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1
Institute of Parasitology, McGill University, Ste. Anne de Bellevue, Quebec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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released by LPS-stimulated U937 human macrophages in the
regulation of COX-2. Macrophages exposed to LPS showed a rapid and
sustained expression of COX-2 mRNA and protein for up to 48 h,
whereas PGE2 production was notably enhanced only after
12 h. LPS increased COX-2 gene transcription and
activation of the transcription factor NF-
B in a transient manner.
LPS-treated macrophages produced high levels of TNF- and moderate
amounts of IL-1ß protein. However, neutralizing Abs against these
cytokines had no effect on COX-2 mRNA and protein expression, nor did
they affect the stability of COX-2 mRNA. Interestingly, in the presence
of LPS or exogenous IL-1ß, COX-2 transcripts were stabilized, and
actinomycin D inhibited their degradation. Only when LPS or IL-1ß was
removed did COX-2 mRNA decay with a t1/2 of
5 h. In contrast, dexamethasone promoted a faster decay of the
LPS-induced COX-2 transcripts (t1/2 =
2.5 h). These results clearly demonstrate that LPS can regulate
COX-2 at both transcriptional and posttranscriptional levels
independently from endogenous IL-1ß and TNF- in human
macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß, and
important secondary mediators such as PGs and platelet-activating
factor (1). PGs are lipid mediators involved in
vasodilation, pain, and fever (2, 3). A major source of
PGs during sepsis are macrophages (1). After
stimulation with LPS, these cells secrete PGs (mainly
PGE2) and proinflammatory cytokines, which, in
turn, act in an autocrine or paracrine manner to regulate the host
response (4).
A rate-limiting step in the synthesis of PGs is the enzyme
cyclooxygenase (COX).3 To date,
two isoforms of this enzyme have been described: COX-1, which is
constitutively expressed in most human tissues (5), and
COX-2, whose expression is readily induced by inflammatory stimuli such
as LPS and cytokines in a variety of cells (6, 7).
Interestingly, COX-2 is regulated by cytokines in a rather cell
type-specific manner. IL-1 is a potent stimulus for the induction of
COX-2 expression in human endothelial cells, human synovial
fibroblasts, and rat mesangial cells (8, 9, 10). In contrast,
TNF- stimulates COX-2 mRNA expression in mouse osteoblasts and
bovine endothelial cells (11, 12), but has no effect by
itself on human monocytes (13). IFN-
, the main
macrophage-activating cytokine (14), can potentiate the
effect of LPS on COX-2 expression in mouse macrophages
(15), but has no effect on COX activity when
simultaneously administered with LPS to human monocytes
(16). Furthermore, we have reported that IFN-
down-regulates COX-2 expression induced by IL-1ß in human macrophages
(17).
The induction of COX-2 expression by IL-1 occurs at the transcriptional
level (8, 17, 18). However, this cytokine also has the
ability to stabilize COX-2 transcripts by a still poorly understood
mechanism (19, 20). The transcriptional regulation of
COX-2 by IL-1ß in human cells and by TNF- in murine osteoblasts
involves activation of the transcription factor NF-B (11, 18, 21). Recent studies have shown that NF-B is also involved in
the induction of COX-2 expression in response to LPS in human and mouse
macrophages (22, 23), indicating a transcriptional
mechanism for this potent stimulus. LPS-treated macrophages release the
proinflammatory cytokines IL-1ß and TNF-. However, the roles these
proinflammatory cytokines play in the regulation of COX-2 are not
known. We hypothesized that these cytokines may participate in an
autocrine manner in the induction or maintenance of COX-2 expression in
human macrophages.
To test this hypothesis, we determined the effect of neutralizing
polyclonal Abs against IL-1ß and TNF- on COX-2 expression in
LPS-stimulated macrophages. LPS induced COX-2 gene
transcription in a transient manner and stabilized the mRNA. In the
presence of neutralizing Abs against IL-1ß and TNF- there was no
effect on COX-2 mRNA and protein expression, and the stability of COX-2
mRNA was unaffected. These results clearly demonstrate that LPS can
trigger COX-2 mRNA and protein expression and maintain high production
of PGs by macrophages without the influence of the released
proinflammatory cytokines, IL-1ß and TNF-.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS (from Escherichia coli, serotype 0111, 4% phenol
extract), phorbol 12-myristate 13-acetate (PMA), dexamethasone (DEX),
3-(4,5-dimethyl-thiazol-2-yl-)2,5-di-phenyltetrazolium bromide (MTT),
and 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) diammonium
salt (ABTS) were purchased from Sigma (St. Louis, MO). Actinomycin D
(AD) was obtained from Life Technologies (Burlington, Canada).
Recombinant human IL-1ß and TNF-, goat anti-human IL-1ß,
goat anti-human TNF-, rabbit anti-human IL-1ß, and normal
goat IgG Abs were purchased from R&D Systems (Minneapolis, MN). Rabbit
anti-human TNF- Ab was purchased from Endogen (Woburn, MA). RPMI
1640 with L-glutamine was obtained from Life
Technologies and was completed with 100 U/ml of penicillin, 100 µg/ml
streptomycin sulfate, 20 mM HEPES (Sigma), and 10% heat-inactivated
FCS (HyClone, Logan, UT).
Cell culture
The human macrophage cell line U937 was grown in complete RPMI 1640. Cells were kept at 37°C in 5% CO2-95% air and harvested at the log phase of growth. To differentiate the cells into adherent macrophages, they were adjusted to 1 x 106 cells/ml and incubated in 24-well plates (1 x 106 cells/well) for 3 days in the presence of 10 nM PMA. Macrophages were made quiescent in fresh complete RPMI without PMA for 24 h before stimulation with LPS or cytokines. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2-yl-)2,5-di-phenyltetrazolium bromide (MTT) method (24) and was 90% after 12 h of incubation with AD (10 µg/ml).
Northern blot and immunoblot analyses
The quantification of COX-2 and COX-1 mRNA was performed by Northern blot analysis as previously described (17), using actin as a housekeeping gene to normalize the values obtained for COX-2. The expression of COX-2 and COX-1 proteins was assessed by immunoblot as previously reported (17), using polyclonal Abs prepared against the complete ovine COX-1 and COX-2 proteins. The Abs were gifts from Dr. G. O’Neill (Merck Frosst, Kirkland, Canada).
Nuclear run-on assays
The protocol was previously described (17).
Briefly, the cells (5 x 107) were lysed,
and the nuclear pellets were obtained by centrifugation. The nuclei
were resuspended in 100 µl of storage buffer and frozen in liquid
nitrogen until used. The nascent chains of newly synthesized mRNA were
elongated in vitro and labeled with
[-32P]UTP. The radiolabeled RNA was then
extracted with Trizol (Life Technologies) and hybridized onto
nitrocellulose membranes containing the following linearized plasmids:
pcDNACOX-1, pcDNACOX-2, pBA-1, pcDNA1Amp, and pBR322. The latter two
plasmids were the controls without the COX and actin fragments,
respectively. The membranes were prehybridized for 3 h and then
hybridized for 48 h; both steps were conducted at 65°C. The
membranes were exposed for autoradiography for 15 days. The films were
scanned, and densitometric analysis was performed as previously
reported (17).
EMSAs
The protocol for nuclear protein extraction was as described
previously (25) with the modifications reported by Zhang
et al. (26). For the DNA-protein binding reaction, nuclear
extracts (10 µg) were incubated for 30 min at room temperature with
1–2 ng (200,000–600,000 cpm) of 32P-labeled
double-stranded oligonucleotides in a binding buffer (20 µl final
volume) containing 10 mM Tris-HCl (pH 7.4), 40 mM NaCl, 1 mM EDTA, 1 mM
2-ME, 0.1% Nonidet P-40, 4% glycerol, 1 µg/µl BSA, and 0.2
µg/µl poly(dI/dC) (Pharmacia Biotech, Piscataway, NJ). For cold
competition binding assays, the nuclear extracts were first incubated
with a 10- or 50-fold excess of unlabeled double-stranded
oligonucleotides for 20 min, followed by the addition of the
radiolabeled oligonucleotides, and incubated for 30 min more at room
temperature. Reactions were stopped with 5 µl of loading buffer (25
mM Tris-HCl (pH 7.5), 50% glycerol, and bromophenol blue). Samples
were subjected to electrophoresis in 5% polyacrylamide gels in 0.25x
Tris-borate/EDTA (TBE) buffer at 150 V for 2 h at 4°C. Gels were
pre-electrophoresed for 1 h at 140 V. The synthetic
single-stranded oligonucleotides (Life Technologies) were annealed by
boiling the complementary oligonucleotides for 10 min in a buffer
containing 10 mM Tris-HCl (pH 7.5) and 50 mM NaCl, and were cooled to
room temperature overnight. The sequences of the oligonucleotides used
were as previously described (18), and the consensus
sequences for NF-B are underlined: distal (upstream) NF-B site
forward, 5'-GGA GAG GGG ATT CCC TGC GCC-3'; distal NF-B
site reverse, 5'-CAG GGA ATC CCC TCT CCC GCC G-3'; proximal NF-B
site forward, 5'-AGT GGG GAC TAC CCC CTC TGC TCC-3'; and
proximal (downstream) NF-B site reverse, 5'-CAG AGG GGG TAG TCC CCA
CTC TCC T-3'. Once annealed, the double-stranded oligonucleotides were
labeled by filling the overhangs with [32P]GTP
(>3000 Ci/mmol; ICN, Costa Mesa, CA) and 2 U of Klenow polymerase
(Life Technologies) in a low salt buffer (50 mM Tris-HCl (pH 7.5), 10
mM MgCl2, 1 mM DTT, and 50 µg/ml BSA) plus a
mixture of dATP, dCTP, and dTTP.
ELISA for IL-1ß and TNF- determination
The amounts of IL-1ß and TNF- released in the supernatants
of stimulated and nonstimulated macrophages were measured by ELISA as
previously described (27) with minor modifications. We
used 3% BSA in PBS to block the nonspecific binding sites and 1% BSA
in PBS to dilute the Abs instead of FCS in PBS. The standard curves
were prepared with twofold dilutions of rIL-1ß (4–500 pg/ml) or
TNF- (8–1000 pg/ml) in 3% BSA-PBS. The supernatants were
diluted 1:3 in 3% BSA-PBS for the TNF- determination,
whereas undiluted supernatants were used to quantify IL-1ß.
PGE2 production
PGE2 production was measured by enzyme immunoassay (Cayman Chemicals, Ann Arbor, MI) after different times of incubation with LPS.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We and others (7, 17) have shown that IL-1ß can
stimulate a sustained accumulation of COX-2 mRNA for up to 12 h.
To determine whether LPS can induce a similar expression of COX-2 mRNA,
we investigated the kinetics of COX-2 mRNA and protein expression in
human macrophages stimulated with LPS for different times. LPS induced
a time-dependent increase in COX-2 mRNA levels (Fig. 1
A). COX-2 transcripts were
detected as early as 2 h, peaked at 6–12 h, and started to
decline slightly after 24 h. Detectable levels of COX-2 protein
appeared after 4–6 h and remained stable up to 24 h (Fig. 1B). Regardless of conditions, COX-1 protein remained stable
throughout the incubation period (Fig. 1B) and correlated
with the levels of COX-1 mRNA (Fig. 1A). The amounts of
PGE2 released in the supernatants of stimulated
cells were 395 and 760 pg/ml after 12 and 24 h, respectively,
whereas nonstimulated cells released 280 pg/ml after 6 h. These
results indicate that LPS is a potent stimulus for the induction and
sustained expression of COX-2 mRNA and protein and for
PGE2 production by human macrophages.
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Nuclear run-on analysis was performed to determine whether the
sustained expression of COX-2 mRNA induced by LPS was the result of a
steady increase in gene transcription. Macrophages were exposed to LPS
for 1 or 12 h, and newly synthesized mRNA chains were radiolabeled
and hybridized to specific probes as described in Materials and
Methods. As shown in Fig. 2A, LPS caused a 5.4-fold
increase in the transcription rate of the COX-2 gene after
1 h, which returned to basal levels after 12 h of
stimulation. In contrast, the transcription rate of COX-1
remained unaltered following LPS treatment. To determine whether the
transcriptional induction correlated with the activation of the
transcription factor NF-B, EMSA was performed using the distal
(upstream) NF-B sequence in the COX-2 promoter as a probe
(18). A DNA-protein complex was formed by the nuclear
protein extracts of cells treated with LPS for 1 h, but
disappeared by 12 h of incubation. The complex was specific, as
its formation was inhibited by preincubation of the nuclear extracts
with a 10- or 50-fold excess of unlabeled NF-B probe (Fig. 2B). Under these conditions we did not observe any
DNA-protein complex when the proximal NF-B sequence was used as a
probe (data not shown). These data indicate that the induction of
COX-2 gene transcription by LPS is rapid but transient and
correlates with the activation of the transcription factor NF-B.
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production by LPS-stimulated human macrophages
The kinetics of IL-1ß and TNF- protein released were
determined by ELISA on the supernatants of macrophages stimulated with
LPS for different times. TNF- was secreted early (400 pg/ml after
2 h; data not shown), reached maximum levels by 6 h, and
remained high even after 48 h (Table I). In contrast, IL-1ß increased
significantly only after 12 h and continued to accumulate for up
to 48 h. These results indicated that in response to LPS, U937
human macrophages release proinflammatory cytokines (IL-1ß and
TNF-) in a similar fashion as blood-derived monocytes
(27). Based on the kinetics of the release of these
cytokines, it is unlikely that they participate in the early
transcriptional activation of COX-2. However, they may play a role in
the maintenance of high COX-2 levels following LPS stimulation.
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Exogenous IL-1ß stimulates a dose- and time-dependent increase
in COX-2 mRNA and protein expression in human macrophages
(17). To determine whether IL-1ß released by
LPS-stimulated macrophages could have an autocrine effect on the
expression of COX-2 mRNA, the following experiment was performed.
Macrophages were stimulated for different times with LPS (6, 12, and
24 h) or with IL-1ß (only for 12 h) in the presence or the
absence of neutralizing anti-IL-1ß Abs. As shown in Fig. 3A, neutralizing Abs to
IL-1ß did not affect COX-2 mRNA expression induced by LPS regardless
of the time of stimulation. In contrast, the neutralizing Abs inhibited
the expression of COX-2 induced by IL-1ß after 12 h. As
expected, the control Ab (normal goat IgG) had no effect on either LPS-
or IL-1ß-induced COX-2 mRNA expression. Similar results were observed
when the levels of COX-2 protein were evaluated by immunoblot (Fig. 3B). These data clearly suggest that endogenous IL-1ß does
not contribute to the maintenance of the steady state levels of COX-2
mRNA induced by LPS.
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does not participate in the
expression of COX-2 induced by LPS
We next determined whether endogenous TNF- secreted by
LPS-stimulated cells could contribute to the expression of COX-2.
Macrophages were treated with LPS for 12, 24, and 48 h in the
presence or the absence of neutralizing Abs against TNF-. As shown
in Fig. 4, Abs against TNF- did not
alter COX-2 mRNA expression regardless of the time examined. Even when
the cells were exposed to LPS and anti-TNF- Abs for shorter
periods (1 and 3 h), the expression of COX-2 mRNA induced by LPS
was not affected (data not shown). In contrast to IL-1ß, exogenous
TNF- (up to 50 ng/ml) did not stimulate COX-2 mRNA expression in
human macrophages. Taken together, these results suggest that the
proinflammatory cytokines IL-1ß and TNF- secreted in response to
LPS stimulation do not play a costimulatory role in the maintenance of
COX-2 mRNA.
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We and others have shown that IL-1ß can increase the rate of
transcription of the COX-2 gene (17, 18).
Moreover, IL-1ß stabilizes the transcripts, inhibiting their rapid
turnover by an unclear mechanism (19, 20). The data in
Fig. 4
clearly show that COX-2 mRNA levels were elevated after 24 and
48 h of LPS stimulation even though the transcription rate
declined to basal levels by 12 h (Fig. 2). Accordingly, we
investigated whether LPS can regulate COX-2 expression at the
posttranscriptional level. To determine the stability of LPS-induced
COX-2 mRNA, macrophages were stimulated with LPS for 12 h followed
by the addition of AD, and the fate of the COX-2 mRNA was evaluated at
different times. In a second group of cells, LPS was removed from the
cell medium after 12-h stimulation, AD was added or not, and the levels
of COX-2 mRNA were evaluated thereafter. As shown in Fig. 5A, only when LPS was removed
during the chase period and in the absence of AD did the levels of
COX-2 mRNA decrease rapidly and significantly over time. Fig. 5B shows similar results from experiments in which
macrophages were stimulated with IL-1ß for 12 h. The half-lives
of the COX-2 transcripts following the removal of LPS and IL-1ß were
5.04 and 5.30 h, respectively. These results suggest that LPS can
stabilize COX-2 mRNA and that active transcription is necessary for its
decay.
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do not affect the
stability of COX-2 mRNA
To assess whether the proinflammatory cytokines IL-1ß and
TNF- released by LPS-stimulated cells can modulate the stability of
the COX-2 transcripts, macrophages were exposed to LPS for 12 h
followed by the addition of neutralizing Abs against the cytokines. As
shown in Fig. 6, the presence of the Abs
during the chase period (up to 8 h) did not affect the stability
of COX-2 transcripts, clearly indicating that endogenous IL-1ß and
TNF- do not participate in the posttranscriptional regulation of
COX-2 by LPS.
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Recent evidence indicates that DEX can inhibit COX-2 expression at
the transcriptional level (22), but also increases the
turnover rate of the COX-2 transcripts induced by IL-1ß
(28). To determine whether DEX could promote the decay of
COX-2 mRNA induced by LPS, macrophages were stimulated for 12 h
followed by the addition of DEX or DEX plus AD, and the levels of COX-2
mRNA were evaluated by Northern blot. Fig. 7 shows that COX-2 transcripts were
rapidly degraded in the presence of DEX, whereas their level
remained high throughout the chase period in the absence of DEX or in
the presence of DEX plus AD. The half-life of LPS-induced COX-2
transcripts was 2.5 h. These results indicate that DEX promotes
the rapid decay of the stable COX-2 transcripts induced by LPS and that
such an effect depends on active transcription.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß released by LPS-stimulated human macrophages on the
expression of COX-2. We found that neutralizing Abs against TNF- and
IL-1ß did not block the induction or the prolonged expression of
COX-2 mRNA and protein after LPS stimulation. Furthermore, LPS
transiently activated COX-2 gene transcription and
stabilized the COX-2 transcripts. Interestingly, COX-2 mRNA decay was
inhibited by AD, but was promoted by DEX. Removal of LPS from the
medium also promoted the degradation of COX-2 mRNA with a similar
turnover rate as IL-1ß-induced transcripts.
We have previously shown that LPS can stimulate the expression of COX-2
mRNA in U937 human macrophages (17, 29). In the present
report we showed a sustained expression of COX-2 mRNA even after
48 h. Likewise, the COX-2 protein was detected after 6 h, and
high levels were still observed after 24 h (Fig. 1A)
and 48 h (data not shown). However, high
PGE2 levels were not observed until 12 and
24 h. The lag period between the presence of the protein and the
product may reflect the lack of substrate availability or a downstream
enzyme. This may be due to a delayed expression of either the cytosolic
PLA2 (30, 31) or the putative
inducible PGE2 synthase, respectively (30, 32).
The presence of polyclonal Abs against TNF- and IL-1ß had no
effect on the expression levels of COX-2 mRNA and protein in
LPS-stimulated cells. This is in contrast with other studies that have
shown that neutralizing Abs against TNF- and IL-1ß can
significantly inhibit COX activity measured by the production of
6-oxo-PGF2 in bovine endothelial cells
(12). In that system TNF- played a major role in the
induction of COX-2 after LPS stimulation. Moreover, exogenous TNF-
enhanced COX activity. In contrast to IL-1ß, TNF- alone did not
induce COX-2 expression in our study. Even though large quantities of
TNF- were produced by U937 macrophages following LPS stimulation,
there was no apparent participation of this cytokine in COX-2 induction
or in the maintenance of the high mRNA levels. Thus, these results
indicate that proinflammatory cytokines can exert distinct and specific
effects on different cell types. For example, TNF- stimulates COX-2
expression in human mesothelial cells (33) and synovial
fibroblasts (34), while it has no effect on human
macrophages (29) and monocytes (13) unless
used at very high concentrations (35). This difference in
species and cell type may be derived from the diversity in the
sequences of the promoter region of the COX-2 gene and the
class and number of receptors expressed on the cells. Similarly, the
lack of effect of neutralizing Abs against IL-1ß on COX-2 mRNA levels
also indicated that endogenous IL-1ß does not play a role in the
regulation of COX-2. Although exogenous IL-1ß easily induces COX-2
expression in our system (17), the lag time for IL-1ß
release may hamper the contribution of this cytokine to the rapid
transcriptional activation by LPS. Likewise, the amount of endogenous
IL-1ß released in response to LPS may not be high enough to
participate in the induction of COX-2 mRNA or protein expression. We
have found that a concentration of at least 100–500 pg/ml of IL-1ß
is necessary to induce detectable levels of COX-2 mRNA. Moreover, we
did not observe a significant synergistic effect when the macrophages
were treated simultaneously with LPS plus IL-1ß (our unpublished
observations).
At present we cannot rule out the participation of other endogenous
cytokines in COX-2 regulation. Recent evidence suggests that endogenous
GM-CSF is involved in LPS-stimulated COX-2 protein expression in human
monocytes (36). This effect occurs through the delayed
activation of STAT5 (90 min). Although endogenous GM-CSF may be playing
a role in the maintenance of high levels of COX-2 mRNA or protein, our
run-on assays clearly indicated that LPS promptly activates the
transcription of the COX-2 gene within 60 min. This was
consistent with the activation of NF-B as shown by EMSA after 1
h of LPS stimulation (Fig. 2B). The involvement of two
NF-B motifs present in the COX-2 promoter, after LPS or
IL-1ß stimulation, has been reported in mouse macrophages
(23), human pulmonary cells (18), and U937
human macrophages (22). In previous studies, the presence
of p50 and p65 NF-B proteins in the complexes was shown by
supershift assays (18, 22). Inoue and Tanabe
(22) observed a DNA-protein complex using the proximal
NF-B motif as a probe; interestingly, we did not detect any complex
using that same probe. A likely explanation for this is the different
binding affinities of the p50/p65 NF-B heterodimer for the two DNA
motifs (18). The involvement of other
cis-elements, such as NF for IL-6 expression and cAMP
response element sites, in the regulation of the human COX-2
gene by LPS has also been reported (37). The contribution
of other endogenous cytokines to LPS-induced COX-2 expression and
the transcription factors involved requires further investigation.
The nuclear run-on assays indicated that LPS exerts a rapid but
transient transcriptional activation of the COX-2 gene. The
high levels of COX-2 transcripts observed after 48 h of
stimulation, however, also suggested a posttranscriptional modulation.
COX-2 was discovered as an immediate-early gene that can be
induced rapidly and transiently by serum and mitogens (38, 39). Moreover, the 3'-UTR of COX-2 mRNA has 22 copies of the
pentamer AUUUA (40), which is considered to be involved in
the instability of mRNA for some cytokines and oncogenes (41, 42). Despite these findings, several reports have demonstrated
that IL-1 can stabilize COX-2 transcripts (8, 17, 19). Our
results indicated that LPS is able to stabilize COX-2 mRNA in a similar
manner as IL-1ß. We also presented evidence showing that endogenous
IL-1ß and TNF- released after LPS stimulation did not contribute
to the stabilization of COX-2 transcripts. The addition of AD, which in
other systems allows determination of the mRNA turnover rate, inhibited
the degradation of COX-2 transcripts induced by LPS or IL-1ß. Only
when the stimuli were removed did the levels of COX-2 mRNA decline. A
potential explanation for this phenomenon is that both LPS and IL-1ß
induce the expression or activation of labile molecule(s) involved in
COX-2 mRNA stabilization. When the stimulus is removed, this unstable
molecule(s) is degraded or inactivated, and therefore, COX-2 mRNA is
also degraded, probably by a short-lived but constitutively transcribed
RNase. However, following removal of the stimulus in the presence of
AD, the basal transcription of the RNase is inhibited, and
consequently, the degradation of COX-2 transcripts is suppressed.
This hypothesis is consistent with evidence indicating the
existence of proteins that bind to the 3'-UTR and have the potential
ability to stabilize different mRNAs (43, 44, 45). This is
supported by the identification of stabilizing cis elements
in the 3'-UTR (20) and of cytosolic proteins that bind to
COX-2 mRNA after IL-1ß stimulation (19). These elements
may be acting in concert to prolong the half-life of the transcripts.
The inhibition of COX-2 mRNA decay by AD is not specific for our
system, because a similar effect has been observed for the
urokinase-type plasminogen activator and GM-CSF transcripts, although
no mechanism has been proposed for it (46, 47).
In contrast to the stabilizing effect of LPS or IL-1ß, DEX may be
responsible for the induction or activation of RNases or other proteins
that mediate COX-2 mRNA turnover. The destabilizing effect of DEX on
IL-1ß-induced COX-2 transcripts has been previously shown
(28). Interestingly, the half-life of the 4.6-kb COX-2
mRNA isoform in the presence of DEX is 
3 times shorter in lung
fibroblasts (28) than in IL-1ß or LPS-stimulated
macrophages (Ref. 17 and this report). Although we have no
explanation for this, the machinery for mRNA degradation may be
differentially expressed depending on the cell type. Recently, it has
been reported that the faster degradation of COX-2 transcripts
induced by DEX in epithelial cells depends on active transcription and
translation, and involves shortening of the COX-2 poly(A) tail
(48).
In summary, we have demonstrated that LPS can regulate the
COX-2 gene by transcriptional and posttranscriptional
mechanisms in human macrophages. First, we showed that the
proinflammatory cytokines IL-1ß and TNF- produced by
LPS-stimulated cells did not contribute to either the maintenance or
the stability of COX-2 mRNA. Second, LPS promoted the transcription of
the COX-2 gene and the stability of COX-2 mRNA in a similar
manner as IL-1ß. Finally, active transcription in the cells or the
presence of DEX was required for the fast degradation of COX-2 mRNA.
Elucidation of the molecular mechanisms underlying the regulation of
COX-2 at both transcriptional and posttranscriptional levels
may open new avenues in the design of more specific strategies for the
treatment of inflammatory diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kris Chadee, Institute of Parasitology, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, P.Q., Canada H9X 3V9. E-mail address:
3 Abbreviations used in this paper: COX, cyclooxygenase; PMA, phorbol 12-myristate 13-acetate; AD, actinomycin D; DEX, dexamethasone; 3'-UTR, 3'-untranslated region.
Received for publication December 23, 1998. Accepted for publication May 4, 1999.
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 R. Horrillo, A. Planaguma, A. Gonzalez-Periz, N. Ferre, E. Titos, R. Miquel, M. Lopez-Parra, J. L. Masferrer, V. Arroyo, and J. Claria Comparative Protection against Liver Inflammation and Fibrosis by a Selective Cyclooxygenase-2 Inhibitor and a Nonredox-Type 5-Lipoxygenase Inhibitor J. Pharmacol. Exp. Ther., December 1, 2007; 323(3): 778 - 786. [Abstract] [Full Text] [PDF]
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 Y.-J. Kang, B. A. Wingerd, T. Arakawa, and W. L. Smith Cyclooxygenase-2 Gene Transcription in a Macrophage Model of Inflammation J. Immunol., December 1, 2006; 177(11): 8111 - 8122. [Abstract] [Full Text] [PDF]
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 Y. Shibata, J. Gabbard, M. Yamashita, S. Tsuji, M. Smith, A. Nishiyama, R. A. Henriksen, and Q. N. Myrvik Heat-killed BCG induces biphasic cyclooxygenase 2+ splenic macrophage formation--role of IL-10 and bone marrow precursors J. Leukoc. Biol., September 1, 2006; 80(3): 590 - 598. [Abstract] [Full Text] [PDF]
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 T Reding, D Bimmler, A Perren, L-K Sun, F Fortunato, F Storni, and R Graf A selective COX-2 inhibitor suppresses chronic pancreatitis in an animal model (WBN/Kob rats): significant reduction of macrophage infiltration and fibrosis Gut, August 1, 2006; 55(8): 1165 - 1173. [Abstract] [Full Text] [PDF]
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 G. K. Harris, Y. Qian, S. S. Leonard, D. C. Sbarra, and X. Shi Luteolin and Chrysin Differentially Inhibit Cyclooxygenase-2 Expression and Scavenge Reactive Oxygen Species but Similarly Inhibit Prostaglandin-E2 Formation in RAW 264.7 Cells J. Nutr., June 1, 2006; 136(6): 1517 - 1521. [Abstract] [Full Text] [PDF]
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 M. Joo, Y. S. Hahn, M. Kwon, R. T. Sadikot, T. S. Blackwell, and J. W. Christman Hepatitis C Virus Core Protein Suppresses NF-{kappa}B Activation and Cyclooxygenase-2 Expression by Direct Interaction with I{kappa}B Kinase {beta} J. Virol., June 15, 2005; 79(12): 7648 - 7657. [Abstract] [Full Text] [PDF]
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Y. Shibata, A. Nishiyama, H. Ohata, J. Gabbard, Q. N. Myrvik, and R. A. Henriksen Differential effects of IL-10 on prostaglandin H synthase-2 expression and prostaglandin E2 biosynthesis between spleen and bone marrow macrophages J. Leukoc. Biol., April 1, 2005; 77(4): 544 - 551. [Abstract] [Full Text] [PDF]
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 S. Fiorucci, A. Mencarelli, A. Meneguzzi, A. Lechi, B. Renga, P. del Soldato, A. Morelli, and P. Minuz Co-administration of nitric oxide-aspirin (NCX-4016) and aspirin prevents platelet and monocyte activation and protects against gastric damage induced by aspirin in humans J. Am. Coll. Cardiol., August 4, 2004; 44(3): 635 - 641. [Abstract] [Full Text] [PDF]
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 D. E. Shifflett, F. G. Bottone Jr., K. M. Young, A. J. Moeser, S. L. Jones, and A. T. Blikslager Neutrophils augment recovery of porcine ischemia-injured ileal mucosa by an IL-1{beta}- and COX-2-dependent mechanism Am J Physiol Gastrointest Liver Physiol, July 1, 2004; 287(1): G50 - G57. [Abstract] [Full Text] [PDF]
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C. Hou, T. Kirchner, M. Singer, M. Matheis, D. Argentieri, and D. Cavender In Vivo Activity of a Phospholipase C Inhibitor, 1-(6-((17{beta}-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), in Acute and Chronic Inflammatory Reactions J. Pharmacol. Exp. Ther., May 1, 2004; 309(2): 697 - 704. [Abstract] [Full Text] [PDF]
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 S. J. Cok, S. J. Acton, A. E. Sexton, and A. R. Morrison Identification of RNA-binding Proteins in RAW 264.7 Cells That Recognize a Lipopolysaccharide-responsive Element in the 3-Untranslated Region of the Murine Cyclooxygenase-2 mRNA J. Biol. Chem., February 27, 2004; 279(9): 8196 - 8205. [Abstract] [Full Text] [PDF]
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C. C. Bowman and K. L. Bost Cyclooxygenase-2-Mediated Prostaglandin E2 Production in Mesenteric Lymph Nodes and in Cultured Macrophages and Dendritic Cells after Infection with Salmonella J. Immunol., February 15, 2004; 172(4): 2469 - 2475. [Abstract] [Full Text] [PDF]
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 T. G. Tessner, F. Muhale, S. Schloemann, S. M. Cohn, A. R. Morrison, and W. F. Stenson Ionizing radiation up-regulates cyclooxygenase-2 in I407 cells through p38 mitogen-activated protein kinase Carcinogenesis, January 1, 2004; 25(1): 37 - 45. [Abstract] [Full Text] [PDF]
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 B.-S. Sheu, H.-B. Yang, S.-M. Sheu, A.-H. Huang, and J.-J. Wu Higher Gastric Cycloxygenase-2 Expression and Precancerous Change in Helicobacter pylori-Infected Relatives of Gastric Cancer Patients Clin. Cancer Res., November 1, 2003; 9(14): 5245 - 5251. [Abstract] [Full Text] [PDF]
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M. Demasi, L. G. Cleland, R. J. Cook-Johnson, G. E. Caughey, and M. J. James Effects of Hypoxia on Monocyte Inflammatory Mediator Production: DISSOCIATION BETWEEN CHANGES IN CYCLOOXYGENASE-2 EXPRESSION AND EICOSANOID SYNTHESIS J. Biol. Chem., October 3, 2003; 278(40): 38607 - 38616. [Abstract] [Full Text] [PDF]
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T. G. Tessner, F. Muhale, S. Schloemann, S. M. Cohn, A. Morrison, and W. F. Stenson Basic fibroblast growth factor upregulates cyclooxygenase-2 in I407 cells through p38 MAP kinase Am J Physiol Gastrointest Liver Physiol, February 1, 2003; 284(2): G269 - G279. [Abstract] [Full Text] [PDF]
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Y.-Q. Zhou, Y.-Q. Chen, J. H. Fisher, and M.-H. Wang Activation of the RON Receptor Tyrosine Kinase by Macrophage-stimulating Protein Inhibits Inducible Cyclooxygenase-2 Expression in Murine Macrophages J. Biol. Chem., October 4, 2002; 277(41): 38104 - 38110. [Abstract] [Full Text] [PDF]
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K. J. Claycombe, D. Wu, M. Nikolova-Karakashian, H. Palmer, A. Beharka, K. E. Paulson, and S. N. Meydani Ceramide Mediates Age-associated Increase in Macrophage Cyclooxygenase-2 Expression J. Biol. Chem., August 16, 2002; 277(34): 30784 - 30791. [Abstract] [Full Text] [PDF]
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 S. Eligini, M. Brambilla, C. Banfi, M. Camera, L. Sironi, S. S. Barbieri, J. Auwerx, E. Tremoli, and S. Colli Oxidized phospholipids inhibit cyclooxygenase-2 in human macrophages via nuclear factor-{kappa}B/I{kappa}B- and ERK2-dependent mechanisms Cardiovasc Res, August 1, 2002; 55(2): 406 - 415. [Abstract] [Full Text] [PDF]
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M. M. Hayes, B. R. Lane, S. R. King, D. M. Markovitz, and M. J. Coffey Peroxisome Proliferator-activated Receptor gamma Agonists Inhibit HIV-1 Replication in Macrophages by Transcriptional and Post-transcriptional Effects J. Biol. Chem., May 3, 2002; 277(19): 16913 - 16919. [Abstract] [Full Text] [PDF]
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S. B. Flohe, J. Bruggemann, C. Herder, C. Goebel, and H. Kolb Enhanced proinflammatory response to endotoxin after priming of macrophages with lead ions J. Leukoc. Biol., March 1, 2002; 71(3): 417 - 424. [Abstract] [Full Text] [PDF]
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D. J. Berg, J. Zhang, D. M. Lauricella, and S. A. Moore IL-10 Is a Central Regulator of Cyclooxygenase-2 Expression and Prostaglandin Production J. Immunol., February 15, 2001; 166(4): 2674 - 2680. [Abstract] [Full Text] [PDF]
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 M. Lasa, M. Brook, J. Saklatvala, and A. R. Clark Dexamethasone Destabilizes Cyclooxygenase 2 mRNA by Inhibiting Mitogen-Activated Protein Kinase p38 Mol. Cell. Biol., February 1, 2001; 21(3): 771 - 780. [Abstract] [Full Text]
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 R. Newton Molecular mechanisms of glucocorticoid action: what is important? Thorax, July 1, 2000; 55(7): 603 - 613. [Full Text]
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 J. C. G. Blanco, C. Contursi, C. A. Salkowski, D. L. DeWitt, K. Ozato, and S. N. Vogel Interferon Regulatory Factor (Irf)-1 and Irf-2 Regulate Interferon {gamma}-Dependent Cyclooxygenase 2 Expression J. Exp. Med., June 19, 2000; 191(12): 2131 - 2144. [Abstract] [Full Text] [PDF]
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