| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Oral Microbiology, Meikai University School of Dentistry, Keyakidai, Sakado City, Saitama, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It is well known that PGs are multipotential mediators in many biological responses. In the case of bacterial infection, these mediators play a predominant role in inflammatory responses caused by Gram-negative infection. Interestingly, recent studies (8, 9, 10) have shown that several Gram-negative bacteria stimulate PG production by interacting with several kinds of cells. In contrast, because many studies (11, 12, 13, 14, 15) have demonstrated that PG, acting in a paracrine fashion, is an important regulator of macrophages, the epithelial cell-derived PG formed after these bacterial infections may act as a potent modulator of macrophages in a given organ.
CD14 is a 55-kDa glycoprotein that binds to LPS via the lipid A moiety of the latter. Therefore, it had been suggested by many investigators (16, 17, 18, 19, 20) that CD14 serves as one of the LPS receptors and contributes to the LPS-stimulated responses of CD14-positive cells such as macrophages and neutrophils. Interestingly, recent studies (21, 22, 23) suggested that CD14 associates with a Toll-like receptor, which is a signaling component of LPS and consequently triggers its cellular transduction. In light of these findings, because CD14 plays a functional role in the initial event of bacterial cell infection, it is very important to investigate the regulation of CD14 expression in macrophages. Therefore, in the present study, we focused on the question as to whether PGs are able to regulate CD14 expression in macrophages. Our present study demonstrates that PGE2 stimulated AP-1-mediated expression of CD14 in mouse macrophages and did so via cAMP-dependent protein kinase A.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RPMI 1640 was obtained from Nissui Pharmaceutical (Tokyo,
Japan); and FCS was obtained from HyClone (Logan, UT).
PGE1, PGE2,
PGA1, PGF2
, and
PGD1 were obtained from Sigma (St. Louis, MO).
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide
(H-89),3
N-[2-(N-formyl-p-chlorocinnamyl-amino)ethyl]isoquinolinesulfonamide
(H-85), dibutyryl (dBt)-cAMP, forskolin, and
3-{1-[3-dimethylamino)propyl]-1H-indol-3-yl}-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione
(GF109203X) were also obtained from Sigma.
5'-[-32P]dCTP, megaprime DNA labeling
system, and [
-32P]ATP were purchased from
Amersham Pharmacia Biotech (Tokyo, Japan). Opti-MEM and lipofectin were
obtained from Life Technologies (Gaithersburg, MD).
Preparation of mouse peritoneal macrophages
BALB/c mice, 7 wk of age, were each injected i.p. with 3 ml of thioglycollate medium (Difco, Detroit, MI). Peritoneal macrophages were prepared from the resulting peritoneal exudate as described earlier (24). The prepared macrophages were treated for selected times with test samples.
RAW 264 macrophage cell line
RAW 264 cells from Riken Cell Bank (Saitama, Japan) were used in transient expression assay as follows. The cells were maintained in RPMI 1640 supplemented with 10% FCS.
Western blot analysis
Macrophage monolayers in 9-cm diameter dishes (1 x 107 peritoneal exudate cells) were incubated in the presence or absence of test samples at various concentrations. Thereafter, the cells were solubilized with lysis buffer (10 mM Tris-HCl, pH 7.9, 1% sodium deoxycholate, 1% Nonidet P-40, 150 mM NaCl, 0.1% SDS, 20 mM EDTA, 0.25 µM PMSF). The samples (20 µg protein) were electrophoresed on 10% polyacrylamide gels by SDS-PAGE using a Tris-glycine buffer system (0.025 M Tris, 0.192 M glycine, 0.1% SDS). The protein was transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) by use of the semidry transblot system (Atto, Tokyo, Japan). Blots were blocked with 5% skim milk in TBS including 0.1% Tween 20 (TBST) for 1 h at room temperature and washed with TBST. Then, the membrane was incubated overnight at 4°C with the primary Ab diluted 1:1000 in 5% BSA in TBST. Protein was detected with a Phototope-HRP Western blot detection kit (New England Biolabs, Beverly, MA). The blots were exposed to X-Omat film (Eastman Kodak, Rochester, NY).
Northern blot analysis
Macrophage monolayers in 9-cm diameter dishes (1 x
107 peritoneal exudate cells) were incubated in
the presence or absence of test samples at various concentrations.
Thereafter, each total cellular RNA was extracted by the guanidine
isothiocyanate procedure (25). As described previously
(26), the RNA was subjected to 1% agarose electrophoresis
and blotted onto nylon membranes (Magnagraph; Micron Separations,
Westboro, MA). The membranes were subsequently baked, prehybridized,
and then hybridized with mouse CD14 (provided by Dr. S. Yamamoto, Oita
Medical University, Oita, Japan), mouse c-fos cDNA (Oncor,
Gaithersburg, MD), mouse c-jun cDNA (American Type Culture
Collection, Manassas, VA), mouse IL-1ß cDNA (provided by T. Hamilton,
Cleveland Clinic Foundation, Cleveland, OH), or mouse IL-6 (American
Type Culture Collection) probes labeled with
5'-[-32P]dCTP by use a megaprime DNA
labeling system. After hybridization, the membranes were washed, dried,
and exposed to x-ray film at -70°C. ß-Actin was used as an
internal standard for quantification of total RNA in each lane of
the gel.
Run-on assay
Macrophage monolayers in 15-cm diameter dishes (2 x
107 peritoneal exudate cells) were treated with
the desired test sample, and thereafter their nuclei were isolated as
described previously (26). Transcription initiated in
intact cells was allowed to proceed for 30 min at 30°C in the
presence of 5'-[-32P]UTP and the RNA was
isolated and hybridized to slot-blotted cDNA probes (5 µg/slot).
Blots were hybridized for 72 h and autoradiographed for 3 days.
The ß-actin gene was used as an internal standard.
Gel mobility-shift assay
This assay was conducted as described previously
(27). Binding reactions were performed for 20 min on ice
with 10 µg of nuclear protein in 20 µl of binding buffer (2 mM
HEPES, pH 7.9, 8 mM NaCl, 0.2 mM EDTA, 12% (v/v) glycerol, 5 mM DTT,
0.5 mM PMSF, 1 µg poly(dI-dC)) containing 20,000 cpm of
32P-labeled oligonucleotide in the absence or
presence of nonlabeled oligonucleotide. Poly(dI-dC) and nuclear extract
were first incubated at 4°C for 10 min before addition of the labeled
oligonucleotide. Then, 30-mer double-stranded oligonucleotides
containing the -TGACTCA- sequence (Oncogene Science, Manhasset, NY) of
the AP-1 binding site were end-labeled by the oligonucleotide 5'
end-labeling [-32P]ATP method. Reaction
mixtures for the binding were incubated for 15 min at room temperature
after addition of the labeled oligonucleotide. Unlabeled
double-stranded oligonucleotide was used as the competitor. DNA-protein
complexes were electrophoresed on native 6% polyacrylamide gels in
0.25x TBE buffer (22 mM Tris, 22 mM boric acid, and 0.5 mM EDTA, pH
8.0). The gels were subsequently vacuumed, dried, and exposed to Kodak
x-ray film at -70°C.
Plasmid construction for transient expression assay
The plasmid pAP1-Luc (Clontech, Palo Alto, CA) was constructed by inserting a synthetic oligonucleotide containing four copies of the 12-tetradecanoylphorbol-13-acetate-responsive element (TRE) sequence into the corresponding sites of pTAL-Luc (Clontech), which contains the HSV thymidine kinase (HSV-TK) promoter enhancer region located upstream of the firefly luciferase gene. pRL-TK conteins the HSV-TK promoter located region upstream of Renilla luciferase (Promega, Madison, WI).
Transient expression assay
RAW 264 cells (1 x 106 cells) were washed three times with Opti-MEM (Life Technologies) and incubated for 1 h in 5-cm diameter dishes with serum-free Opti-MEM. Then, the cells were transfected with reporter plasmid at 2 µg and pRL-TK at 0.2 µg by use of Lipofectin (Life Technologies) and thereafter incubated for 24 h in serum-free Opti-MEM. Next, the transfected cells were treated or not with PGE2 for 12 h in serum-free RPMI 1640. Then, the cellular extracts were prepared with reporter passive lysis buffer (Promega) and examined for firefly luciferase activity after determination of Renilla luciferase activity (pRL-TK). The latter was used as an internal control to normalize for variations in transfection efficiency. The results was expressed as percentage of maximum.
Preparation of oligonucleotides
Antisense c-fos (5'-TGC-GTT-GAA-GCC-CGA-GAA-3') and c-jun (5'-CGT-TTC-CAT-CTT-TGC-AGT-3') unsubstituted oligodeoxynucleotides and their corresponding sense oligonucleotides were synthesized and purified as described previously (27). These nucleotide sequences were complementary to the first 18 nucleotides following the AUG sequence of fos and jun mRNAs.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Because PGs are produced by several kinds of cells and
consequently play an important role in inflammatory responses, it is of
considerable interest to investigate whether these prostanoids
stimulate expression of CD14, a major receptor involved in triggering
LPS responses in the host. Therefore, first we examined the effect of
several PGs on the expression of the CD14 gene in mouse macrophages.
Although PGE2-untreated macrophages exhibited a
low basal level of CD14 gene expression, as shown in Fig. 1
, PGE1,
E2, and A1 clearly
stimulated the expression of the gene in the cells. However, such
stimulatory action was not observed in PGF2-
and PGD1-treated cells. Therefore, in the
following experiments we used PGE2, the most
important PG under physiological conditions, and investigated the
mechanism of the PG-stimulatory action on CD14 expression.
|
To define the PGE2 stimulation of CD14
expression in mouse macrophages, we examined in detail the stimulatory
action of PGE2 on CD14 expression. As shown in
Fig. 2A,
PGE2 stimulated the CD14 gene expression in a
dose-related fashion. Marked stimulation was observed as early as
1 h after the initiation of the PG treatment (Fig. 2B).
Furthermore, we looked for the presence of CD14 protein in the cells by
carrying out a Western blotting assay. As shown in Fig. 2C,
the stimulated production of CD14 having a molecular mass of 55 kDa was
confirmed in the cells. These results indicate that
PGE2 is a potent stimulator of CD14 expression in
mouse macrophages.
|
Forskolin activates adenylate cyclase directly, resulting in an
increase in the intracellular cAMP level. Fig. 3A shows that forskolin
stimulated expression of the CD14 gene in the macrophages. The
membrane-permeable cAMP analogue dBt-cAMP also had the same effect
(Fig. 3B). PGE2-stimulated expression
of the CD14 gene in the cells in the combined presence of the
phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX)
increased to a greater degree than in its absence (Fig. 3C).
Thus, these results show that cAMP elevation was clearly capable of
stimulating expression of the CD14 gene in mouse macrophages.
|
Stimulation of CD14 expression by cAMP-elevating agents in the
cells suggested to us that the PGE2 stimulation
of the receptor expression might be mediated by protein kinase A. Thus,
using the kinase inhibitor, H-89, we examined this point. H-89 at 20
µM clearly inhibited the PGE2 stimulation of
CD14 gene expression, though H-85, a negative control, did not exhibit
such action (Fig. 4A). In
contrast, GF109203X, a potent inhibitor of protein kinase C, did not
manifest such inhibitory effect (Fig. 4B). These results
suggested the involvement of cAMP-dependent protein kinase A in
PGE2 stimulation of CD14 expression in mouse
macrophages.
|
To ascertain whether PGE2-stimulated
expression of CD14 gene in the cells depends on transcriptional
activation, we tested the transcriptional activity by a run-on assay
using nuclei isolated from the cells treated with
PGE2 (200 ng/ml). Fig. 5 shows that PGE2
stimulated the transcriptional activity of the CD14 gene and also that
the stimulated transcriptional activity was clearly inhibited by H-89.
No significant change was detected in the transcriptional level of
ß-actin gene mRNA, and no hybridization with the vector plasmid was
evident.
|
The promoter sequence of mouse CD14 gene has been known
(28). The promoter contains TRE, which binds to the
transcription factor AP-1, which is a heterodimer of c-Fos and c-Jun.
Thus we assumed the possibility that AP-1 may play an important role as
a transcriptional factor in PGE2 stimulation of
CD14 expression in the cells. Therefore, we examined the inducing
effect of PGE2 on expression of c-fos
and c-jun protooncogenes in the cells. As shown in Fig. 6A, marked expression of these
oncogenes by PGE2 appeared at 1 h after
initiation of the PG treatment. Although the data are not shown, we
observed that PGE2 stimulation of both
protooncogenes was inhibited by H-89. Futhermore, this
PGE2 stimulation suggested to us the increased
binding of AP-1 to TRE. In this regard, we examined by the gel
mobility-shift assay whether PGE2 is able to
stimulate binding of AP-1 to TRE in the cells. The cells were treated
or not with the PG. As shown in Fig. 6B, increased binding
of nuclear protein to TRE was observed in
PGE2-treated cells. This increased nuclear
protein may be AP-1, because the increased binding was completely
prevented by a specific competitor of TRE. In addition, using an
AP-1-promoted luciferase assay, we explored whether the PG actually is
able to stimulate AP-1 transcriptional activity in RAW 264 cells. As
expected, we observed that the PG had this ability (Fig. 6C). Taken together, these results show that
PGE2 is a potent stimulator of AP-1 in mouse
macrophages.
|
The above results suggested to us that
PGE2-stimulated expression of CD14 may be
mediated via up-regulation of AP-1. Thus, we investigated whether the
PGE2 stimulation could be inhibited by the
addition of antisense c-fos and c-jun
oligonucleotides. The cells were pretreated or not for 6 h with
antisense or sense c-fos and c-jun
oligonucleotides and then incubated in the presence or absence of
PGE2. Thereafter, RNA was isolated and employed
in a Northern blot assay. As shown in Fig. 7, PGE2-stimulated
expression of the CD14 gene in the cells was inhibited by the addition
of antisense c-fos and c-jun oligonucleotides.
However, such an inhibitory effect was not observed with their sense
oligonucleotides.
|
Our interest was to address whether PGE2 is
actually able to amplify the LPS response in the macrophages. The cells
were pretreated or not 6 h with PGE2 at 200
ng/ml and then incubated in the presence or absence of LPS at the doses
indicated in Fig. 8. Thereafter, gene
expression of IL-1ß and IL-6 was analyzed by Northern blot assay. As
expected, Fig. 8 shows that PGE2 and LPS
synergistically stimulated gene expression of both cytokines. These
results suggest that PGE2 has the ability to
augment an LPS response through up-regulation of CD14 expression.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The present study shows that PGE2 stimulated CD14 expression in mouse macrophages via an AP-1-dependent mechanism through protein kinase A. This demonstration suggests a pathogenic role of PGs in the initial stage of CD14-mediated septic shock caused by LPS.
Because macrophages are the predominant cell of CD14-positive cells, these cells are thought to play an important role as a signal cell in the early stage of the host inflammatory response in LPS-induced septic shock. Therefore, for understanding the role of PGE2 in the early stage of LPS-induced septic shock, it was of interest to us to explore whether PGE2 stimulates CD14 expression in the cells. Several studies (11, 12, 13, 14) have shown that PGE2 down-regulates the production of some proinflammatory cytokines by macrophages. In contrast, PG was able to stimulate the expression of cytokines in other cells (32). Our previous study (33) showed PGE2 stimulation of IL-1ß expression in a mouse calvarial cell system. In view of these observations, this PG may be able to act as a positive or negative regulator, depending on the experimental system. We observed here that PGE2 clearly stimulated the expression of CD14 in mouse macrophages in vitro and also is able to stimulate its gene expression in spleen, kidney, and liver in mice (our unpublished data). Thus, we demonstrated the stimulatory action of PGE2 on CD14 expression both in vitro and in vivo. Also, our present study supports the observation that PGE2 was able to stimulate CD14 expression of cells of the human Mono Mac 6 cell line described previously (34).
Because the diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids (35, 36, 37), we examined the PGE receptor subtype, i.e., EP1, EP2, EP3, and EP4, in mouse macrophages. Our RT-PCR showed the presence of the EP2 and EP4 but not that of EP1 and EP3 receptor subtypes. In particular, although the EP2 expression was very strong, only a trace amount of EP4 expression was detected (our unpublished data). This observation is supported by previous studies (38, 39) that EP2 expression increases in macrophages treated with certain stimulants, because we used thioglycollate-stimulated macrophages in this study. Although PGE2 stimulation of CD14 expression in the cells may be primarily mediated via EP2, we do not yet understand the precise role of this EP2 mediation at this point.
It is well known that PGE2 signaling is mediated by cAMP-dependent protein kinase A. As expected, cAMP-elevating agents, forskolin, IBMX, and dBt-cAMP were able to stimulate expression of the CD14 gene in mouse macrophages, suggesting involvement of cAMP-dependent protein kinase A in the CD14 gene expression. This suggestion was supported by our experimental result that the PGE2-stimulated expression of the CD14 gene was significantly inhibited by H89, a potent inhibitor of protein kinase A, but not by GF109203X, a specific inhibitor of protein kinase C.
Several studies (40, 41, 42) have suggested that
PGE2 signaling is transduced through AP-1 via a
cAMP-dependent or -independent mechanism. In contrast, it has also been
shown that PGE2 acts as an inhibitor of NF-
B.
In this regard, because our run-on assay showed that
PGE2 stimulated the CD14 expression at a
transcriptional level in mouse macrophages, we addressed which
transcriptional factor was involved in PGE2
stimulation of the CD14 expression via cAMP-dependent protein kinase A.
We observed that the PG dramatically induced expression of
c-fos and c-jun genes as early as 1 h after
the initiation of the treatment. This stimulatory expression of the
both protooncogenes clearly was inhibited by H-89 (our unpublished
data). In addition, the gel mobility-shift assay indicated that the PG
increased AP-1 binding to its consensus sequence and, in addition,
stimulated AP-1-promoted luciferase activity. These observations
suggest AP-1 mediation in PGE2 stimulation of the
CD14 expression via cAMP-dependent protein kinase A. This suggestion
was supported by the interesting observation that
PGE2 stimulation of the CD14 expression was
considerably inhibited by simultaneous addition of c-fos and
c-jun antisense oligonucleotides.
In contrast, several inflammatory cytokines act as pathogenic factors in CD14-mediated septic shock of Gram-negative bacterial infection. Therefore, it was of interest to address whether LPS is able to synergistically stimulate the expression of inflammatory cytokines in PGE2-pretreated macrophages. For this point, we examined LPS-stimulated expression of IL-1ß and IL-6 genes in PGE2-pretreated cells. As expected, PGE2 pretreatment synergistically stimulated LPS-induced gene expression of both cytokines in the cells. These results suggest that PGE2 may be considerably involved in LPS-induced septic shock through up-regulation of inflammatory cytokines via CD14.
In conclusion, the present study is first to demonstrate that PGE2 stimulates AP-1-mediated expression of CD14 via cAMP-dependent protein kinase A and acts as a signal molecule involved in the early stage of Gram-negative bacterial septic shock.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Shigemasa Hanazawa, at the current address: Department of Microbiology, Faculty of Dentistry, Kyushu University, Higashiku Maidashi 3-1-1, Fukuoka City, Fukuoka 812-0054, Japan.
3 Abbrevriations used in this study: H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; H85, N-[2-(N-formyl-p-chlorocinnamylamino)ethyl]isoquinolinesulfonamide; TRE, 12-tetradecanoylphorbol-13-acetate-responsive element; GF109203X, 3-{1-[3-(dimethylamino)propyl]-1H-indol-3-yl}-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione; TK, thymidine kinase; IBMX, 3-isobutyl-1-methylxanthine; dBt, dibutyryl.
Received for publication November 30, 1999. Accepted for publication March 6, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production. J. Clin. Invest. 100:296.[Medline]
B activation in J774 macrophages. FEBS Lett. 440:76.[Medline]
and IL-1ß in murine peritoneal macrophages. J. Immunol. 145:3333.[Abstract]
gene expression by low doses of prostaglandin E2 and cyclic GMP. Immunobiology 182:44.[Medline]
induces expression of monocyte chemoattractant JE via fos and jun genes in clonal osteoblastic MC3T3–E1 cells. J. Biol. Chem. 268:9526.-induced monocyte chemoattractant JE/MCP-1 in clonal osteoblastic MC3T3–E1 cells. J. Biol. Chem. 269:21379.This article has been cited by other articles:
|
|
 |
|
  K. Chakraborty, P. C. Maity, A. K. Sil, Y. Takeda, and S. Das cAMP Stringently Regulates Human Cathelicidin Antimicrobial Peptide Expression in the Mucosal Epithelial Cells by Activating cAMP-response Element-binding Protein, AP-1, and Inducible cAMP Early Repressor J. Biol. Chem., August 14, 2009; 284(33): 21810 - 21827. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Hazan-Eitan, Y. Weinstein, N. Hadad, A. Konforty, and R. Levy Induction of Fc{gamma}RIIA expression in myeloid PLB cells during differentiation depends on cytosolic phospholipase A2 activity and is regulated via activation of CREB by PGE2 Blood, September 1, 2006; 108(5): 1758 - 1766. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Kobayashi, T. Mizoguchi, I. Take, S. Kurihara, N. Udagawa, and N. Takahashi Prostaglandin E2 Enhances Osteoclastic Differentiation of Precursor Cells through Protein Kinase A-dependent Phosphorylation of TAK1 J. Biol. Chem., March 25, 2005; 280(12): 11395 - 11403. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. K. Roach, S.-B. Lee, and J. S. Schorey Differential Activation of the Transcription Factor Cyclic AMP Response Element Binding Protein (CREB) in Macrophages following Infection with Pathogenic and Nonpathogenic Mycobacteria and Role for CREB in Tumor Necrosis Factor Alpha Production Infect. Immun., January 1, 2005; 73(1): 514 - 522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mitsuhashi, J. Liu, S. Cao, X. Shi, and X. Ma Regulation of interleukin-12 gene expression and its anti-tumor activities by prostaglandin E2 derived from mammary carcinomas J. Leukoc. Biol., August 1, 2004; 76(2): 322 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Yadav, S. K. Roach, and J. S. Schorey Increased Mitogen-Activated Protein Kinase Activity and TNF-{alpha} Production Associated with Mycobacterium smegmatis- but Not Mycobacterium avium-Infected Macrophages Requires Prolonged Stimulation of the Calmodulin/Calmodulin Kinase and Cyclic AMP/Protein Kinase A Pathways J. Immunol., May 1, 2004; 172(9): 5588 - 5597. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
