-2 promoter sequences spanning )831 and
)170 to +103, kindly provided by L. J. Crofford, Division
of Rheumatology, University of Michigan [11], were cloned
into the pXP2 luciferase vector in the HindIII and XhoI
sites (pXP2 ⁄ COX-2). An expression vector for the mouse
PEA3 gene downstream of the CMV promoter
(pCANMycPEA3) was a gift from L. Howe, Strang Cancer
Research Laboratory, Rockefellar University.
EMSA
In vitro transcription ⁄ translation was performed in TNT
rabbit reticulocyte lysate (Promega) using the pCI ⁄ ESE-1
vector as described [23]. EMSAs were performed using 2 lL
of in vitro translation product and [
32
P]-labeled double-
stranded oligonucleotide probes [40]. Supershift assays were
performed by preincubating the in vitro translated protein
20 min at room temperature with 2 lL antibody.
Oligonucleotides used as probes and for competition
studies were: (a) COX-2 promoter Ets site #1, 5¢-GCA
CGTCCAGGAACTCCTCAGC-3¢; (b) COX-2 promoter

Cotransfections were carried out in 6-well plates containing
3–8 · 10
5
cells per well using 600 ng of reporter gene con-
struct DNA and 200 ng expression vector DNA using
LipofectAMINE PLUS (Gibco-BRL) for 16 h as described
[23]. Transfections were performed independently in dupli-
cate, repeated three to four times with different plasmid
preparations and gave similar results. Cotransfection of a
second plasmid for determination of transfection efficiency
was omitted, because potential artifacts with this technique
have been reported [41] and many commonly used viral
promoters contain binding sites for Ets factors.
Adenovirus infection
Adenoviruses encoding the dominant negative forms of
ESE-1 were constructed using the Adeno-X expression
system from Clontech. TC ⁄ 28a2 cells were infected with
adenovirus for 1 h in serum-free medium using a multiplicity
of infection of 300. After infection the cells were washed with
medium and incubated for 16 h in DMEM containing 10%
fetal calf serum in the absence or presence of IL-1
(500 pgÆmL
)1
) (R & D Systems, Minneapolis, MN, USA).
Western blot analysis
RAW 264.7 cells were plated at 4 · 10
5
cells per well 16 h
before being exposed to LPS (100 ngÆmL
)1

Chromatin immunoprecipitation (ChIP)
ChIP was conducted as previously reported [21]. Briefly,
TC28 ⁄ a2 chondrocytes cells (2 · 10
7
) were plated on 150-mm
dishes and transfected with either pcDNA3Flag ⁄ ESE-1
or pcDNA3Flag and after 24 h stimulated with IL-1
(500 pgÆmL
)1
) for an additional 3 h. A 10-min formaldehyde
cross-linking step was stopped by adding glycine (0.125 m,
5 min at room temperature). After two washes, the cells were
resuspended in 0.3 mL lysis buffer, sonicated and then centri-
fuged at 4 °C. Supernatants were collected and 100 lLof
chromatin preparation were aliquoted as the input fraction.
The remainder of the supernatants was diluted 1 : 10 in dilu-
tion buffer for immunoclearing with sheared salmon sperm
DNA, normal rabbit serum and protein A–Sepharose for 2 h
at 4 °C. Immunoprecipitation was performed overnight at
4 °C with 80 lL M2 Agarose (anti-Flag Ig at 50% slurry in
TE) (Sigma) or with 5 lL rabbit IgG and 80 lL protein A–
Sepharose as a negative control. Precipitates were washed se-
quentially for 10 min each in 1 mL of TSE buffers [21]. Preci-
pitates were then extracted three times with 1% SDS, 0.1 m
NaHCO
3
. Eluates were pooled and heated at 65 °C overnight
to reverse the formaldehyde cross-linking, without proteinase
digestion. DNA fragments were purified with QIAquick
PCR purification Kit (Qiagen). PCR was performed using

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FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS 1687