Abstract
PGs play a functional role in the early stage of Gram-negative bacterial infections, because this prostanoid is produced rapidly by epithelial cells after a bacterial infection. CD14, one of the LPS receptors, is a key molecule in triggering the response to bacterial LPS in association with a Toll-like molecule. Therefore, in this study, we investigated the effect of PG on CD14 expression in mouse macrophages. PGE1, PGE2, and PGA1 among the PGs tested strongly stimulated the expression of the CD14 gene in the cells. The stimulatory action also was observed by Western blot analysis. cAMP-elevating agents stimulated expression of CD14 gene as well. Protein kinase A inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), but not protein kinase C inhibitor 3-{1-[3-(dimethylamino)propyl]-1H-indol-3-yl}-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (GF109203X), abolished the stimulated expression of CD14. A run-on assay showed that PGE2 stimulated the CD14 gene expression at the transcriptional level via protein kinase A. PGE2 also stimulated activation of AP-1, a heterodimer of c-Jun and c-Fos, because the prostanoid increased specific binding of nuclear proteins to the AP-1 consensus sequence and stimulated AP-1-promoted luciferase activity. PGE2-stimulated expression of CD14 was inhibited by antisense c-fos and c-jun oligonucleotides, but not by their sense oligonucleotides. Finally, PGE2 pretreatment synergistically stimulated LPS-induced expression of IL-1β and IL-6 genes in mouse macrophages. Therefore, the present study demonstrates that PGE2 has the ability to stimulate AP-1-mediated expression of CD14 in mouse macrophages via cAMP-dependent protein kinase A.
Pathogenic Gram-negative bacteria, such as Shigella and enteroinvasive Escherichia coli, invade and destroy the colonic epithelium (1, 2, 3, 4, 5, 6, 7). The ability of these bacteria to invade epithelial cells is important for initiation of the disease caused by these organisms.
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
Reagents
RPMI 1640 was obtained from Nissui Pharmaceutical (Tokyo, Japan); and FCS was obtained from HyClone (Logan, UT). PGE1, PGE2, PGA1, PGF2α, 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′-[α-32P]dCTP, megaprime DNA labeling system, and [γ-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 × 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 × 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-1β cDNA (provided by T. Hamilton, Cleveland Clinic Foundation, Cleveland, OH), or mouse IL-6 (American Type Culture Collection) probes labeled with 5′-[α-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. β-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 × 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′-[α-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 β-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 [γ-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 0.25× 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 × 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
Effect of several PGs on expression of the CD14 gene in mouse macrophages
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 PGF2α- 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.
Inducing effect of PGs on CD14 gene expression in mouse macrophages. Macrophages from BALB/c mice were treated or not with each PG at 200 ng/ml, and total RNA was prepared at 1 h after the initiation of the treatment. Northern blot analysis was performed with CD14 and β-actin cDNAs used as probes. An identical experiment independently performed gave similar results.
PGE2 stimulates CD14 expression in mouse macrophages
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. 2⇓A, 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. 2⇓B). Furthermore, we looked for the presence of CD14 protein in the cells by carrying out a Western blotting assay. As shown in Fig. 2⇓C, 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.
PGE2 stimulates CD14 expression of mouse macrophages. A, Macrophages from BALB/c mice were treated or not with PGE2 at the selected doses, and total RNA was prepared at 1 h after the initiation of the treatment. B, The cells from BALB/c mice were treated or not with PGE2 at 200 ng/ml, and total RNA was prepared at various times after the initiation of the treatment. Northern blot analysis in A and B was performed with CD14 and β-actin cDNAs used as probes. C, The cells from BALB/c mice were treated or not with PGE2 at 200 ng/ml, and cell extracts were prepared at 6 h after the initiation of the treatment. Western blotting analysis using anti-mouse CD14 Ab was conducted to test for CD14 expression at the protein level in the cells. An identical experiment independently performed gave similar results.
cAMP-elevating agents stimulate expression of CD14 gene in mouse macrophages
Forskolin activates adenylate cyclase directly, resulting in an increase in the intracellular cAMP level. Fig. 3⇓A 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. 3⇓B). 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. 3⇓C). Thus, these results show that cAMP elevation was clearly capable of stimulating expression of the CD14 gene in mouse macrophages.
cAMP-elevating agents stimulate expression of the CD14 gene in mouse macrophages. Macrophages from BALB/c mice were treated or not with forskolin (A), dBt-cAMP (B), or IBMX (C) at the selected doses, and total RNA was prepared at 1 h after the initiation of the treatment. Northern blot analysis was performed with CD14 and β-actin cDNAs used as probes. An identical experiment independently performed gave similar results.
Protein kinase A inhibitor, but not protein kinase C inhibitor, abolishes PGE2-stimulated expression of 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. 4⇓A). In contrast, GF109203X, a potent inhibitor of protein kinase C, did not manifest such inhibitory effect (Fig. 4⇓B). These results suggested the involvement of cAMP-dependent protein kinase A in PGE2 stimulation of CD14 expression in mouse macrophages.
Protein kinase A inhibitor, but not protein kinase C inhibitor, abolishes PGE2-stimulated expression of the CD14 gene in mouse macrophages. Macrophages from BALB/c mice were pretreated or not for 1 h with H-89 at 20 μM, H-85 at 20 μM (A), or GF109203X (B) at the indicated doses (micrograms per milliliter) and then were treated in the presence or absence of PGE2 at 200 ng/ml, and then total RNA was prepared at 1 h after the initiation of the treatment. Northern blot analysis was performed with CD14 and β-actin cDNAs used as probes. An identical experiment independently performed gave similar results.
PGE2 stimulates CD14 gene expression at the transcriptional level 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 β-actin gene mRNA, and no hybridization with the vector plasmid was evident.
PGE2 stimulates CD14 gene expression at transcriptional levels in mouse macrophages. Macrophages from BALB/c mice were treated or not with PGE2 at 200 ng/ml in the presence or absence of H-89 at 20 μM, and then their nuclei were prepared at 30 min after the initiation of the treatment. A transcriptional activity assay was performed with CD14 and β-actin cDNAs as described in Materials and Methods. pBR322 was used as a negative control. An identical experiment independently performed gave similar results.
PGE2 stimulates the transcriptional factor AP-1 activity in mouse macrophages
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. 6⇓A, 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. 6⇓B, 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. 6⇓C). Taken together, these results show that PGE2 is a potent stimulator of AP-1 in mouse macrophages.
PGE2 stimulates transcriptional factor AP-1 activity in mouse macrophages. A, Macrophages from BALB/c mice were treated or not with PGE2 at 200 ng/ml, and then total RNA was prepared at various times after the initiation of the treatment. Northern blot analysis was performed with c-fos, c-jun, and β-actin cDNAs used as probes. B, The cells were treated or not with PGE2 at 200 ng/ml. The nuclear proteins were prepared 3 h later. The gel mobility-shift assay was performed with 32P-labeled oligonucleotide or unlabeled oligonucleotide (10 ng) as the competitor, containing the AP-1 consensus sequence in the presence of the nuclear proteins. C, RAW 264 cells were cotransfected with pRL-TK and the reporter plasmid (pAP-1-LUC) and then were washed three times. The transfected cells were incubated in serum-free RPMI 1640 supplemented or not with PGE2 at 200 ng/ml. The cellular extracts were prepared 12 h later and subsequently subjected to the luciferase assay. The pRL-TK plasmid, a luciferase expression plasmid, was used as an internal control to normalize for variations in transfection efficiency. The luciferase activity is expressed as percentage of maximum. An identical experiment independently performed gave similar results.
PGE2-stimulated expression of the CD14 gene in mouse macrophages is inhibited by antisense c-fos and c-jun oligonucleotides
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.
PGE2-stimulated expression of the CD14 gene in mouse macrophages is inhibited by antisense c-fos and c-jun oligonucleotides. Macrophages from BALB/c mice were pretreated or not for 6 h with a combination of c-fos and c-jun antisense or of their sense oligonucleotide (2 μM, respectively) and subsequently treated or not with PGE2 at 200 ng/ml. Their total RNA was prepared 1 h later. Northern blot analysis was performed with CD14 and β-actin cDNAs used as probes. An identical experiment independently performed gave similar results.
PGE2 synergistically stimulates expression of LPS-induced IL-1β and IL-6 genes in mouse macrophages
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-1β 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.
PGE2 synergistically stimulates expression of LPS-stimulated expression of IL-1β and IL-6 genes in mouse macrophages. Macrophages from BALB/c mice were pretreated or not for 6 h with PGE2 at 200 ng/ml, and subsequently treated or not with LPS at 10 or 100 ng/ml. Their total RNA was prepared 1 h later. Northern blot analysis was performed with IL-1β, IL-6, and β-actin cDNAs used as probes. An identical experiment independently performed gave similar results.
Discussion
CD14 plays an important role as a trigger of LPS-induced biological responses in monocytes/macrophages and neutrophils, because CD14 transgenic mice that overexpress human CD14 are highly sensitive to LPS response (29, 30) and because, in contrast, CD14 knockout mice are dramatically less sensitive to LPS (31). These findings suggest that LPS-induced septic shock may be closely related to the level of CD14 expression in vivo. Therefore, it is very important to investigate CD14 expression after bacterial infection.
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-1β 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-κB. 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-1β 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
We thank Dr. S. Yamamoto for providing mouse CD14 cDNA probe and Dr. T. Hamilton for a providing mouse IL-1β cDNA probe. We also thank Dr. T. Arakawa for useful discussion.
Footnotes
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↵1 This work was supported by a grant-in-aid for scientific research (11470380) from the Ministry of Education, Science, and Culture of Japan.
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↵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.
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↵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 November 30, 1999.
- Accepted March 6, 2000.
- Copyright © 2000 by The American Association of Immunologists
References
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