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*
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan;
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo, Japan; and
Discovery Research Laboratory I, Minase Research Institute, Ono Pharmaceutical, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IL-12 production were equipotent with PGE2 at any time
point, but the inhibitory effects of butaprost were only seen from
14 h after stimulation. PGE2 or dibutyryl cAMP alone,
but not butaprost, reduced EP4 expression, and indomethacin
reversed the LPS-induced down-regulation of EP4, indicating
that the down-regulation of EP4 is mediated by LPS-induced
PG synthesis and EP4 activation. Indeed, when we used
C3H/HeJ (LPS-hyporesponsive) macrophages, such reduction in
EP4 expression was found in the cells treated with
PGE2 alone, but not in LPS-treated cells. In contrast,
up-regulation of EP2 expression was again observed in
LPS-treated C3H/HeJ macrophages. These results suggest that
EP4 is involved mainly in the inhibition of cytokine
release, and that the gene expression of EP2 and
EP4 is differentially regulated during macrophage
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1
, and IL-12 by macrophages (8, 9).
A number of reports have stated that such inhibitory effects of
PGE2 on cytokine production are mediated by an
increase in intracellular cAMP. The effects of
PGE2 are exerted by specific receptors on the
plasma membrane of target cells (10, 11). Based on
pharmacological and cDNA cloning studies, four subtypes of PGE
receptor, EP1, EP2,
EP3, and EP4, have been
identified and have been shown to differ in their signal transduction
pathways (12, 13, 14). Previous investigations have strongly
suggested the coupling of PGE receptors to adenylate cyclase in
activated macrophages (9). We and other groups have
revealed that EP2 and EP4
receptors, both of which couple to the stimulation of adenylate
cyclase, are expressed in cultured murine macrophage-like cell lines
such as J774.1 and RAW264.7 (15, 16). However, it is not
known whether the two receptors contribute equally to the inhibition of
activated macrophage function. Furthermore, recent studies using
receptor gene knockouts have focused on the regulation of PG receptor
gene expression. In contrast, recent findings in the field of local
mediators have led us to consider the possibility that gene expression
of the receptors could also be induced by various kinds of stimuli
(17). For example, Matsuoka et al. found that PGD
receptor expression induced in alveolar epithelial cells upon Ag
challenge plays a pivotal role in the appearance of allergic responses
(18). Therefore, it is possible that PGE receptor gene
expression changes during macrophage activation. However, there have
been no reports stating such a point of view regarding PGE receptors in
macrophages. Here we designed experiments for two main purposes. First, to identify which receptor is mainly responsible for the inhibitory action of PGE2 on cytokine release, we investigated time-dependent mRNA expression of PGE receptors during LPS-induced macrophage activation, and examined the effects of EP-specific agonists on cytokine production. Second, to explore the possible mechanisms underlying regulation of receptor gene expression, we examined the effects of cAMP-elevating agents, an inhibitor of PG synthesis, and a spontaneous deficiency in LPS perception, which was recently identified to be a genetic mutation in one of the receptors, for a bacterial cell wall components. This study demonstrates that gene expression of EP4 is down-regulated via LPS-induced PG synthesis and EP4 activation, whereas that of EP2 is up-regulated by LPS stimulation via a Toll receptor-independent mechanism. Although the expression profiles suggested the possibility of a receptor switch from EP4 to EP2, the EP4 expressed initially appears mainly to contribute to the inhibition of cytokine production in macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS from Escherichia coli O55:B5, dibutyryl cAMP
(dbcAMP) and indomethacin were obtained from Sigma (St. Louis, MO).
Cycloheximide was purchased from Wako Chemicals (Osaka, Japan). The
125I-labeled cyclic AMP assay system was
purchased from Amersham (Arlington Heights, IL), and ELISA kits
for mouse TNF- and for mouse IL-12 were purchased from BioSource
International (Camarillo, CA) and R&D Systems (Minneapolis, MN),
respectively. PGE2 in the medium was quantified
using an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI).
PGE2 was purchased from Funakoshi (Tokyo, Japan).
Butaprost, an EP2-specific agonist, and ONO-604,
an EP4-specific agonist, were generated, and
their specificities were analyzed by measuring their binding affinities
to the respective EPs expressed in Chinese hamster ovary (CHO) cells
(Table I
) (19).
PGE2 bound to all EPs. Based on the results
obtained from dose dependence analyses, we used 10 nM of ONO-604 and 1
µM of butaprost. These agonists at these concentrations selectively
activate EP4 and EP2,
respectively.
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Six-week-old C3H/HeN and C3H/HeJ female mice were obtained from
Japan SLC (Hamamatsu, Japan) as specific pathogen-free animals. The
mice were killed, and their peritoneal resident macrophages were
collected by washing the peritoneal cavity with 5 ml of ice-cold
saline. After washing with PBS, the macrophages (2 x
106 cells) were seeded onto plastic Petri dishes
in Ham’s F-12 medium (LPS < 10 pg/ml; Flow Laboratories, McLean,
VA), supplemented with 10% (v/v) heat-inactivated FBS (LPS < 30
pg/ml; Life Technologies, Gaithersburg, MD). After incubation at 37°C
for 1 h, nonadherent cells were removed by repeated washing. More
than 95% of the cells were macrophages as identified by their
phagocytic activity toward zymosan, by Fc-rosette formation to the
immune complex between sheep RBC and the specific Ab (SRBC-Ab), and by
the nonspecific esterase reaction that catalyzes -naphtyl
butylate. The purity of the cell population was consistent throughout
the experiments, and the viability of the macrophages remained greater
than 98%.
The culture medium was replaced with fresh medium either with or without 100 ng/ml LPS, and the cells were then incubated at 37°C for the indicated number of hours. The cells were washed twice with PBS, scraped with a cell scraper (Costar) in ice-cold PBS, and pelleted by centrifugation (1200 rpm) at 4°C. The resultant cell pellet was washed with 0.5 ml of ice-cold PBS and stored at -80°C until use.
Immunoblotting of COX-2
Western blotting was performed as described (20). Fifty-microgram aliquots of the cell extracts were loaded onto 7.5% SDS gels (21) and electrotransferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). The proteins on the membrane were treated with a polyclonal anti-COX-2 Ab (Oxford Biomedical Research, Oxford, MI). The immune complexes on the membrane were then treated with 125I-labeled protein A at 37°C for 1 h. After repeated washing, the images were visualized with the BAS-2000 bioimage analyzer (Fuji, Tokyo, Japan).
Northern blot analysis
Total RNA was isolated from 1–1.5 x 107 macrophages by the acid guanidinium thiocyanate-phenol-chloroform method (22). The RNA (10 µg) was separated by electrophoresis on a 1.5% agarose gel and transferred onto a nylon membrane (BIODYNE; Pall, East Hills, NY). For the detection of EP2 mRNA, the EcoRI insert DNA (1.7 kb) of ML202 (23) was used as a hybridization probe. For EP4 mRNA detection, a 970-bp fragment of the mouse EP4 cDNA containing the putative first to seventh transmembrane domains was used as a probe (15). The cDNA probes used for mouse COX-1 and COX-2 were as reported previously (24). Hybridization was conducted under the conditions described previously (25), and RNA bands were visualized by autoradiography. The blots were then stripped and rehybridized with a 32P-labeled DNA probe for GAPDH. Quantification of the hybridized signals was conducted with the BAS-2000 Bio-image analyzer. Northern blot experiments were independently repeated three times. Representative results are shown in the photographs, and EP mRNA levels were normalized to GAPDH mRNA levels as the mean ± SEM of three independent experiments.
cAMP assay, PGE2 quantification, and ELISAs
cAMP levels in peritoneal macrophages were determined as reported previously (26). Cells cultured in 24-well plates (1 x 106 cells/well) were washed with 0.5 ml of Krebs-HEPES buffer (pH 7.4) with 10 µM indomethacin, and preincubated for 10 min. Reactions were started by the addition of test reagents along with 100 µM Ro-20-1724 and 10 µM indomethacin. After incubation for 10 min at 37°C, reactions were terminated by the addition of 10% trichloroacetic acid. Then the cAMP content of the cells was measured by the cAMP radioimmunoassay kit.
For the quantification of PGE2, peritoneal macrophages collected from C3H/HeN mice were preincubated in 24-well plates at a density of 1 x 106 cells/well and then incubated with 0.5 ml of medium with or without 100 ng/ml LPS for the indicated time periods. The concentration of PGE2 in the medium was then determined by an enzyme immunoassay kit, according to the manufacturer’s protocol.
For the quantification of cytokine levels, cells were seeded at
0.5 x 106 cells/well and then incubated
with 0.5 ml of medium containing 100 ng/ml LPS in the presence or
absence of PGE2 (1 µM), butaprost (1 µM), and
ONO-604 (10 nM). The culture supernatants were transferred to new tubes
for the indicated time periods, followed by the examination of TNF-
and IL-12 levels by ELISA kits, according to the manufacturer’s
protocol.
Statistical analysis
For Northern analyses, data were expressed as the mean ± SEM of three independent experiments. For the determination of PGE2, cAMP, and cytokine levels, representative data were expressed as the mean ± SEM of triplicate determinants. These experiments were independently repeated three times, and similar results were obtained. Statistical analyses were performed using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we designed experiments to 1) clarify the mode of
PGE2 action in macrophages with respect to the
PGE receptor subtypes, and 2) investigate possible changes in receptor
gene expression during macrophage activation. Peritoneal resident
macrophages isolated from C3H/HeN mice were used for this purpose.
Macrophages themselves produce a large amount of
PGE2 in response to stimuli such as LPS, and such
endogenous PGE2 is thought to affect functions of
macrophages in an autocrine manner. Therefore, we thought it is
necessary to examine the time-dependent production of
PGE2 from LPS-stimulated cells in our system
(Fig. 1A). Basal
PGE2 production in the absence of LPS treatment
for 3 h was 0.441 ng/ml. In LPS-treated macrophages, a large
amount of PGE2 was detected;
PGE2 production 3 and 7 h after LPS
treatment was 30.7 ± 1.58 and 286 ± 13 ng/ml, respectively,
which corresponds to 0.087 and 0.81 µM of PGE2,
respectively. When we examined the expression of COX isozyme mRNAs in
these cells, faint expression of COX-1 mRNA was detected in
nonstimulated macrophages, and expression levels did not change upon
LPS treatment (data not shown). In contrast, LPS treatment rapidly
induced the expression of COX-2 mRNA (Fig. 1B). Western blot
analyses again showed that COX-2 proteins were induced when cells were
treated with LPS (Fig. 1C). LPS treatment stimulates
PGE2 production in C3H/HeN macrophages, resulting
in exposure of the cells to micromolar order concentrations of
PGE2. Transcriptional induction of COX-2 may be a
possible mechanism for LPS-induced PGE2
production.
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and
IL-12 production in LPS-stimulated macrophages
PGE2 regulates a variety of functions in
macrophages, including cytokine production. This study demonstrated
that LPS-stimulated macrophages are exposed to micromolar order
concentrations of PGE2. To explore the possible
involvement of endogenous PGE2 in cytokine
release, we examined the effects of indomethacin, an inhibitor of PG
synthesis, on production of the most representative macrophage
cytokines, TNF- and IL-12. As shown in Fig. 2, TNF- was released immediately after
LPS-treatment, reaching a peak after 3 h, and then diminished,
possibly due to its rapid degradation (rapid response). In contrast,
IL-12 production gradually increased until 14 h after treatment
(slow response). When indomethacin was added simultaneously with LPS,
both TNF- and IL-12 production was enhanced; a 5.7-fold increase in
TNF- at 3 h and a 1.9-fold increase in IL-12 at 14 h was
observed compared with treatment with LPS alone at the corresponding
times. These results indicated that endogenous PGs are involved in the
suppression of cytokine production. Even in this system, exogenous
PGE2 (1 µM) was effective for the inhibition of
cytokine production, resulting in 17% of the value obtained with LPS
alone for TNF- production (3 h) and 48% for IL-12 production (14 h)
(Fig. 2). Because PGE2 is known to be coupled to
cAMP production in a number of macrophage-like cells, we further
examined the effects of dbcAMP on cytokine production. In both cases,
dbcAMP completely mimicked the effects of PGE2,
suggesting that suppression of cytokine production by
PGE2 is mediated by PGE receptors coupling to the
stimulation of adenylate cyclase, possibly by the
EP2 and/or EP4
receptors.
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To investigate whether EP2 and
EP4 receptors are expressed in nonstimulated and
LPS-stimulated macrophages, we examined the expression of
EP2 and EP4 mRNA (Fig. 3). In macrophages just before treatment
(time 0), a significant amount of EP4 mRNA was
found, but EP2 mRNA could not be detected in the
Northern blot. When these macrophages were stimulated with LPS, the
expression of EP2 mRNA transiently increased to a
maximum 3 h after treatment, then decreased by 6 h, but did
not return to values before the detection level. A slight but
significant induction of EP2 mRNA was also
observed in macrophages treated in a similar manner in the absence of
LPS. The expression level after LPS treatment is 3-fold that after
medium change only. These results indicate that the expression levels
of EP2 mRNA in peritoneal macrophages is below
detection levels before any treatment, is slightly induced upon medium
change, possibly due to other induction factors in the serum, and is
strongly enhanced by LPS. In contrast, the expression of
EP4 mRNA was down-regulated 3 h after LPS
treatment, resulting in <10% of the control level. This suppression
was still observed 6 h after LPS treatment, whereas no suppression
was observed upon medium change only. To test the expression of
EP1 and EP3 mRNA, we
performed RT-PCR analyses on the RNA samples from freshly prepared
macrophages without any treatment, macrophages treated with LPS for up
to 6 h, and macrophages subjected to medium change only (data not
shown). However, we failed to detect significant signals for
EP1 and EP3 mRNA in all
preparations. Peritoneal macrophages are unlikely to express the
EP1 and EP3 subtypes of PGE
receptors.
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To confirm the dynamic changes in the expression of these
receptors, it was considered best to investigate the
EP2 and EP4 receptors at
the protein level using Western blot analyses. However, unfortunately,
there are currently no EP2 or
EP4 receptor Abs available for Western blot
detection, and we chose to study the expression of the
EP2 and EP4 receptors using
functional analyses by examining the effects of
PGE2 and EP-selective agonists on cAMP production
in C3H/HeN macrophages at the indicated time points after LPS treatment
(Fig. 4). As shown in Fig. 1
, because
these macrophages produce PGE2 at micromolar
order concentrations, we used 1 µM of
PGE2 to induce cAMP production. We used 1 µM of
butaprost, an EP2-specific agonist, and 10 nM of
ONO-604, an EP4-selective agonist, both of which
have equipotent efficacies for inducing cAMP production as that of 1
µM of PGE2, without affecting other receptor
functions as evaluated in the CHO expression system (Table I). In
macrophages without LPS treatment, both PGE2 and
ONO-604, but not butaprost increased cAMP production, suggesting that
the PGE2-induced cAMP production is mediated via
EP4, but not by EP2 in this
preparation. Similar results were obtained 1 and 3 h after LPS
treatment. In contrast, in macrophages stimulated with LPS for 6
h, butaprost increased cAMP to levels generated by
PGE2, but ONO-604 did not. These results indicate
that the PGE receptor responsible for cAMP switches from
EP4 to EP2 by 6 h
after LPS treatment. In this assay, basal cAMP production increased and
the response generated by agonists decreased with time after LPS
treatment despite the presence of indomethacin in the assay solution.
One of the reasons could be because a variety of responses occur in
LPS-stimulated macrophages, and other cAMP-producing factors may be
released during the assay or the sensitivity of
PGE2 in PGE receptors may be altered. Thus, this
assay may not reflect the exact amount of each receptor expressed at
the protein level.
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and IL-12
in LPS-stimulated macrophages
To investigate the contribution of the EP2
and EP4 receptors in macrophage functions, we
examined the effects of EP-selective agonists on the LPS-induced
production of TNF- and IL-12. Regarding TNF- production measured
3 h after LPS treatment, PGE2 inhibited TNF-
production by 83% and ONO-604 by 61%, but inhibition by butaprost
was only by 9.6% (Fig. 5). As suggested
in the cAMP formation assays, EP4 works as a
predominant PGE receptor against TNF- production around 3 h
after stimulation. Regarding IL-12 production, both
PGE2 and ONO-604 showed inhibitory effect at
every time point examined, and the inhibitory potency of ONO-604 was
close to that of PGE2. This is an interesting
observation, as the EP4 receptor is likely not to
work around 6 h after stimulation, as deduced from the cAMP
formation experiments. Activation of EP4 for just
the first several hours after LPS treatment may be enough to cause an
inhibition of this cytokine to near maximum levels. On the contrary,
the inhibitory effect of butaprost was not significant at 3 and 7
h, but apparent at 14 h (29.3% inhibition), indicating that the
EP2 receptor works only during late time points.
These results indicated that the effects of PGE2
are mediated by both the EP2 and
EP4 receptors, but that each receptor contributes
to the inhibition of activated macrophage function in a different
manner via changes in their expression patterns.
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We next focused on the regulatory mechanisms underlying the
changes in expression of EP2 and
EP4 mRNAs upon LPS treatment. LPS stimulation was
able to influence the transcriptional expression of various proteins in
a direct or indirect manner in macrophages. To explore whether the
up-regulation of the EP2 gene and the
down-regulation of the EP4 gene require general
protein synthesis, we examined the effect of cycloheximide on the
EP2 and EP4 mRNA expression
(Fig. 6). Cycloheximide (0.1 µg/ml)
failed to affect LPS-induced EP2 gene expression,
but completely reversed the LPS-induced down-regulation of
EP4 gene expression. These results suggest that
the up-regulation of EP2 gene expression does not
require the protein synthesis, and is possibly directly activated by
LPS signals, but that the down-regulation of EP4
gene expression requires protein synthesis, possibly indirectly
controlled by LPS via the synthesis of other proteins. COX-2 is
undoubtedly one of the proteins synthesized rapidly in response to LPS
treatment in macrophages. PGE2 release is
believed to be a result of COX-2 protein induction, as shown in Fig. 1.
Thus we hypothesized that the LPS-induced PGE2
may affect EP4 gene expression itself in a
negative-feedback manner. Based on this hypothesis, we examined the
effect of PGE2 alone or indomethacin with LPS on
EP4 mRNA expression (Fig. 7). Incubation of
PGE2 (1 µM) without LPS for 3 h inhibited
the basal expression of EP4 mRNA, and
indomethacin added simultaneously with LPS stimulation reversed the
LPS-diminished expression of EP4 mRNA, suggesting
that the decrease in EP4 mRNA expression was a
result of feedback regulation by PGE2. dbcAMP (1
mM) and ONO-604 (10 nM, data not shown) mimicked the effects of
PGE2, but butaprost (1 µM) failed to inhibit
EP4 mRNA expression. These results suggested that
EP4-induced cAMP formation negatively regulates
the expression of the EP4 gene itself, and the
effect of cycloheximide may be due to inhibition of COX-2 protein
synthesis. In contrast, none of the reagents tested affected the
expression levels of EP2 mRNA, confirming that
the increase in EP2 mRNA expression upon LPS
treatment is independent of PG synthesis.
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One of the reasons we chose macrophages from the C3H/HeN strain is
that this strain has a genetically comparable mutant strain, C3H/HeJ,
in which the macrophages hardly respond to LPS. Recently, C3H/HeJ was
found to have a point mutation within the coding region of the
Toll-like receptor 4 (TLR4), a number of a protein family containing
proteins that have been implicated in LPS-induced cell signaling
(27). Indeed, C3H/HeJ macrophages did produce a
significant amount of PGE2, but much less than
that in C3H/HeN; the PGE2 contents in the medium
was <1 nM even after 7 h of LPS stimulation (Fig. 8A). COX-2 expression levels
were analyzed by Northern and Western analyses, showing that COX-2
induction was faint in LPS-treated C3H/HeJ macrophages (data not
shown). If the down-regulation of EP4 gene
expression requires more than a nanomolar order concentration of
PGE2, it should not occur in this strain. As
expected, no suppression of EP4 mRNA expression
was observed after LPS treatment (Fig. 8B). In addition,
when C3H/HeJ macrophages were stimulated with
PGE2 or dbcAMP, there was a decrease in
EP4 mRNA, as was seen in the C3H/HeN macrophages
(Fig. 8C). These results again indicate that the expression
of EP4 mRNA is down-regulated via an
EP4-mediated cAMP-dependent pathway. In contrast,
surprisingly and unexpectedly, a slight induction of
EP2 mRNA was again seen upon just medium change,
and this induction was greatly enhanced by LPS treatment, as was seen
in C3H/HeN macrophages. These results suggest that the mechanism
underlying the induction and up-regulation of EP2
gene expression may be independent of TLR4-mediated signals induced by
LPS treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although there have been many reports on the mechanism of COX-2
expression after LPS stimulation and the effect of
PGE2 on LPS-activated macrophages, there have
been very few reports that focus on the expression and function of the
PGE receptors in monocytes/macrophages (28, 29, 30). Here we
examined the time-dependent expression of EP2 and
EP4 mRNA during activation in resident peritoneal
macrophages, and found that the EP2 gene is
transiently up-regulated, whereas the EP4 gene is
down-regulated upon LPS stimulation. Based on the results obtained from
the cAMP assays using subtype-selective agonists, we conclude that
EP4 is the dominant PGE receptor in nonstimulated
macrophages or macrophages up to 3 h after LPS treatment. This is
supported by our results showing that TNF- production 3 h after
LPS treatment is completely inhibited by an EP4
agonist, but not an EP2 agonist. However, the
cAMP assay also indicated that the EP4 receptor
is no longer active by 6 h after LPS treatment, and, in turn,
EP2 seems to become the dominant receptor by
6 h. This is also supported by the result that the inhibitory
effect of an EP2 agonist on IL-12 production was
apparent at this time point. In the presence of an
EP2 agonist, EP2 is likely
to work by 6 h after LPS treatment, resulting in no increase
observed in production of IL-12 between 6 and 14 h. In contrast,
the inhibitory effect of an EP4 agonist on IL-12
production was observed for a longer time than we expected, even beyond
6 h after LPS treatment. Because IL-12 production initiated by LPS
includes de novo synthesis, the EP4-induced cAMP
increase during the initial few hours after LPS addition may exert
continuous inhibition on IL-12 production. It is possible that the
EP4-induced cAMP increase and the resultant
activation of cAMP-dependent kinase during initiation of IL-12 gene
activation may have a crucial effect on the following gene expression
of this cytokine. A number of reports have established that the
cAMP-dependent pathway inhibits IL-12 production at the transcriptional
level, possibly by affecting cAMP response element (CRE) binding
complexes (31, 32, 33). Thus, EP4 gene
expression is down-regulated upon stimulation with LPS, but this
receptor appears to have a pivotal role in autocrine regulation of
cytokine release, and EP2 might contribute to
extend the inhibitory effect of PGE2 on a
day-scale duration. In any case, these two receptors are likely to
cooperate elaborately with each other with regard to inhibition of net
responses of macrophages. Such transcriptional switching from
EP4 to EP2 may have more
profound roles other than inhibition of cytokine production. The
precise physiological significance of EP2 and
EP4 in macrophages currently remains unknown, but
should be addressed by receptor-knockout studies in the near
future.
Mechanisms underlying the down-regulation of EP4 gene expression and the up-regulation of EP2 gene expression in LPS-treated macrophages
One of the remarkable findings in this study is that both
EP2 and EP4 gene expression
are affected by LPS stimulation, but that the mechanisms of their
regulation appear quite different. This study demonstrated that the
down-regulation of EP4 gene expression is
mediated by PGE2 itself, produced upon
LPS-stimulation. Because an EP4 agonist, as well
as dbcAMP, but not an EP2 agonist affected
EP4 expression,
EP4-mediated cAMP accumulation is thought to be
involved in its own down-regulation. Moreover, the reversal action of
cycloheximide may be due to its inhibitory effect on the endogenous
synthesis of COX-2 protein. Such effects of LPS on
EP4 expression were different from our
previous observations in the J774.1 macrophage-like cell line, in which
EP4 mRNA was slightly induced upon
LPS-stimulation. One possibility for this difference is that J774.1
cells produce a smaller amount of PGE2 in
response to LPS treatment. Indeed, the amount of
PGE2 production in the current experiment 14
h after LPS treatment (Fig. 1) is 10 times greater than the value
obtained from J774.1 cells in the same condition (34). One
of the reasons we chose peritoneal resident macrophages in this study
is that these cells maintain characteristics close to that of native
macrophages. It is interesting in this respect that peritoneal
macrophages are able to produce a much larger amount of
PGE2 compared with the J774.1 cell line. In
addition, it should be noted that the concentration of
PGE2 that was added exogenously is in the same
range as that derived from LPS-stimulated macrophages. This suggests
that down-regulation of EP4 gene expression in
macrophages may take place in the peritoneal cavity when they are
subjected to inflammatory conditions. It has indeed been reported that
PGE2 is present at greater than nanomolar
order concentrations in exudates from mice with peritonitis
(35). However, at present, it remains unknown as to what
kinds of transcription factors are involved in the regulation of
EP4 receptor gene expression, regarding both its
constitutive expression as well as its expression upon induction by
stimuli. The EP4 gene contains a NF-
B site in
its 5' flanking region (16), and interaction between
cAMP-induced CRE and NF-B is a likely regulatory mechanism because
cAMP-elevating agents reduced NF-B binding through stabilization of
IB in several cell types (36). However,
EP4 gene expression is already present in
macrophages and other macrophage-like cells in the absence of LPS
treatment (37). Therefore, it is unlikely that NF-B is
involved in the basal expression of the EP4
gene.
We previously reported that the EP2 gene has
potential NF-B, NF-IL6, progesterone response element, and
CRE binding sites in its promoter region, and that this gene has two
transcriptional start sites specific to macrophages and uterine
epithelial cells (38). These results suggested that
EP2 gene expression may be regulated by many
kinds of stimuli in a cell type-specific manner. This study
demonstrated that EP2 gene expression is induced
upon medium change, and that this induction is up-regulated by LPS
stimulation. A slight induction of EP2 gene
expression was also observed in C3H/HeJ macrophages. This induction
upon medium change could be due to growth factors present in the fetal
serum. Indeed, we and other groups have found that
EP2 gene expression is affected by stimulation
with not only LPS but also by hormones and cytokines (15, 39). In addition, the most remarkable finding in this study is
that an up-regulation of EP2 by LPS stimulation
was observed in C3H/HeJ macrophages. A mutation in the gene for TLR4
has been found to be the main cause of LPS hyporesponsiveness in
C3H/HeJ mice (27), and TLR4 is now thought to be one of
the receptors recognizing LPS in vivo (40). However, this
study indicates that TLR4 is not necessary for LPS-induced
up-regulation of EP2 gene expression. As shown by
PGE2 synthesis, C3H/HeJ macrophages were still
sensitive to LPS even though the response was much less than that of
C3H/HeN macrophages, indicating that some mechanism that responds to
LPS treatment exists other than that via TLR4 in C3H/HeJ macrophages.
Such mechanisms of LPS-induced signaling may play a role in the
up-regulation of the EP2 gene. Indeed, the
activation of NF-B upon LPS treatment in C3H/HeJ macrophages has
been reported (41), and this event may be involved in the
up-regulation of the EP2 gene.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Atsushi Ichikawa, Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
3 Abbreviations used in the paper: COX, cyclooxygenase; dbcAMP, dibutyryl cAMP; TLR4, Toll-like receptor 4; CRE, cAMP response element; CHO, Chinese hamster ovary.
Received for publication December 9, 1999. Accepted for publication January 22, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. J. Leukocyte Biol. 57:174.[Abstract]This article has been cited by other articles:
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B. B. Moore, M. N. Ballinger, E. S. White, M. E. Green, A. B. Herrygers, C. A. Wilke, G. B. Toews, and M. Peters-Golden Bleomycin-Induced E Prostanoid Receptor Changes Alter Fibroblast Responses to Prostaglandin E2 J. Immunol., May 1, 2005; 174(9): 5644 - 5649. [Abstract] [Full Text] [PDF]
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 S. Kubo, H. K. Takahashi, M. Takei, H. Iwagaki, T. Yoshino, N. Tanaka, S. Mori, and M. Nishibori E-Prostanoid (EP)2/EP4 Receptor-Dependent Maturation of Human Monocyte-Derived Dendritic Cells and Induction of Helper T2 Polarization J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 1213 - 1220. [Abstract] [Full Text] [PDF]
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H. Harizi, C. Grosset, and N. Gualde Prostaglandin E2 modulates dendritic cell function via EP2 and EP4 receptor subtypes J. Leukoc. Biol., June 1, 2003; 73(6): 756 - 763. [Abstract] [Full Text] [PDF]
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R. P. Charbeneau, P. J. Christensen, C. J. Chrisman, R. Paine III, G. B. Toews, M. Peters-Golden, and B. B. Moore Impaired synthesis of prostaglandin E2 by lung fibroblasts and alveolar epithelial cells from GM-CSF-/- mice: implications for fibroproliferation Am J Physiol Lung Cell Mol Physiol, June 1, 2003; 284(6): L1103 - L1111. [Abstract] [Full Text] [PDF]
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T. Hoshino, S. Tsutsumi, W. Tomisato, H.-J. Hwang, T. Tsuchiya, and T. Mizushima Prostaglandin E2 Protects Gastric Mucosal Cells from Apoptosis via EP2 and EP4 Receptor Activation J. Biol. Chem., April 4, 2003; 278(15): 12752 - 12758. [Abstract] [Full Text] [PDF]
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 L. Walch, E. Clavarino, and P. L. Morris Prostaglandin (PG) FP and EP1 Receptors Mediate PGF2{alpha} and PGE2 Regulation of Interleukin-1{beta} Expression in Leydig Cell Progenitors Endocrinology, April 1, 2003; 144(4): 1284 - 1291. [Abstract] [Full Text] [PDF]
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 R. L. Konger, G. A. Scott, Y. Landt, J. H. Ladenson, and A. P. Pentland Loss of the EP2 Prostaglandin E2 Receptor in Immortalized Human Keratinocytes Results in Increased Invasiveness and Decreased Paxillin Expression Am. J. Pathol., December 1, 2002; 161(6): 2065 - 2078. [Abstract] [Full Text] [PDF]
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L. Walch and P. L. Morris Cyclooxygenase 2 Pathway Mediates IL-1{beta} Regulation of IL-1{alpha}, -1{beta}, and IL-6 mRNA Levels in Leydig Cell Progenitors Endocrinology, September 1, 2002; 143(9): 3276 - 3283. [Abstract] [Full Text] [PDF]
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E. Scandella, Y. Men, S. Gillessen, R. Forster, and M. Groettrup Prostaglandin E2 is a key factor for CCR7 surface expression and migration of monocyte-derived dendritic cells Blood, July 30, 2002; 100(4): 1354 - 1361. [Abstract] [Full Text] [PDF]
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S. Uematsu, M. Matsumoto, K. Takeda, and S. Akira Lipopolysaccharide-Dependent Prostaglandin E2 Production Is Regulated by the Glutathione-Dependent Prostaglandin E2 Synthase Gene Induced by the Toll-Like Receptor 4/MyD88/NF-IL6 Pathway J. Immunol., June 1, 2002; 168(11): 5811 - 5816. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) 100 ng/ml LPS for the indicated time periods.
The concentrations of PGE2 in the medium were then
determined as described in Materials and Methods. The
values obtained from the medium only were used as the value at 0
h. Values are expressed as the mean ± SEM for triplicate
determinants. The experiments were independently repeated three times,
and similar results were obtained. B, Expression of
COX-2 mRNA in LPS-stimulated C3H/HeN macrophages. Peritoneal
macrophages were exposed to fresh medium with (LPS +) or without 100
ng/ml LPS (LPS -) for the indicated times, and total RNA was isolated.
Ten micrograms of each RNA sample was subjected to Northern blot
analysis. The blots were hybridized with a probe for COX-2, and then
rehybridized with a probe for GAPDH. C, Detection of the
COX-2 protein in C3H/HeN macrophages. Peritoneal macrophages were
exposed to medium with (LPS +) or without 100 ng/ml LPS (LPS -) for
4 h. The cells were collected, and the cell lysates were subjected
to immunoblot analysis for the COX-2 protein.
), 1 µM PGE2 (
)
for the indicated times. The supernatants were recovered 3, 7, and
14 h after LPS stimulation, and the TNF-
), 1 µM butaprost
(
), or 10 nM ONO-604 (
) for 10 min. Test reagents contain 10 µM
indomethacin and 100 µM Ro-20-1724. The cAMP contents were then
determined as described in Materials and Methods. Values
are expressed as the mean ± SEM of triplicate determinants. The
experiments were independently repeated three times, and similar
results were obtained (*, p < 0.005 for PGE
analog-treated vs PG-untreated cells).
), or 10 nM ONO-604 (
) for the indicated time
periods. The supernatants were recovered, and the TNF-
