Tài liệu Báo cáo khoa học: The Ets transcription factor ESE-1 mediates induction of the COX-2 gene by LPS in monocytes doc

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

Fax: +1 617 975 5299
Tel: +1 617 667 3393
E-mail:
(Received 21 July 2004, revised 19 January
2005, accepted 2 February 2005)
doi:10.1111/j.1742-4658.2005.04592.x
Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prosta-
glandins that are major inflammatory agents. COX-2 production is trig-
gered by exposure to various cytokines and to bacterial endotoxins. We
present here a novel role for the Ets transcription factor ESE-1 in regula-
ting the COX-2 gene in response to endotoxin and other pro-inflammatory
stimuli. We report that the induction of COX-2 expression by lipopoly-
saccharide (LPS) and pro-inflammatory cytokines correlates with ESE-1
induction in monocyte ⁄ macrophages. ESE-1, in turn, binds to several E26
transformation specific (Ets) sites on the COX-2 promoter. In vitro analysis
demonstrates that ESE-1 binds to and activates the COX-2 promoter to
levels comparable to LPS-mediated induction. Moreover, we provide
results showing that the induction of COX-2 by LPS may require ESE-1,
as the mutation of the Ets sites in the COX-2 promoter or overexpression
of a dominant-negative form of ESE-1 inhibits LPS-mediated COX-2
induction. The effect of ESE-1 on the COX-2 promoter is further enhanced
by cooperation with other transcription factors such as nuclear factor-jB
and nuclear factor of activated T cells. Neutralization of COX-2 is the goal
of many anti-inflammatory drugs. As an activator of COX-2 induction,
ESE-1 may become a target for such therapeutics as well. Together with
our previous reports of the role of ESE-1 as an inducer of nitric oxide syn-
thase in endothelial cells and as a mediator of pro-inflammatory cytokines
in vascular and connective tissue cells, these results establish ESE-1 as an
important player in the regulation of inflammation.
Abbreviations

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

CREB and cJun as well as USF-1 for the E-box [12].
Some Ets factors have also been suggested to play a
role in the regulation of COX-2 such as Ets-1 [14,15],
PEA3 [16–18] and Pu.1 [19].
The multiple activation modalities observed across
the different studies as well as the similarity of the
recognition sites of the NFAT and Ets factors led us to
investigate the potential involvement of the epithelial
specific Ets factor 1 (ESE-1 ⁄ ESX ⁄ ELF3 ⁄ ERT ⁄ JEN) in
this process. This factor is a likely regulator of COX-2
expression, as we recently discovered that ESE-1 plays
an important role in the responses of various cell types
to inflammatory mediators [20,21]. We reported the
induction of ESE-1 in response to IL-1, TNF-a and
LPS in vascular and connective tissue cells. This induc-
tion was mediated through the activation of NF-jB
[21]. We showed that ESE-1 could activate the expres-
sion of another important player in the inflammatory
response: inducible nitric oxide synthase (iNOS) [20].
Although ESE-1 was initially described as exclusively
expressed in epithelial cells in a variety of tissues
[22–25], we subsequently observed a broader expres-
sion pattern for ESE-1 under inflammatory conditions
[21].
We now report that ESE-1 can bind to the promoter
of COX-2 and that the integrity of the Ets sites is
required for LPS-induced COX-2 expression. ESE-1
can activate the COX-2 promoter in the monocyte cell
line RAW 264.7 as well as chondrocytic cells where it
acts synergistically with NF-jB and NFAT. Moreover,

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-

tein induction upon stimulation by LPS correlated
with the expression pattern of ESE-1 (Fig. 1). Thus,
COX-2 may be a potential target for ESE-1 during
inflammation.
ESE-1 transactivates the human COX-2 promoter
To elucidate whether the COX-2 gene could be an
ESE-1 target gene in inflammatory processes, we
screened the regulatory region of the COX-2 promoter
for potential ESE-1 binding sites. Inspection of the
COX-2 promoter sequence (GeneBank accession num-
ber AY229989) revealed the presence of five possible
Ets binding sites within the first 200 bp upstream of
the transcription initiation site, one of which overlaps
with a potential NFAT site (Fig. 2A).
To determine whether the COX-2 gene may be
regulated by ESE-1, we constructed two human
F. T. Grall et al. ESE-1 activates COX-2 expression
FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS 1677
COX-2 promoter luciferase reporter plasmids, pXP2 ⁄
COX-2–170 and pXP2 ⁄ COX-2–831, starting at )170
and )831, respectively, upstream of the tran-
scription start site, which we transiently transfected
into RAW 264.7 cells. Cotransfections of these pro-
moter plasmids together with the ESE-1 expression
vector, pCI ⁄ ESE-1, enhanced COX-2 promoter activ-
ity 10- and 30-fold, when the long or the short pro-
moter constructs, respectively, were used (Fig. 2B).
Stimulation with LPS resulted in a more than
20-fold induction of the COX-2 promoter (Fig. 2B).
This ESE-1-mediated transactivation of the COX-2

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

promoter by performing an EMSA. We used as probes
the five putative Ets binding sites of the COX-2 pro-
moter present within the )170 COX-2 promoter. We
tested their ability to form a complex with in vitro
translated ESE-1 protein. As shown in Fig. 3A, sites 1
and 3 formed strong protein ⁄ DNA complexes with
ESE-1. The specificity of this complex was confirmed
in supershift assays using two different anit-ESE-1 Igs
(Fig. 3). Site 4, which overlaps with the NFAT binding
site, did not appear to bind specifically to ESE-1 in
this assay. The mobility of the ESE-1 complex was
consistent with that reported in Rudders et al. and
was absent when unprogrammed reticulocyte lysate
was used.
To further determine whether ESE-1 binds to the
COX-2 promoter in vivo, we performed a chromatin
immunoprecipitation experiment in chondrocyte cells
which express both ESE-1 and COX-2 in response
to IL-1 (Fig. 3B). T ⁄ C28a2 chondrocyte cells were
transfected with either pcDNA3-1⁄ Flag-ESE-1 or
pcDNA3-1 ⁄ Flag. After cross-linking the proteins
bound to DNA with formaldehyde followed by soni-
cation, the cell extracts were immunoprecipitated
using the anti-Flag Ig or nonspecific serum. As shown
in Fig. 4B, a precipitate specifically retaining the
COX-2 promoter region spanning all five Ets sites
shown in Fig. 2 ()186 to +56 of the transcription
start site) was only obtained in cells transfected with
the plasmid containing ESE-1. This experiment most
clearly demonstrates that ESE-1 directly binds to the

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

suggesting that ESE-1 acts on the COX-2 promoter via
multiple Ets sites (Fig. 4A), but that sites 2, 3 and 4
are crucial for inducibility, since their mutations gave
the strongest reductions of activity compared to other
isolated mutations. This is in line with the findings of
Liu et al. [18] who showed that the Ets site number 3
was critical f or COX-2 induction by NO. No inducibility
A
B
C
DE
Fig. 2. The COX-2 promoter is a target for ESE-1. (A) Sequence of the COX-2 promoter. The five putative Ets binding sites present in the
)170 COX-2 construct (starting at the asterisk) are highlighted as well as additional NFAT and NF-jB sites within the COX-2 promoter
sequence. Two extra Ets sites described in Howes et al. [16] are underlined. (B) Transcriptional activation of the COX-2 promoter by ESE-1
and LPS. RAW cells were cotransfected with the pXP2 luciferase construct containing the COX-2 promoter (pXP2 ⁄ COX-2) starting either at
)831 or at )170 and the pCI ⁄ ESE-1 expression vector and incubated in the absence or presence of LPS. Luciferase activity in the lysates
was determined 16 h later, as described. Data shown are means of duplicate measurements from one representative transfection. The
experiment was repeated three times with different plasmid preparations with comparable results. Error bars represent the SD for the two
replicates. (C) Transcriptional activation of the COX-2 promoter by ESE-1 in chondrocytes. T ⁄ C28a2 cells were cotransfected with the pXP2
luciferase construct containing the )170 COX-2 promoter (pXP2 ⁄ COX-2) and the pCI ⁄ ESE-1 expression vector. Luciferase activity in the
lysates was determined 16 h later, as described. Data shown are means of duplicate measurements from one representative transfection.
Error bars represent SD of the two replicates. (D) Transcriptional activation of the COX-2 promoter by ESE-1 and PEA3. RAW cells were
cotransfected with the COX-2 promoter luciferase construct (pXP2 ⁄ COX-2–170) and different amounts of expression vectors for ESE-1 or
PEA3 maintaining constant (700 ng) the total amount of DNA with pCI vector. Luciferase activity in the lysates was determined 16 h later,
as described. Data shown are means of duplicate measurements from one representative transfection. The experiment was repeated three
times with different plasmid preparations with comparable results. Error bars represent SD for the two replicates. (E) Protein expression
levels of ESE-1 and PEA3 after transfection into 293ft cells. Myc-tagged Ets factors were transfected into 293 cells. The cells were lysed
16 h later and equal amounts of lysate were loaded on a gel for a western blot analysis using anti-myc Ig.
F. T. Grall et al. ESE-1 activates COX-2 expression
FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS 1679

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

DNA sides at the same sites and may actually cooper-
ate in transactivation of the COX-2 promoter.
ESE-1 cooperates with NF-jB in the
transactivation of the COX-2 promoter
In addition to the Ets binding sites the COX-2 promo-
ter contains at least four putative NF-jB binding sites.
As NF-jB has been demonstrated previously to play a
role in regulating COX-2 promoter activity in at least
some cell types [7,11] and NF-jB cooperates with
ESE-1 in transactivating the iNOS promoter in endo-
A
B
Fig. 3. ESE-1 binds to several Ets sites in the COX-2 promoter. (A) Interaction of ESE-1 with Ets binding sites in the COX-2 promoter. EMSA
using either unprogrammed reticulocyte lysate [23] or in vitro translated ESE-1 (ESE-1) and five labeled oligonucleotide probes containing dif-
ferent COX-2 promoter Ets sites. The in vitro translated proteins were alternatively preincubated with antibody (Ab1, east-acres Biological;
Ab2, QED; C, negative control, normal rabbit serum). The white arrow indicates the specific ESE-1 DNA–protein complex and the black
arrow shows the supershift form with the antibody. (B) Binding of ESE-1 to endogenous human COX-2 promoter by ChIP. The anti-Flag Ig
was used to specifically enrich COX-2 promoter DNA sequences in a ChIP assay. Chromatin proteins from IL-1 treated T ⁄ C28a2 cells trans-
fected with either pcDNA3 ⁄ Flag (lanes 1–3) or pcDNA ⁄ Flag-ESE-1 (lanes 4–6) were crosslinked to DNA with formaldehyde, and purified
nucleoprotein complexes were immunoprecipitated using either anti-Flag Ig or nonspecific rabbit serum. The precipitated DNA fractions were
analyzed by PCR for the presence of the COX-2 promoter region encompassing the Ets sites. The input and genomic DNA (gDNA) were
used as a positive control, and water was used as a negative control for PCR.
ESE-1 activates COX-2 expression F. T. Grall et al.
1680 FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS
thelial cells [20], we evaluated whether ESE-1 and
NF-jB cooperate in the context of the COX-2 promo-
ter as well. The )831 COX-2 promoter luciferase
construct was cotransfected into RAW cells together
with either ESE-1, NF-jB p50 or p65 alone as well as
with various combinations thereof (Fig. 5B). Whereas

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

with the pCI ⁄ ESE-1 expression vector, the NF-jB p50 and p65
expression vectors or a combination thereof and the )831 or )170
COX-2 promoter wild-type (B) or )170 mut ets 1 +2 + 3 +4 + 5 (C)
luciferase constructs. Luciferase activity in the lysates was deter-
mined 16 h later, as described elsewhere [23]. Data shown are
means of duplicate measurements from one representative trans-
fection. The experiment was repeated twice with different plasmid
preparations with comparable results. Error bars represent the
standard deviation of the two replicates.
F. T. Grall et al. ESE-1 activates COX-2 expression
FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS 1681
p50 alone did not significantly enhance COX-2 promo-
ter activity, p65 increased promoter activity by three
to fourfold. The p50⁄ p65 combination enhanced trans-
activation of the COX-2 promoter to 10-fold, similar
to the effect of ESE-1 alone. ESE-1 cooperated with
both p50 and p65, which enhanced COX-2 promoter
activity by 30-fold in cotransfection with ESE-1. This
cooperativity was most striking when ESE-1 was
cotransfected with the p50 ⁄ p65 heterodimer, which
increased transactivation of the COX-2 promoter by
70-fold. Mutation of the Ets sites within the COX-2
promoter did not significantly affect NF-jB mediated
transactivation, but strongly reduced cooperativity
between ESE-1 and NF-jB (Fig. 5C).
Dominant-negative ESE-1 mutants inhibit LPS
and IL-1 mediated induction of COX-2 gene
expression
To evaluate whether ESE-1 is indeed involved in regu-
lation of inducible COX-2 gene expression we used

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

the dominant-negative ESE-1 mutants in RAW cells
transfected with the pXP2 ⁄ COX-2 promoter luciferase
construct. ESE-1 DN1 expression completely blocked
LPS-mediated induction of the COX-2 promoter, again
confirming the involvement of ESE-1 in LPS-mediated
activation of the COX-2 promoter (Fig. 6B).
These data most vividly indicate that ESE-1 may
play a critical role in induction of the COX-2 gene by
pro-inflammatory stimuli.
Discussion
The regulation of COX-2 expression during inflamma-
tion has been the focus of numerous studies. The het-
erogeneity of parameters such as the cell type and the
stimulus used has made it difficult to describe precisely
Fig. 6. Dominant-negative mutants of ESE-1 reduce expression of
the endogenous COX-2 gene in response to LPS and inhibit the
transactivation its promoter. (A) T ⁄ C28a2 chondrocyte cells were
infected with the adenoviruses Ad ⁄ ESE-1 DN1, DN2 or b-galac-
tosidase (beta-gal) and 16 h later treated with IL-1 (500 pgÆmL
)1
).
The RNA was harvested 28 h later and used for real-time PCR
using COX-2 specific primers. Data shown are means of duplicate
measurements from one representative transfection representing
the ratio of the measurements of COX-2 to GAPDH mRNA. Error
bars represent the SD of the two replicates. (B) RAW cells were
cotransfected with the pCI expression vector containing the dom-
inant-negative mutant of ESE-1 (ESE-1 DN1) and the COX-2 pro-
moter Luciferase ()170) construct. Cells were grown in the
absence or presence of LPS (500 ngÆmL

)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

promoter activation by LPS, since mutation of two or
more sites markedly attenuates the response. This is
true even when the NFAT site is left intact (identical to
Ets site number 4). ESE-1 mediated transactivation of
the COX-2 promoter is synergistic with NFAT and
NF-jB, since ESE-1 can enhances the activity of COX-2
promoter due to NFAT or NF-jB transactivation more
than additive. This cooperativity can be due to the pre-
viously demonstrated [20] direct binding of ESE-1 to
the NF-jB family members p50 and p65. NF-jB itself
also upregulates endogenous ESE-1 expression in
response to pro-inflammatory stimuli via a high affinity
NF-jB binding site within the ESE-1 promoter [20,21].
Thus, ESE-1 may be involved in a positive feedback
loop during the inflammatory response to enhance the
transactivation effect of NF-jB on some of its target
genes by cooperating with ESE-1. The mutation of the
Ets site 4 decreases the ESE-1 activation of COX-2,
even though no binding could be detected in vitro by
EMSA. This site may be functionally mixed or it is
possible that these two factors interact with each other
and bind as a complex on this site. That would explain
the further increase of COX-2 activation when ESE-1 is
transfected together with NFAT.
The involvement of an Ets factor in the regulation
of COX-2 was reported by Howe et al. [16], who dem-
onstrated that overexpression of another member of
the Ets family, PEA3, could activate the COX-2 pro-
moter. When we compared the activities of PEA3 to
ESE-1, we found that ESE-1 is a more effective trans-

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

IL-1 or TNF-a [32–36]. Many additional cytokine-
responsive genes contain putative Ets binding sites
within their regulatory regions, including COX-2,
iNOS, and MMP-13.
We demonstrate here that endogenous COX-2 gene
expression can be inhibited by using dominant negative
forms of ESE-1. The inhibitory effect of these domin-
ant negative ESE-1 mutants on COX-2 expression,
confirm the importance of ESE-1 or a related factor
that would bind to these Ets sites. These results give
some insight in the potential use of new therapeutic
approaches manipulating the activity of ESE-1 or
other Ets factors as a tool to reduce inflammation.
Indeed, several pro-inflammatory agents such as IL-1
[21], and TNF-a [21] can also induce ESE-1, and
ESE-1 target genes with regard to inflammation so far
identified by us include iNOS, COX-2 and potentially
MMP-1 and MMP-13 (X Gu, F Grall, M Joseph,
L Zerbini & T Libermann, unpublished data). The kin-
etic of activation of ESE-1 seems to implicate ESE-1
at later stages of inflammation. These results suggest
that ESE-1 regulates a subset of the genes whose inhi-
bition could be of significant interest in the manage-
ment of the inflammatory reaction.
F. T. Grall et al. ESE-1 activates COX-2 expression
FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS 1683
In conclusion, this report demonstrates further that
ESE-1 is a relevant player in the inflammatory process.
ESE-1 is upregulated in several cell types in response to
pro-inflammatory stimuli through the NF-jB pathway.

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

Pleasanton, CA, USA), 0.5 lm each PCR primer, 0.5 ·
SYBR Green I, 5% dimethylsulfoxide, 1 U Taq DNA poly-
merase (Promega, Madison, WI, USA) with 2 lL cDNA
in a final volume of 25 lL. The samples were loaded into
wells of Low Profile 96-well microtiter plates. After an ini-
tial denaturation step at 95 °C for 2 min, conditions for
cycling were 38 cycles of denaturation (95 °C, 30 s),
annealing (54 °C, 30 s), and extension (72 °C, 1 min).
Then, the fluorescence signal was measured immediately
following incubation at 78 °C for 5 s that follows each
extension step, thereby eliminating possible primer dimer
detection. At the end of the PCR cycles, a melting curve
was generated to identify the specificity of the PCR prod-
uct. For each run, serial dilutions of human GAPDH plas-
mids were used as standards for quantitative measurement
of the amount of amplified cDNA. For normalization of
each sample, hGAPDH primers were used to measure the
amount of hGAPDH cDNA. All samples were run as
duplicates and the data were presented as ratios of Cox-2 ⁄
hGAPDH. The primers used for real time PCR are as
follows. For hGAPDH forward, 5¢-CAAAGTTGTCATG
GATGACC-3¢; reverse, 5¢-CCATGGAGAAGGCTGGG
G-3¢, which will amplify 195 bp of human GAPDH. For
Cox2 forward, 5¢-TTCAAATGAGATTGTGGGAAAA
TTGCT-3¢; reverse, 3¢-ATATCATCTCTGCCTGAGTAT
CTT-3¢, which will amplify 304 bp of human Cox2.
Expression vector and luciferase reporter gene
constructs
Full-length and dominant negative mutant ESE-1 cDNAs
were inserted into the EcoRI site of the pCI (Promega) euk-

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

Ets site #2, 5¢-GAGAGAACCTTCCTTTTTATAA-3¢; (c)
COX-2 promoter Ets site #3, 5¢-CGAAAAGGCGGAAAG
AAACAGT-3¢; (d) COX-2 promoter Ets site #4, 5¢-GAGA
GGAGGGAAAAATTTGTGG-3¢;3¢-CTCTCCTCCCTTT
TTAAACACC-5¢; (5) COX-2 promoter Ets site #5,
5¢-TCTCATTTCCGTGGGTAAAAA-3¢.
Site-directed mutagenesis
Mutations in the different COX-2 promoter ETS sites
were generated by site-directed mutagenesis with the
QuikChange Site-directed Mutagenesis kit (Stratagene,
Cedar Creek, TX, USA) and confirmed by sequencing.
ESE-1 activates COX-2 expression F. T. Grall et al.
1684 FEBS Journal 272 (2005) 1676–1687 ª 2005 FEBS
The following primers were used (the mutated bases are
underscored): (a) COX-2 promoter Ets site #1, 5¢-GCT
GAGGAGT
AGCTGGACGTGCTCCTGAC-3¢;(b)COX-2
promoter Ets site #2, 5¢-CAGTCTTATAAAAA
CCAA
GGTTCTCTCGGTTAGCGACC-3¢; (c) COX-2 promoter
Ets site #3, 5¢-GACGAAATGACTGTTTCTTT
GAGCC
TTTTCGTACCCC-3¢; (d) COX-2 promoter Ets site #4,
5¢-AGGGGAGAGGAGGG
TTAAATTTGTGGGGGGTA
CGAAAAGGCGG-3¢; (e) COX-2 promoter Ets site #5:
5¢-GGGTTTTTTACCCACG
CTAATGAGAAAATCGGAA
ACC-3¢.
DNA transfection assays

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

) in fresh med-
ium for different periods of time. The cells were rinsed with
NaCl ⁄ P
i
, harvested in 200 lL RIPA lysis buffer containing
protease inhibitors (Roche) and frozen and thawed once
before being sonicated. Forty microliters of lysate were loa-
ded on a 10% polyacrylamide gel containing SDS. Proteins
were transferred to a poly(vinylidene difluoride) (PVDF)
membrane and blocked with 5% milk in NaCl ⁄ P
i
⁄ Tween
(0.2%). A polyclonal antibody directed against the amino-
terminal half of ESE-1 (East Acres Biologicals, South-
bridge, MA, USA) or an anti-COX-2 Ig (Santa-Cruz, Santa
Cruz, CA, USA) were used to detect the presence of these
proteins. A secondary antibody labeled with horseradish
peroxidase (HRP) was used for detection by ECL. The
signal intensity was determined with the alphaease soft-
ware (AlphaInnotech, San Leandro, CA, USA) and then
divided for normalization by the protein concentration for
each lane.
293ft cells (3 · 10
5
; Invitrogen, Carlsbad, CA, USA) were
transfected with 3 lg ESE-1 or PEA3 expression plasmid
using lipofectamine and lysed 16 h later. Cell lysate (225 lg)
was loaded onto a SDS ⁄ PAGE gel. Anti-myc ⁄ HRP conju-
gated Ig (Santa-Cruz) was used at 1 : 200 dilution for 4 h to
detect myc-tagged ESE-1 and PEA3.

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

5 lLofa60lL DNA extraction in TE buffer with Hi-Fi
Taq polymerase (Invitrogen) for 28 cycles (95 °C for 30 s,
52 °C for 30 s and 68 °C for 1 min). The primers used were
5¢-CTGGGTTTCCGATTTTCTCA-3¢ and 5¢-CTGCTG
AGGAGTTCCTGGAC-3¢ which amplify 200 bp of the
human COX-2 promoter.
Acknowledgements
This study was supported by National Institutes of
Health Grant RO1 ⁄ AI49527 and a Brain Tumor Soci-
ety Research grant to T. A. L. and by National Insti-
tutes of Health Grant KO8 ⁄ CA 71429 to P. O. K.
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