From the Department of Surgery, Weill Medical College
of Cornell University and The New York Presbyterian Hospital, New York,
New York 10021, § Strang Cancer Prevention Center, New York,
New York 10021, and the
Department of Pharmacology, National
Cardiovascular Center Research Institute, Osaka 565, Japan
Received for publication, June 12, 2000, and in revised form, October 5, 2000
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ABSTRACT |
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Macrophage expression of cyclooxygenase-2
(COX-2), the inducible isoform of COX, is up-regulated by
pro-inflammatory stimuli both in vivo and in
vitro. Here we investigated the mechanisms regulating
COX-2 gene expression in macrophage/monocytic cells. Lipopolysaccharide (LPS) is known to induce de novo COX-2
mRNA expression in these cells. Transient cotransfections with a
COX-2 promoter-luciferase construct and
different expression vectors showed that LPS up-regulates
COX-2 transcription through both mitogen-activated protein
kinase (MAPK) and protein kinase C (PKC) pathways. Cotransfections with
expression vectors for dominant negative mutants of MAPK and PKC
isoforms did not suppress the effects of LPS on COX-2.
Electrophoretic mobility shift assays and transient transfection
experiments with deleted and mutated variants of a COX-2
promoter-luciferase construct showed that NF Cyclooxygenase (COX)1
can be a rate-limiting step in the synthesis of prostaglandins (PGs),
lipid mediators that contribute to the development of inflammatory
responses (1). In this process, phospholipase A2 catalyzes
the release of arachidonic acid from membrane phospholipids, while COX
catalyzes the conversion of arachidonic acid into PGs (2, 3). There are
two isoforms of COX, COX-1 and COX-2, the product of two different
genes. COX-1 is expressed constitutively in most tissues and may be
responsible for housekeeping functions (4). In contrast, COX-2 is not
detectable in most normal tissues or resting immune cells, but its
expression can be induced by endotoxin, cytokines, growth factors, and
carcinogens (5-7).
Macrophage activation is accompanied by a significant increase in COX-2
expression, whereas levels of COX-1 remain unchanged (7, 8). In
vivo macrophage COX-2 immunoreactivity is a characteristic finding
in the synovium of patients with osteoarthritis as well as in other
forms of inflammation, whereas COX-1 expression typically
remains unchanged (9, 10). Nonsteroidal anti-inflammatory drugs inhibit
PG synthesis through the inhibition of COX activity, which confers on
them anti-inflammatory and analgesic properties (11). Studies with
newly developed selective COX-2 inhibitors also have shown suppression
of PG production and acute tissue inflammation (12). Moreover,
homozygous deletion of the Cox-2 gene in mice led to a
striking mitigation of endotoxin-induced hepatocellular cytotoxicity
(13). Therefore, COX-2 may play a critical role in the development of
local and systemic inflammatory responses.
The different responses of the genes encoding COX-1 and COX-2 reflect,
at least in part, differences in the regulatory elements in the 5'
flanking regions of these two genes. In the COX-2 gene (Fig.
1), promoter elements for nuclear factor
B, NF-IL6,
and CRE promoter sites mediate gene transcription independently in
response to LPS treatment. In these experiments, isolated NF
B,
NF-IL6, and CRE promoter sites were less effective than the intact
promoter in mediating COX-2 transcription. Cotransfections with mutated COX-2 promoter-luciferase
constructs and expression vectors showed that each one of these
promoter elements can be activated by LPS through both MAPK and PKC
pathways to induce gene expression. In summary, there is redundancy in
the signaling pathways and promoter elements regulating
COX-2 transcription in endotoxin-treated cells of
macrophage/monocytic lineage.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (NF
B,
223/
214) and nuclear factor interleukin-6 (NF-IL6,
132/
124) and a cAMP-responsive element (CRE,
59/
53) have been
found to be important in regulating transcription (14-16). The CRE
appears to be the crucial site in epithelial cells (17, 18), whereas
other promoter elements, such as those for NF
B and NF-IL6, seem to
have a role in regulating COX-2 gene transcription in
macrophage-like cells (19-21). However, the relative contribution of
the different promoter elements in mediating COX-2
transcription in macrophages has not been completely elucidated.
View larger version (21K):
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Fig. 1.
Schematic of the COX-2
promoter and mutant reporter constructs.
In the present work, we have investigated the regulation of
COX-2 gene expression in endotoxin-treated RAW 264.7 macrophages and THP-1 monocytic cells. Our data show that there is
transcriptional redundancy among the NFB, NF-IL6, and CRE promoter
sites although maximal transcriptional activity requires cooperation
among these elements. Also in a redundant manner, mitogen-activated
protein kinase (MAPK) and protein kinase C (PKC) signaling pathways are capable of inducing COX-2 transcription through NF
B,
NF-IL6, or CRE promoter sites. These results are important for
understanding why COX-2 expression is up-regulated during macrophage activation.
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EXPERIMENTAL PROCEDURES |
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Materials--
RPMI and fetal bovine serum were from Life
Technologies, Inc. Escherichia coli (strain 055:B5)
lipopolysaccharide, DEAE-dextran, and
o-nitrophenyl--D-galactopyranoside were from
Sigma. [32P]ATP was from PerkinElmer Life Sciences.
Endotoxin-free plasmid DNA was prepared using Qiagen DNA purification
kits (Chatsworth, CA). Reagents for the luciferase assay were from
Analytical Luminescence (San Diego, CA). Mutagenesis kits were from
Stratagene (La Jolla, CA). Oligonucleotides were synthesized by Genosys
Biotechnologies Inc. (The Woodlands, TX). T4 polynucleotide kinase was
from New England Biolabs, Inc. (Beverly, MA).
Cell Lines-- Murine macrophage-like RAW 264.7 cells and human THP-1 monocytic cells were maintained in RPMI supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B).
Plasmids--
Expression vectors for Ras, MAPK kinase-1,
mitogen-activated protein kinases (ERK-1 and -2, p38, and JNK),
PKC isoforms (,
, and
), and their dominant negative
mutants were provided by Dr. Andrew J. Dannenberg (Weill Medical
College of Cornell University, New York, NY). Human COX-2
promoter-luciferase deletion constructs (
327/+59,
220/+59,
124/+59, and
52/+59) and mutant constructs (NF
B
mutant (designated KBM), NF-IL6 mutant (designated ILM), CRE mutant
(designated CRM), the double mutant ILM/CRM, and the triple mutant
KBM/ILM/CRM) have been described previously (14, 15, 20). The double
mutants KBM/ILM and KBM/CRM were created using site-directed
mutagenesis kits. Briefly, primers that incorporated mutations (lowercase letters) for NF-IL6 (sense:
5'-CCCTGCCCCCACCGGGCTTAgtacATTTTTTTAAGGGGAGAG-3') or CRE (sense:
5'-GGCGGAAAGAAACAGTCATTTgaTCtCATGGGCTTGGTTTTCAGT-3') were
used to amplify the KBM construct. This was achieved with the use of
pfu Turbo-DNA polymerase and sequential cycling in a 480 PerkinElmer Life Sciences thermocycler as per the protocol of the
mutagenesis kit. The parental DNA was then digested with DpnI, and the new constructs harboring the KBM/ILM or
KBM/CRM mutations were used to transform E. coli competent
cells. Incorporation of the desired mutations was confirmed by DNA
sequencing (Fig. 1).
Transient Transfection Assays--
RAW 264.7 or THP-1 cells
(5 × 106 per treatment group) were washed twice in
serum-free RPMI and then suspended in 0.5 ml of transfection solution
containing 50 mM Tris and 500 mg/ml DEAE-dextran. Subsequently, 2 µg of a COX-2
promoter-luciferase construct, 2 µg of either an
expression vector or empty plasmid, and 0.5 µg of the control plasmid
pSV--galactosidase were added, and the mixture
was incubated at 37 °C and 5% CO2 for 30 min.
Me2SO (100 µl/ml of transfection mixture) was added for 1 min at room temperature, and the reaction was stopped by adding 10 volumes of RPMI. After pelleting by centrifugation, transfected cells
were plated in 100-mm dishes and incubated in 10% fetal calf
serum RPMI for 24 h. Subsequently, cells were treated with
fresh 3% fetal bovine serum RPMI with or without LPS (50 ng/ml).
Luciferase and
-galactosidase activities were measured in cellular
extracts 6 h later as described previously (22).
Electrophoretic Mobility Shift Assays--
Cells were plated in
100-mm dishes at a density of 3 × 106 cells/dish and
allowed to attach for 24 h prior to experiments. Cells were then
treated with fresh 3% fetal bovine serum RPMI with or without LPS (50 ng/ml). Nuclear extracts were obtained 30 min later as described
previously (23). Briefly, cells were lysed by incubation in buffer A
(10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride) on ice for 15 min,
followed by vortexing for 10 s. Nuclei were pelleted, and nuclear
extracts were obtained by high salt extraction after incubating nuclei
in buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride) for 20 min. Nuclei were
pelleted again by centrifugation for 5 min, and the supernatant
fraction (nuclear extract) was stored at 70 °C.
Double-stranded DNA oligonucleotides containing the consensus binding sites for the NF
B (sense: 5'-GGAGAGTGGGGACTACCCCCTCTGCT-3'), NF-IL6 (sense: 5'-CACCGGGCTTACGCAATTTTTTTAA-3'), or CRE (sense: 3'-AACAGTCATTTCGTC ACATGGGCTTG-5') elements found in the
COX-2 promoter were labeled with [32P]ATP
using T4 kinase. 4 µg of nuclear extract were incubated with 1 µl
of DNA probe in a total of 10 µl containing 4% glycerol, 50 mM NaCl, 10 mM Tris, pH 7.5, 1.5 mM
MgCl2, 0.5 mM EDTA, 0.5 mM
dithiothreitol, and 1 µg of poly(dI·dC). Cold chase was carried out
with a 50 molar excess of the same, unlabeled probe or with a probe
that contained mutated binding sequences (lowercase letters) for NF
B
(cccgggACCC), NF-IL6 (TTAgtacAT), or CRE (TTTgaTCt). The nuclear
extract-DNA complexes were resolved in 4% polyacrylamide gels using
0.5 × TBE at 150 V. The gels were then dried and autoradiographed.
Statistics--
Comparisons among groups were made by Student's
t test. A difference among groups of p < 0.05 was considered significant.
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RESULTS |
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LPS Induces COX-2 Transcription through MAPK and PKC Signaling
Pathways--
RAW 264.7 and THP-1 cells were transiently cotransfected
with a 327/+59 COX-2 promoter-luciferase
reporter construct and different MAPK (ERK-1 and -2, p38, and JNK) and
PKC (PKC-
, PKC-
, and PKC-
) expression vectors. As seen in Fig.
2, overexpression of some MAPK
(A and D) and PKC isoforms (B and
E) increased both basal and LPS-mediated luciferase
activity. There was a synergistic effect among ERK-2, p38, JNK, and
PKC-
overexpression and LPS treatment, whereas overexpression of
ERK-1, PKC-
, and PKC-
had no significant effect. Overexpression
of dominant negative mutants for the same mitogen-activated
protein kinases and PKC isoforms did not abrogate LPS-mediated
induction of luciferase activity (C and F).
Therefore, LPS can induce COX-2 transcription through the
activation of ERK-2, p38, JNK, and PKC-
, although none of these
pathways is absolutely necessary to mediate this effect. This denotes
redundancy in the signaling mechanisms by which LPS induces
COX-2 transcription.
|
NFB, NF-IL6, and CRE Promoter Elements Regulate LPS-mediated
COX-2 Transcription--
To characterize the promoter region(s)
regulating LPS-mediated transcription, we performed transient
transfection experiments using reporter constructs harboring deleted
and mutated variants of the COX-2 promoter (Fig. 1).
Transfection experiments with deletion constructs (Fig.
3, A and D) showed
that promoter elements between
327 and
52 were necessary to induce
luciferase activity after LPS treatment. Because NF
B, NF-IL6, and
CRE binding sites can be found in the
327/
52 region (14), we
performed transient transfections with mutant variants of a
327/+59
COX-2 promoter construct in which the NF
B, NF-IL6, CRE,
or all three sites had been mutated (KBM, ILM, CRM, and KBM/ILM/CRM
constructs, respectively). Fig. 3, B and E shows
that none of these promoter elements were essential to induce
luciferase activity after LPS treatment. However, LPS did not increase
luciferase activity when the KBM/ILM/CRM construct was used; therefore,
the combination of at least two promoter elements appears to be
necessary to activate transcription. The ability of the NF
B, NF-IL6,
and CRE sites to individually support transcription was assessed in
transfection experiments with COX-2 promoter constructs
containing only one functional promoter element (i.e. double
mutant constructs). As seen in Fig. 3, C and F,
there were differences in the basal luciferase activity level sustained
by each of these promoter sites, but all of them mediated induction of
luciferase activity after LPS treatment. Remarkably, none of these
individual promoter elements, not even the sum of their effects, were
as effective in inducing luciferase activity after LPS treatment as a
promoter with three functional sites. Therefore, the coexistence of
NF
B, NF-IL6, and CRE sites in the intact promoter has synergistic
rather than additive effects.
|
LPS Induces Binding of Nuclear Proteins to the NFB, NF-IL6, and
CRE Promoter Sites of the COX-2 Gene--
For LPS to induce
COX-2 gene transcription through NF
B, NF-IL6, and CRE
promoter sites, it should induce binding of nuclear proteins
(i.e. transcription factors) to these promoter elements. As
seen by electrophoretic mobility shift assay, treatment with LPS
induces binding of nuclear proteins to all three promoter sites (Fig.
4). In combination, the data from
electrophoretic mobility shift assay and transfection experiments
indicate that maximal LPS induction of COX-2 transcription
is achieved through the activation and binding of transcription factors
to any combination of two promoter sites. Because the presence of all
three promoter elements is not necessary to mediate this
effect, but only any combination of two, it appears
that there is redundancy also at the promoter level in LPS-mediated
COX-2 transcription.
|
LPS Activates MAPK and PKC Signaling Pathways to Induce COX-2 Gene
Transcription through NFB, NF-IL6, or CRE Promoter Sites--
We
further investigated the signaling pathways by which LPS induces
NF
B-, NF-IL6-, and CRE-mediated COX-2 transcription. We
performed transient cotransfections with those MAPK and PKC expression
vectors that induced COX-2 gene transcription (Fig. 2) and
double mutant reporter constructs that contained only a functional
NF
B (Fig. 5A), NF-IL6 (Fig.
5B), or CRE (Fig. 5C) promoter site. As shown in
Fig. 5A, ERK-2, p38, JNK, and PKC-
overexpression had a
synergistic effect with LPS to induce luciferase activity, indicating
that these pathways participate in LPS-mediated gene transcription
through the NF
B site. Overexpression of upstream signaling
intermediates, such as Ras and MEKK-1, had the same effect (data not
shown). Fig. 5B shows that p38 and PKC-
overexpression synergize with LPS to induce transcription through the NF-IL6 promoter
site. In Fig. 5C, overexpression of ERK-2, JNK, and PKC-
are synergistic with LPS in inducing transcription through the CRE
site. Overexpression of dominant negative mutants for Ras, MEKK, MAPKs,
or PKC isoforms did not inhibit the stimulatory effect of LPS on gene
transcription through the NF
B, NF-IL6, or CRE sites (data not
shown). Therefore, it appears that COX-2 gene transcription
through each promoter element (i.e. NF
B-, NF-IL6-, or
CRE-mediated transcription) is supported by at least two different signaling pathways.
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DISCUSSION |
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COX-2 expression and PG synthesis in macrophages may be important in eliciting local and systemic inflammatory responses. When exposed to LPS and other pro-inflammatory stimuli, macrophages secrete PGs as a result of increased arachidonic acid release and COX-2 enzyme activity. Increased COX-2 activity may result from increased enzymatic activity or mRNA stability (26-28), but in endotoxin-treated macrophages it results mainly from augmented rates of transcription of the COX-2 gene. Therefore, the signaling mechanisms governing COX-2 gene transcription could be a potential target of strategies designed to suppress local or systemic inflammatory responses. However, the inter-relation between signaling pathways and promoter sites in the COX-2 gene and the individual role of these cis-acting elements in mediating transcription are not well understood.
Our data show that in both RAW 264.7 macrophages and THP-1 monocytic
cells there is redundancy in the transcriptional activation of the
COX-2 gene both at the extranuclear signaling level and at
the promoter level. Overexpression of dominant negative mutants for
different MAPKs and PKC isoforms do not abrogate the effects of LPS on
COX-2 transcription indicating that there is redundancy at
the signaling level. Mutation of a single NFB, NF-IL6, or CRE site
also does not abrogate the LPS effect indicating that there is
redundancy at the promoter level. Therefore, transcriptional repression
of COX-2 could not be accomplished by targeting a single signaling pathway or transcription factor/promoter element.
Our results indicate that LPS induces COX-2 expression in cells of
macrophage/monocytic cell lineage through the binding of transcription
factors to the NFB, NF-IL6, and CRE promoter elements situated
within the first 327 base pairs in the 5' flanking region. The
participation of at least two of these cis-acting elements is necessary
to achieve maximal induction of transcription. We also show that there
is lack of specificity in the signaling pathways that mediate
COX-2 gene expression through each of these promoter sites.
LPS can activate different MAPK pathways to induce COX-2 gene transcription: through NF
B via ERK-2, p38, and JNK pathways, through NF-IL6 via a p38 pathway, and through CRE via ERK-2 and JNK
pathways. Moreover, PKC-
signaling seems to mediate transcription after LPS treatment through all three promoter sites. Therefore, individual signaling pathways, such as ERK-2, p38, JNK, or PKC-
, appear to be sufficient to mediate COX-2 gene transcription
by virtue of their ability to recruit transcription factors to at least
two promoter sites.
Previous studies have shown that pharmacological inhibition of
NFB activation decreases COX-2 expression in macrophage-like cells (19), and COX-2 transcription through the NF
B
promoter site also has been demonstrated (20). The role of other
cis-acting elements and their cooperation in promoting transcription
was not evaluated in those studies. Other investigators have attempted to characterize the signaling pathways leading to COX-2
transcriptional activation. In one study, MAPK signaling was found to
be necessary in LPS-mediated activation of the COX-2 NF
B
promoter site (19), but other studies could not confirm this
finding (29). A recent report on the transcriptional regulation of the
murine COX-2 gene in LPS-treated RAW 246.7 cells showed that
two NF-IL6 sites are necessary for maximal induction of transcription
and that without a functional CRE site transcription cannot occur (21).
In addition, activation of a MEKK-1/JNK pathway was necessary
for induction of murine COX-2 transcription. This study
differs from ours in that specificity rather than redundancy, both at
the signaling pathway and promoter levels, characterizes LPS-mediated
induction of COX-2 transcription. The differences in results
may reflect differences in transcriptional regulation between the
murine promoter, studied by these investigators, and the human
promoter, utilized in our work.
In summary, we have presented experiments with mutant COX-2
promoter reporter constructs and with expression vectors for wild type
or dominant negative mutants for MAPK and PKC isoforms. These experiments demonstrate the redundant rather than specific character of
the cell signaling leading to COX-2 gene transcription in
endotoxin-treated macrophage/monocytic cells. Redundancy in the
signaling pathways and promoter elements regulating COX-2
gene transcription may constitute an important mechanism ensuring
increased levels of COX-2 in macrophage/monocytic cells during inflammation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK50201 (to J. M. D.).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.
¶ Recipient of a Fellowship Award from the Cancer Research Foundation of America.
** To whom correspondence should be addressed: The New York Presbyterian Hospital-Cornell Campus, Dept. of Surgery, Rm. F-739, York Ave., New York, NY 10021. Tel.: 212-746-5143; Fax: 212-746-8753; E-mail: jmdaly@cornell.med.edu.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.M005077200
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ABBREVIATIONS |
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The abbreviations used are:
COX, cyclooxygenase;
PG, prostaglandin;
NFB, nuclear factor
B;
NF-IL6, nuclear
factor interleukin-6;
CRE, cyclic AMP-responsive element;
MAPK, mitogen-activated protein kinase;
PKC, protein kinase C;
ERK, extracellular signal-regulated kinase;
JNK, c-Jun
NH2-terminal kinase;
LPS, lipopolysaccharide.
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