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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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A wide variety of cells produce IL-8 in response to different stimuli
and certain pathogens (1). In respiratory inflammation induced by
Pseudomonas areuginosa, soluble bacterial products were
shown to cause marked induction of IL-8 mRNA expression and protein
production in human transformed bronchial epithelial cells (2, 3).
Epithelial cells are a potential source of various proinflammatory
cytokines, such as IL-1, TNF-
, and IL-8 (4, 5). It is becoming clear
that intestinal mucosal epithelial cells are key mediators for
bidirectional communication between pathogens and host responses. They
also play a crucial role in the pathogenesis of inflammatory bowel
diseases. Local acute inflammation evoked by the induction of IL-8
contributes to tissue lesions during invasive or noninvasive bacterial
infections, such as Salmonella sp. or Helicobacter
pylori (6, 7, 8). Recent studies demonstrated that the colonic
protozoan parasite, Entamoeba histolytica, enhanced IL-8
production in human colonic epithelial cells by secretory components
released from the amebae (9) or through a paracrine effect elicited by
proinflammatory cytokine (IL-1) released from damaged host
cells (10). In inflammatory bowel diseases, such as ulcerative colitis
and Crohn’s disease, IL-8 production from mucosal epithelial cells has
been suggested to play a role in tissue injury (11).
While it is certain that a variety of infectious agents and proinflammatory cytokines can stimulate IL-8, little is known of the role of lipid mediators of inflammation in the regulation of mucosal epithelial cell functions. Low m.w. arachidonic metabolites, particularly PGE(s), are primarily involved in inflammatory reactions (12). Among the PGs, PGE2 is considered to be the most potent mediator of inflammation. PGE2 causes vasodilation and has pyretic effects during host inflammatory responses. Four subtypes of G protein-coupled PGE2 receptors, termed EP1, EP2, EP3, and EP4, mediate PGE2’s function and trigger intracellular signal transduction (13, 14). Recently, it was reported that PGE2 caused osteoblast formation through the induction of IL-1ß by activation of the PKA3 signal transduction pathway (15). A clear role for PGE2 in up-regulating the production of IL-6 has been demonstrated in mouse peritoneal macrophages following injection of mineral oil (16); activation of cyclo-oxygenase (Cox) 2, but not Cox-1 enzyme activity, contributed to the induction of IL-6 (17). There is also evidence that PGE2 has regulatory functions on IL-8 gene expression in various cell types in response to certain stimuli. For example, it enhances IL-8 production in human synovial fibroblasts stimulated with IL-1ß (18), but has no effect on neutrophil-derived IL-8 evoked by LPS (19). Moreover, in human alveolar macrophages and blood monocytes, PGE2 down-regulated IL-8 in response to LPS (20). The role of PGE2 in secretory responses from intestinal epithelial cells has been well addressed (21, 22, 23); however, there are no reports to date documenting PGE2 modulation of IL-8 gene expression in mucosal epithelial cells, and this is the focus of the present study.
Herein, we demonstrate that exogenous PGE2 coupling through EP4 receptors on human colonic epithelial cells up-regulates IL-8 gene expression and protein production by a posttranscriptional mechanism. These results highlight a potential novel role for PGE2 in the exacerbation of intestinal inflammation in the gastrointestinal tract.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PGE2, 13,14-dihydro-15-keto PGE2, 1-hydroxy-PGE1, and sulprostone were purchased from Cayman Chemical (Ann Arbor, MI). Forskolin, PMA, dibutyryl cAMP (dcAMP), actinomycin, and dexamethasone (Dex) were purchased from Sigma (St. Louis, MO). Iloprost was a gift from Dr. F. McDonald (Schering, Berlin, Germany). Butaprost was a gift from Dr. P. Gardiner (Bayer, U.K.). SQ22536 (9-(tetrahydro-2'-furyl)adenine) and H89 (N-[lswb]2-(p-bromocinnamy (amino)ethyl]-5-isoquinolinesulfonamide, HCl) were purchased from Calbiochem (San Diego, CA). M&B28767 was a gift from Rhône-Poulenc Rorer (Dagenham, U.K.). Lipofectamine reagent was purchased from Life Technologies (Burlington, Canada). 2-Nitrophenyl-ß-D-galactopyranoside was purchased from Boehringer Mannheim Canada (Laval, Canada). [14C]chloramphenicol was purchased from Amersham Canada (Oakville, Canada). Acetyl coenzyme A was purchased from Pharmacia Biotech (Baie Durfé, Canada). The pcDNA3 and pRc/CMV plasmids were purchased from Promega (Madison, WI).
Cell culture
Human colonic adenocarcinoma cells, T84, were obtained from American Type Culture Collection (Manassas, VA) and were grown in 24-well plates in 1 ml of a 1/1 mixture of DMEM with 4.5 g of D-glucose/l and Ham’s F-12 nutrient mixture (Life Technologies) containing 5% FBS (HyClone, Logan, UT), 100 U/ml of penicillin, 100 µg/ml of streptomycin sulfate, and 20 mM HEPES (Sigma). Cultures were maintained at 37°C in a humidified 5% CO2 atmosphere.
RNA preparation and semiquantitative RT-PCR
Total RNAs were isolated from T84 cells with TRIzol reagent (Life Technologies), and semiquantitative RT-PCR was performed as previously described (9). A consistent concentration of competitor (644 bp) in pGEM plasmid was used to compete the target IL-8 fragment (284 bp). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
Measurement of IL-8 protein
An IL-8 ELISA was used to measure IL-8 production in the
supernatant of T84 cell (
7 x 105) cultures.
Ninety-six-well ELISA plates (Immunn II, Dynatech, San Louis Obispo,
CA) were coated with anti-human IL-8 neutralizing Ab (200 ng; R&D
Systems, Minneapolis, MN) in 50 µl of carbonate buffer, pH 9.6,
overnight at 4°C. The plates were washed with PBS and were blocked
with 1% BSA overnight at 4°C. Samples (50 µl) were added to each
well and were incubated for 1 h at 37°C, while different
concentrations of human rIL-8 protein (R&D Systems) were used as the
standard. After washing, 50 ng of rabbit anti-human IL-8 polyclonal
Ab (Endogen, Boston, MA) was added for a further 1-h incubation at
37°C. Subsequently, goat anti-rabbit IgG Ab horseradish
peroxidase-conjugate (50 µl; Bio-Rad, Richmond, CA) at a 1/1500
dilution was added and incubated for 1 h at 37°C. The plates
were washed, and 100 µl of ABTS (Bio-Rad) was added to each well to
quantify the results. The OD was measured 30 min later at room
temperature with an ELISA reader at 405 nm. IL-8 protein levels are
expressed as nanograms per milliliter.
Measurement of intracellular cAMP
Confluent T84 cells were stimulated with PGE2 (1 µM) or forskolin (10 µM) for 10 min and extracted with absolute ethanol. The supernatant was dried in a speed vacuum, and the sample was resuspended in phosphate buffer. The cAMP assay was performed using a sensitive protein binding EIA kit (Cayman Chemicals).
Nuclear run-on assay
T84 cells were collected; homogenized in lysis buffer containing
320 mM sucrose, 10 mM Tris-HCl (pH 7.8), 0.1 mM EDTA (pH 8.0), 2 mM
MgCl2, 1 mM DTT, and 0.2% Triton X-100; and kept on ice
for 10 min. After centrifugation at 1500 x g for 15
min at 4°C, nuclei were resuspended in 100 µl of storage buffer
containing 50% glycerol, 50 mM Tris-HCl (pH 8.0), 5 mM
MgCl2, 0.1 mM EDTA, and 1 mM DTT. The reaction was
performed in 200 µl of reaction buffer containing 25% glycerol; 25
mM Tris-HCl (pH 8.0); 5 mM MgCl2; 0.1 mM EDTA; 1 mM DTT;
120 mM KCl; 500 µM each of ATP, GTP, and CTP; 50 µM UTP; and 100
µCi of [-32P]UTP (3000 Ci/mM) for 60 min at 26°C.
Subsequently, -32P-labeled nuclear RNA was extracted
with Trizol reagent and hybridized onto denatured membrane-bound cDNA
probes at 65°C for 48 h in hybridization buffer containing 5x
Denhart solution, 6x SSC, 0.05% SDS, 0.1 mM EDTA, and 100 µg/ml of
salmon sperm DNA. Three micrograms of IL-8 or GAPDH cDNAs generated by
PCR using specific primers as previously described (9) was immobilized
onto nitrocellulose membrane using a Minifold I (Schleicher and
Schuell, Keene, NH). pGEM vector was used as a control for nonspecific
hybridization. After sequential washing with 6x SSC and 1% SDS at
room temperature for 10 min and with 1% SSC at 65°C, autoradiography
was performed. OD was measured using the National Institutes of Health
Image software, and the ratio of IL-8/GAPDH represents the
transcription rate.
Construction of chloramphenicol acetytransferase (CAT) reporter gene plasmids
The IL-8 3' UTR (1902–3710 bp; GenBank accession no. M28130) was amplified by PCR using the human colonic epithelial cell line T84 genomic DNA as a template with the primers gatatcTAAAAAAATTCATTCTCTGTGGTATCC (sense) and ctcgagTGAATTCCGAAGTTCTTTTTGTTC (antisense). This fragment was ligated into the EcoRV and XhoI sites of the plasmid pcDNA3, which contains the CAT reporter gene (pcDNACAT) in the HindIII and BamHI sites, yielding the reporter gene construct pcDNACAT-UTR. The polyadenylation site was determined at 3152 bp, and the sequence from 3153 to 3710 bp was not transcribed into mRNA (our unpublished observations).
Transfections and CAT and ß-galactosidase assays
T84 cells were plated at a density of 7 x 106 in 60-mm dish in complete medium. After 24 h, 3 µg of pcDNACAT-UTR or pcDNACAT plasmids together with 3 µg of pRc/CMV plasmid with ß-galactosidase gene in the NotI site (pRc/CMV-gal) to correct the difference in the transfection efficiency were transfected into cells using the Lipofectamine reagent. To determine the effect of PGE2 on CAT reporter gene expression, 1 µM PGE2, 10 µM forskolin, or 1 µM PMA was added to the culture medium 26 h after transfection. The cytoplasmic extract from each sample after 20-h incubation was then assayed for CAT and ß-galactosidase activity as described previously (24). Briefly, transfected T84 cells were washed with PBS and suspended in 300 µl of CAT buffer containing 40 mM Tris-HCl (pH 7.5), 1 mM NaCl, and 1 mM EDTA. After freeze-thawing, the cell extract was used for ß-galactosidase activity in the presence of 67 mM sodium phosphate (pH 7.5), 1 mM MgCl2, 0.045 mM ß-ME, and 150 µg of 2-nitrophenyl-ß-D-galactopyranoside (ONPG) at 37°C for 24 h. OD was measured at 415 nm. After normalized by ß-galactosidase activity, the cell extract was heated at 65°C for 10 min and was used for measuring CAT activity in reaction buffer containing 100 mM Tris-HCl (pH 8.0), 125 nCi of [14C]chloramphenicol, and 25 µg of acetyl coenzyme A at 37°C for 24 h. After incubation, chloramphenicol and its derivative were extracted with ethyl acetate, and the acetylated chloramphenicol and substrate were separated on a thin layer silica gel plate (VWR Calab, Mississauga, Canada) in the presence of the solvent chloroform/methanol (19/1) and then autoradiographed. The percent conversion of [14C]chloramphenicol to the acetylated form was quantified in comparison to the control groups using National Institutes of Health Program.
Statistical analysis
The data were calculated as the mean ± SD. The results were analyzed by Student’s t test. A value of p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PGE2 has been shown to elevate intracellular cAMP by
interacting with specific EP receptors (13, 14). To determine whether
PGE2 can modulate IL-8 mRNA expression and protein
production through a receptor-coupling mechanism, T84 cells were
exposed to various concentrations of PGE2, and IL-8 mRNA
and protein levels were quantified. As shown in Figure 1
A, IL-8 mRNA accumulation
occurred in a dose-dependent fashion with increasing concentrations
(0.01–10 µM) of PGE2. Two agents that increase
intracellular cAMP, forskolin (0.01–25 µM) and dcAMP (0.1–1000
µM), showed similar dose-dependent effects on IL-8 mRNA expression. A
similar dose- and time-dependent accumulation of IL-8 mRNA was observed
in the well-characterized human colonic adenocarcinoma cell line,
LS174T in response to PGE2 (data not shown). Figure 1, B and C, shows
the kinetics of IL-8 mRNA expression and protein production. Rapid
increases in IL-8 mRNA levels were observed 1 h after treatment
with 1 µM PGE2, peaked after 2 h, and remained
consistently high up to 24 h. The onset of IL-8 mRNA expression
was delayed in response to forskolin (10 µM) and dcAMP (100 µM),
with peak expression occurring at 6 h. Interestingly, IL-8 protein
was not detected before 12 h of incubation with PGE2,
forskolin, or dcAMP, whereas after 6 h, significant production of
IL-8 was observed in response to PMA (1 µM). The effect of
PGE2 on IL-8 protein production was similar to those of
forskolin and dcAMP. These results suggest that PGE2 is
able to elevate not only IL-8 mRNA expression but also protein
production in human colonic cells.
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Receptor coupling is involved in IL-8 mRNA induction in response to exogenous PGE2
It is well known that PGE2 functions through
receptor-coupling events. Among the PGE2 receptors
(EP1, EP2, EP3, and
EP4), EP2 and EP4 were shown to
elevate intracellular cAMP, which leads to the activation of protein
kinase A (13, 14). Accordingly, we determined whether PGE2
receptor agonists could modulate IL-8 mRNA expression. As shown in
Figure 2, PGE2 caused a significant increase in IL-8 mRNA
expression compared with that in unstimulated controls. In comparison,
the PGE2 metabolite, 13,14-dihydro-15-keto-PGE2
had only a weak effect on IL-8 mRNA expression, demonstrating
specificity for PGE2. Iloprost, an EP1 agonist;
sulprostone, an EP1/EP3 agonist; as well as butaprost, an
EP2 agonist, had no significant effect on IL-8 mRNA
expression. In contrast, 1-hydroxy-PGE1, which has been
shown to have high affinity for EP2 and EP4
(26, 27), and M&B28767, an EP3/EP4 agonist,
caused a marked or moderate increase in IL-8 mRNA expression. T84 cells
express three subtypes of PGE2 receptors, EP2,
EP3, and EP4, as revealed by RT-PCR (our
unpublished observations). As butaprost, an EP2 agonist,
and sulprostone, an EP3 agonist, had no significant effect
on IL-8 mRNA expression, these results strongly suggest that
PGE2 coupling through EP4 resulted in IL-8 gene
expression.
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Based on the results presented above, we then determined whether
PGE2 coupling through EP4 receptors could
elevate intracellular cAMP in T84 cells (Table I). PGE2 and forskolin
increased intracellular cAMP similar to those reported in previous
studies (22, 28). However, in cells pretreated with the adenylate
cyclase inhibitor, SQ22536 (100 µM), intracellular cAMP levels in
response to PGE2 or forskolin were markedly inhibited
(p < 0.05).
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, cells pretreated with H89 produced
significantly (p < 0.05) less IL-8 protein
following stimulation with PGE2 or forskolin. In contrast,
IL-8 protein production in response to PMA, a protein kinase C
activator, was not inhibited. Depletion of protein kinase C in cells
following treatment with 1 µM PMA for 24 h also did not affect
IL-8 mRNA expression stimulated by PGE2, forskolin, or
dcAMP, whereas PMA-induced IL-8 mRNA expression was inhibited
(our unpublished observations). Taken together, these data
clearly show that PGE2 coupling through EP4
receptors results in cAMP-dependent PKA signal transduction.
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To determine whether IL-8 gene expression is transcriptionally or
posttranscriptionally regulated by PGE2, nuclear run-on
assays were performed to examine the rate of IL-8 gene transcription.
Unlike PMA, which increased the transcriptional rate of IL-8 twofold,
PGE2 and forskolin had no effect on IL-8 gene transcription
compared with that in the unstimulated controls (Fig. 3). Based on these results, we then
measured the stability of IL-8 mRNA in response to PGE2,
forskolin, or PMA by RT-PCR in the presence of actinomycin D, a
transcription inhibitor. As shown in Figure 4, PGE2 (84% mRNA remaining
after 10 h), forskolin (130%), and PMA (111%) delayed the
degradation of IL-8 mRNA compared with that in the untreated controls
(51%). These results were confirmed by Northern blot analysis. T84
cells were treated with cycloheximide to elevate IL-8 mRNA levels (9)
and washed, and then the fate of IL-8 mRNA was measured following
treatment with actinomycin D in the presence or the absence of the
stimuli. Regardless of the stimuli, IL-8 transcripts remained
consistently higher than those in the untreated controls (our
unpublished observations). Taken together, these data strongly suggest
that the stimuli enhanced IL-8 gene expression by stabilizing IL-8
mRNA.
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). CAT gene expression was dramatically
reduced after 3' UTR was constructed in the 3' flank of CAT, clearly
implicating the down-regulatory effect of IL-8 3' UTR. Moreover,
treatment with PGE2 or forskolin increased CAT gene
expression in cells transfected with pcDNACAT-UTR (157 and 167%,
respectively), while PGE2 (95%) had no effect on CAT
activity in cells transfected with pcDNACAT, and forskolin (119%)
slightly increased CAT activity compared with that in the control
groups (100%). In response to PMA, CAT gene expression was markedly
increased in cells transfected with either pcDNACAT-UTR (267%) or
pcDNACAT (198%). Clearly, the increase in CAT activity in
the cells transfected with pcDNACAT-UTR is much higher than that in the
cells transfected with pcDNACAT in response to PGE2,
forskolin, or PMA. Taken together, these results suggest that
PGE2 caused the accumulation of IL-8 mRNA through
posttranscriptional regulation, and that PGE2-, forskolin-,
or PMA-responsive cis-element(s) in IL-8 3' UTR contributed
to stabilizing the newly synthesized IL-8 mRNA.
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Glucocorticoids suppress IL-8 gene expression in various cell
lines in response to certain stimuli either by inhibiting IL-8 gene
transcription or by decreasing mRNA stability at the
posttranscriptional level (29). We have previously shown (9) that the
accumulation of IL-8 mRNA in response to E. histolytica
secretory components occurs by a posttranscriptional mechanism and is
sensitive to Dex. However, Dex could not alter PMA-induced IL-8 mRNA
expression, which is regulated both transcriptionally and
posttranscriptionally (9). To determine whether Dex could affect
PGE2-induced IL-8 expression, T84 cells were pretreated
with Dex and then stimulated with PGE2, PMA, forskolin, or
dcAMP. After 24-h stimulation, the supernatant was collected and
quantified for IL-8 protein (Fig. 6). Dex
pretreatment significantly inhibited IL-8 protein production in cells
stimulated with PGE2, forskolin, and dcAMP as well as in
control unstimulated cells. However, Dex pretreatment had no effect on
IL-8 production in response to PMA. These data suggest that Dex exerts
an inhibitory action on PGE2-induced IL-8 gene expression
in T84 cells by destabilizing newly synthesized mRNA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our studies demonstrate that exogenous PGE2 coupling through EP4 receptors on T84 human colonic epithelial cells markedly enhanced the expression of IL-8 mRNA and stimulated the production of IL-8 protein. This effect was also observed in another human colonic epithelial cell line, LS174T, whereas in human macrophages, PGE2 inhibited basal expression of IL-8 mRNA (our unpublished observations). The ability of PGE2 to stimulate IL-8 from mucosal epithelial cells may play a central role in the pathogenesis and immunopathology of inflammatory bowel diseases. The accumulation of IL-8 mRNA in response to exogenous PGE2 occurred in a dose- and time-dependent manner. Following PGE2 stimulation, IL-8 mRNA expression was observed as early as 1 h, with peak levels occurring after 2 h. Interestingly, IL-8 secretion was first noted after 12-h stimulation with PGE2, forskolin, or dcAMP, whereas PMA-induced IL-8 protein production occurred as early as 6 h. These results imply that different signal transduction pathways and regulatory mechanisms are involved in IL-8 gene expression and protein production. Even after 24-h stimulation with PGE2, forskolin, or dcAMP, the amount of IL-8 produced was sevenfold less than that from PMA-stimulated cells. Endogenous PGE2, derived from T84 cells, played no role in the expression of IL-8 mRNA, as Cox-1- and Cox-2-specific inhibitors had no effect on basal or PGE2-simulated IL-8 mRNA levels. These results suggest a role for exogenous PGE2 in the up-regulation of IL-8 mRNA expression and protein secretion in human colonic epithelial cells.
Inflammation of the intestinal mucosa is characterized by infiltration
of neutrophils, macrophages, lymphocytes, and plasma cells (30).
Macrophages and neutrophils produce high output PGE2 and
thus, provide the majority of the PGs in the inflamed gut. In animal
models and human cases of intestinal inflammation, elevated levels of
arachidonic acid metabolites have been detected. It is well documented
that exogenous PGE2 causes physiologic changes in
intestinal mucosal epithelial cells, such as secretion of water,
electrolytes, and mucins, in in vivo and in vitro models (21, 22, 23, 28).
Inflammatory bowel disease is associated with the production of high
levels of proinflammatory cytokines. IL-8 released from epithelial
cells, macrophages, and mesenchymal cells is present in the tissues of
the inflamed intestine (11). Proinflammatory cytokines, such as TNF-
and IL-1/ß, have the ability to regulate IL-8 gene expression in
intestinal epithelial cells (11). However, it is not known whether
lipid mediators of inflammation can modulate IL-8 gene expression in
mucosal epithelial cells. PGE2 was shown to have no effect
on IL-8 gene expression in neutrophils (18) and to exert a negative
regulation of IL-8 production in human alveolar macrophages and blood
monocytes (19). In this study we clearly demonstrate that exogenous
PGE2 is a potent mediator of the regulation of IL-8 gene
expression in human colonic epithelial cells. The consequence of
overproduction of IL-8 by intestinal epithelial cells may lead to
exacerbation of tissue injury or damage through the amplification of
mucosal inflammation.
PGE2 exerts its function on target cells through receptor-coupling events. Four subtypes of PGE2 receptors have been identified, namely EP1, EP2, EP3, and EP4 (13, 14). Among them, EP2 and EP4 have the capability of elevating intracellular cAMP (26, 27), which presumably triggers the cAMP-dependent PKA signal transduction cascade. Coupling through EP1 increases intracellular Ca2+, and coupling through EP3 decreases intracellular cAMP (13, 14). The EP1/EP3 agonist, sulprostone, had no effect on IL-8 mRNA expression, and the EP2-selective agonist, butaprost, had only a weak effect. In contrast, 1-hydroxy-PGE1, an EP2/EP4 agonist, and M&B28767, an EP3/EP4 agonist, significantly stimulated the expression of IL-8 mRNA. Although the increase in IL-8 mRNA levels evoked by M&B28767 was not as potent as that caused by PGE2, these results clearly demonstrate the involvement of EP4 receptors. PGE2 coupling through EP4 receptors stimulated intracellular cAMP, which was specifically inhibited by a selective adenylate cyclase inhibitor. This is consistent with previous studies (22, 28). Furthermore, a selective PKA inhibitor reduced IL-8 production in response to PGE2, confirming the contribution of cAMP-dependent PKA signal transduction in this event. Taken together, these results unequivocally show that PGE2 coupling through EP4 receptors plays a major role in the initiation of cAMP-dependent PKA signal transduction resulting in IL-8 gene expression in T84 cells.
Numerous studies have shown that the IL-8 gene can be regulated by
various stimuli, such as LPS, cytokines, PMA, and agents that elevate
intracellular cAMP or calcium in various cell types (1).
Transcriptional regulation of IL-8 gene expression is well defined. A
variety of transcription factors, such as NF-
B, NF-IL-6, activator
protein-1, octamer-1, or CCAAT/enhancer-binding
protein, are responsible for IL-8 gene transcription regulation
(31, 32, 33, 34, 35, 36). However, there is limited information with regard to
posttranscriptional regulation of IL-8 gene expression. Previous
studies have shown that IFN-
can up-regulate IL-8 gene expression in
human monocytic cells (U937) concomitant with stabilization of mRNA
levels (37). As well, IL-1ß caused a delay in the degradation of IL-8
mRNA in human diploid fibroblasts (38). Villarete et al. (39) reported
that transcriptional and posttranscriptional regulations are involved
in IL-8 gene expression in human blood cells stimulated with LPS. The
IL-8 gene contains AU-rich sequences in its 3' UTR that may cause its
mRNA to be more susceptible to degradation (40). We have previously
shown that E. histolytica secretory components can stimulate
IL-8 gene expression in T84 cells by a posttranscriptional mechanism
(9). In the present study, nuclear run-on assays revealed that there
are no differences in the rate of IL-8 gene transcription following
stimulation with PGE2 and forskolin or in the untreated
controls, clearly implicating a posttranscriptional effect. This is in
contrast to PMA stimulation, which markedly increased the rate of IL-8
gene transcription. Interestingly, PMA, PGE2, and forskolin
stabilized the degradation of IL-8 mRNA compared with that in the
untreated groups. This suggests that these stimuli can regulate the
expression of IL-8 mRNA at the posttranscriptional level. The presence
of a PGE2-responsive cis-element(s) in IL-8 3'
UTR was clearly shown by the ability of PGE2 to elevate CAT
gene expression in T84 cells transfected with CAT reporter gene and
IL-8 3' UTR, but not with the CAT reporter gene only. Based on these
results it is clear that the accumulation of IL-8 mRNA elicited by
exogenous PGE2 is caused by a delay in the degradation of
newly synthesized IL-8 mRNA.
Overproduction of IL-8 results in neutrophil-dependent tissue injury (41). IL-8 is considered one of the primary targets for the treatment of intestinal inflammation (11). Thus, suppression of IL-8 production could be beneficial for the control of inflammation. Glucocorticoids are the most effective inhibitors of IL-8 gene expression in several types of cells (1, 42). Dex, a synthetic glucocorticoid, has an inhibitory effect on IL-8 gene expression through transcriptional regulation or by destabilizing mRNA levels (29). However, it had no effect on IL-8 gene expression in peripheral blood monocytes in response to PMA, fibroblasts stimulated with leukoregulin, or airway epithelial cells stimulated with elastase (43, 44, 45). Our results show that Dex can inhibit IL-8 protein production in cells stimulated with PGE2 and forskolin, but has no effect on PMA-induced IL-8 production. Therefore, the signal transduction pathways initiated by PGE2 and forskolin (PKA pathway) and PMA (PKC pathway) are independently regulated by Dex. Dex has an inhibitory effect on IL-8 gene expression in T84 cells stimulated by PGE2 through a posttranscription mechanism that is cAMP dependent (PKA pathway); however, no effect was observed on PKC-dependent signaling with PMA, which resulted in both transcriptional and posttranscriptional regulation of IL-8.
In conclusion, our findings unravel a novel role for exogenous PGE2 in up-regulating IL-8 gene expression and protein production by human colonic epithelial cells. PGE2 coupling through EP4 receptors triggered cAMP-dependent PKA signal transduction cascade for posttranscriptional regulation of IL-8 gene expression. This study highlights a potential mechanism explaining how mucosal epithelial cells exacerbate intestinal inflammation in the inflamed intestine via production of IL-8 in response to exogenous PGE2.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and requests for reprints to Dr. Kris Chadee, Institute of Parasitology, McGill University, Macdonald Campus, 21,111 Lakeshore Rd., Ste. Anne de Bellevue, Quebec. Canada H9X 3V9. E-mail address:
3 Abbreviations used in this paper: PKA, protein kinase A; Cox, cyclo-oxygenase; dcAMP, dibutyryl cAMP; Dex, dexamethasone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAT, chloramphenicol acetyltransferase; UTR, untranslated region.
Received for publication March 24, 1998. Accepted for publication June 1, 1998.
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 N. El-Nefiawy, K. Abdel-Hakim, and N. Kanayama The selective prostaglandin EP4 agonist, APS-999 Na, induces follicular growth and maturation in the rat ovary Eur. J. Endocrinol., February 1, 2005; 152(2): 315 - 323. [Abstract] [Full Text] [PDF]
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 E.-Y. Choi, E.-C. Kim, H.-M. Oh, S. Kim, H.-J. Lee, E.-Y. Cho, K.-H. Yoon, E.-A Kim, W.-C. Han, S.-C. Choi, et al. Iron Chelator Triggers Inflammatory Signals in Human Intestinal Epithelial Cells: Involvement of p38 and Extracellular Signal-Regulated Kinase Signaling Pathways J. Immunol., June 1, 2004; 172(11): 7069 - 7077. [Abstract] [Full Text] [PDF]
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J. Shao, B. M. Evers, and H. Sheng Prostaglandin E2 Synergistically Enhances Receptor Tyrosine Kinase-dependent Signaling System in Colon Cancer Cells J. Biol. Chem., April 2, 2004; 279(14): 14287 - 14293. [Abstract] [Full Text] [PDF]
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 S.-W. Ryoo, D.-U. Kim, M. Won, K.-S. Chung, Y.-J. Jang, G.-T. Oh, S.-K. Park, P.-J. Maeng, H.-S. Yoo, and K.-L. Hoe Native LDL induces interleukin-8 expression via H2O2, p38 Kinase, and activator protein-1 in human aortic smooth muscle cells Cardiovasc Res, April 1, 2004; 62(1): 185 - 193. [Abstract] [Full Text] [PDF]
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 I. Dey, K. Keller, A. Belley, and K. Chadee Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica PNAS, November 11, 2003; 100(23): 13561 - 13566. [Abstract] [Full Text] [PDF]
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 D. L. Gessell-Lee, V. L. Popov, I. Boldogh, J. P. Olano, and J. W. Peterson Role of Cyclooxygenase Enzymes in a Murine Model of Experimental Cholera Infect. Immun., November 1, 2003; 71(11): 6234 - 6242. [Abstract] [Full Text] [PDF]
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 R. Murakami, F. Kambe, H. Mitsuyama, K. Okumura, T. Murohara, S. Niwata, R. Yamamoto, and H. Seo Cyclosporin A Enhances Interleukin-8 Expression by Inducing Activator Protein-1 in Human Aortic Smooth Muscle Cells Arterioscler. Thromb. Vasc. Biol., November 1, 2003; 23(11): 2034 - 2040. [Abstract] [Full Text] [PDF]
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H. Nishihara, S. Kizaka-Kondoh, P. A. Insel, and L. Eckmann Inhibition of apoptosis in normal and transformed intestinal epithelial cells by cAMP through induction of inhibitor of apoptosis protein (IAP)-2 PNAS, July 22, 2003; 100(15): 8921 - 8926. [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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 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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), 1 µM PMA (•), 10
µM forskolin (
), 100 µM dcAMP (
), or medium (
) for various
time periods. The data present the mean ± SD from four separate
experiments.
), 1 µM PMA (
) and incubated with 1 µM PGE2, 10 µM forskolin,
100 µM dcAMP, or 1 µM PMA for 24 h. The cell culture
supernatant was collected and quantified for IL-8 protein by ELISA. The
data present the mean ± SD from four separate experiments. *,
p < 0.05 compared with the untreated control group
(