Departments of 1 Internal Medicine, 2 Physiology and Biophysics, and 3 Pathology, University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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Elevated mucosal
interleukin-1 (IL-1) levels are frequently seen during acute and
chronic intestinal inflammation, and IL-1 neutralization lessens the
severity of inflammation. One major effect of IL-1 is the increased
release of eicosanoid mediators via induction of cyclooxygenase-2
(COX-2). One site of COX-2-derived prostaglandin synthesis during acute
and chronic intestinal inflammation is the intestinal myofibroblast.
COX-2 expression has also been documented in these cells in colonic
neoplasms. Thus an understanding of the regulation of COX-2 expression
in human intestinal myofibroblasts is important. As an initial step
toward this goal we have characterized IL-1 signaling pathways that
induce COX-2 expression in cultured human intestinal myofibroblasts.
IL-1 treatment resulted in a dramatic transcriptional induction of
COX-2 gene expression. Activation of nuclear factor-
B (NF-
B),
extracellular signal-regulated protein kinase (ERK), p38, and protein
kinase C (PKC) signaling pathways was each necessary for optimal COX-2
induction. In contrast to what occurs in other cell types, including
other myofibroblasts such as renal mesangial cells, PKC inhibition did
not prevent IL-1-induced NF-
B or mitogen activated protein kinase/
stress-activated protein kinase activation, suggesting a novel role for
PKC isoforms during this process. The stimulatory effects of PKC,
NF-
B, ERK-1/2, and presumably c-Jun NH2-terminal kinase
activation were exerted at the transcriptional level, whereas p38
activation resulted in increased stability of the COX-2 message. We
conclude that, in intestinal myofibroblasts, IL-1-mediated induction of
COX-2 expression is a complex process that requires input from multiple signaling pathways. Each parallel pathway acts in relative autonomy, the sum of their actions culminating in a dramatic increase in COX-2
transcription and message stability.
prostaglandins; eicosanoids; intestinal inflammation; intestinal carcinogenesis; stromal cells; epithelial-mesenchymal interactions
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INTRODUCTION |
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INCREASED MUCOSAL LEVELS OF the proinflammatory cytokine interleukin-1 (IL-1) are consistently seen during acute and chronic intestinal inflammation in humans (7, 61) and animals (4, 31). Immunoneutralization of IL-1 activity greatly diminishes the severity of disease in a murine model of colitis, which suggests that this cytokine plays an important initiating role in inflammation (4). One major effect of IL-1 is induction of the localized synthesis and release of eicosanoid mediators through the cyclooxygenase (COX) pathways (10). At least two isoforms of COX exist in humans, each encoded by separate unlinked loci. COX-1 is constitutively expressed in many cell types, whereas COX-2 expression is more restricted and usually only observed following stimulation of cells with mitogens or proinflammatory cytokines, or during neoplastic progression (52).
Increased COX-2-derived prostaglandin (PG) synthesis has been repeatedly documented during acute and chronic intestinal inflammation (16, 49). Although epithelial COX-2 expression has been demonstrated following invasion by Salmonella (13) and in individuals suffering from Crohn's disease and ulcerative colitis (51), significant expression of COX-2 in the lamina propria also occurs. For example, in a rat model of colitis, increased COX-2 mRNA levels are found in cells of the lamina propria and muscularis of the colon, in regions occupied by mast cells, neutrophils, smooth muscle cells, and subepithelial myofibroblasts (41). Likewise, recent studies using a rat model of nonsteroidal anti-inflammatory drug (NSAID)-induced gastric ulceration (45), murine colitis (49), or ulcerated human specimens (24) have localized COX-2 expression to cells in the lamina propria that are consistent with myofibroblasts.
Increased COX-2 expression also occurs during the process of colon
carcinogenesis (12). Although epithelial COX-2 expression frequently occurs in carcinomas (48), in nonmalignant
adenomas the enzyme is prevalent in the stroma (8, 49).
With the use of adenomatous polyposis coli (APC)-mutant mice harboring
a COX-2-LacZ transgene (in which -galactosidase expression is driven
by the COX-2 promoter), COX-2 transcription was localized in early
murine adenomas, not in epithelial cells, but to a location directly subjacent to the epithelial cells in the area occupied by intestinal subepithelial myofibroblasts (38). Our laboratory has
presented preliminary evidence that COX-2 is localized in the stromal
cells of adenomatous polyps, cells that turn out to be activated
myofibroblasts (1, 2).
Intestinal subepithelial myofibroblasts (ISEMFs) are members of a family of phenotypically interrelated cells, which include glomerular mesangial cells, renal and pulmonary interstitial fibroblasts, and hepatic stellate (perisinusoidal Ito) cells (39, 40). Located at the interface between the epithelium and lamina propria, ISEMFs modulate information transfer between these tissue compartments and play a pivotal role in mucosal immunophysiology (39, 40). Previous reports from this laboratory and others have developed the concept that ISEMFs can amplify or suppress the effects of inflammatory mediators on epithelial ion transport through COX-2-dependent mechanisms (5, 21).
Given the importance of COX-2 expression to intestinal inflammation and
carcinogenesis, and the observations that intestinal myofibroblasts are
major sites of COX-2 synthesis during these processes, it is important
to determine the key signaling pathways that regulate COX-2 expression
in human intestinal myofibroblasts. Regulation of COX-2 gene expression
is a complex process that varies in different cell types and among
species. COX-2 gene expression is induced by a wide variety of stimuli
such as proinflammatory cytokines, growth factors, differentiation
factors, endotoxin, tumor promoters, reactive oxygen intermediates, and
cell-cell interactions (52). As such, the COX-2 gene is
subject to regulation by numerous signaling pathways, and the relative
contribution of each depends upon the stimulus, the cellular
environment, and the particular cell type. We have characterized a
myofibroblast cell line, 18Co, derived from mucosal biopsy of human
neonatal colon, which exhibits the phenotypic features of primary ISEMF cultures and ISEMFs in situ (56). Herein, using IL-1 as a
model stimulus, we report the first detailed analysis of signaling
pathways important for COX-2 expression in human intestinal
myofibroblasts and how these pathways interact with each other.
Critical pathways mobilized by IL-1 to induce COX-2 expression in
intestinal myofibroblasts include nuclear factor-B (NF-
B);
mitogen-activated protein kinases [MAPKs; extracellular
signal-regulated protein kinase-1 or -2 (ERK-1/2), p38]; c-Jun
NH2-terminal kinase [JNK; stress-activated protein kinase
(SAPK)]; and protein kinase C (PKC). Each parallel pathway acts in
relative autonomy, the sum of their actions culminating in a dramatic
increase in COX-2 transcription and message stability.
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MATERIALS AND METHODS |
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Materials.
Recombinant human IL-1 was purchased from R&D Systems (Minneapolis,
MN). The p38 inhibitor SB-203580, mitogen-activated/extracellular response kinase-1 (MEK1) inhibitor PD-98059, proteasome inhibitor MG-132, and NF-
B transactivation inhibitor PG-490 (triptolide) were
purchased from Biomol (Plymouth Meeting, PA). The PKC inhibitor bisindolylmaleimide I (BIS) was purchased from Calbiochem (San Diego,
CA). The transcriptional inhibitor
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole (DRB) was
purchased from Calbiochem. The protease inhibitors leupeptin, aprotinin, and phenylmethylsulfonyl fluoride (PMSF) were purchased from
Sigma (St. Louis, MO). The COX-2 and COX-1 polyclonal antibodies were
purchased from Cayman Chemical (Ann Arbor, MI). Antibodies to NF-
B
family members were purchased from Santa Cruz Technologies (Santa Cruz,
CA) in concentrated aliquots for electrophoretic mobility shift assay
(EMSA) analysis. Antibodies recognizing phosphorylated and total p38
(phospho-Thr-180/Tyr-182), c-Jun (phospho-Ser-73), and
phospho-activating transcription factor-2 (ATF-2) (phospho-Thr-71) were
purchased from New England Biolabs. The antibody recognizing phosphorylated JNK (phospho-Thr-183/Tyr-185) was purchased from Promega
(Madison, WI). Antibodies recognizing phosphorylated ERK-1 and ERK-2
(phospho-Thr-202/Tyr-204) were purchased from Promega. The antibody
used for detection of total ERK-1/2 was purchased from Santa Cruz
Technologies. The monoclonal antibody to
-smooth muscle actin (clone
1A4) was purchased from Sigma. The human COX-1 and COX-2 cDNA probes
used in Northern analyses were the kind gifts of Dr. Timothy Hla
(22). Custom oligonucleotides were purchased from
Genosys Biotechnologies (Woodlands, TX). Oligonucleotides representing
the consensus NF-
B binding sequence were purchased from Promega.
Cell culture. The human colonic subepithelial myofibroblast cell line, 18Co was obtained from the American Type Culture Collection (CRL-1459) and maintained as described previously (56). Culture media, supplements, and subculturing reagents were purchased from Sigma. Cells were cultured in Eagle's minimum essential medium supplemented with 10% NuSerum (Becton-Dickinson) at 37°C in a humidified incubator containing 5% CO2. For experiments, cells between passages 10 and 14 were used at confluence. Cells were fed with fresh medium 24 h before stimulation. Unless otherwise stated, all agents were supplied at time 0 in fresh, serum-containing medium.
Northern analysis of COX-2 and COX-1 mRNA levels. Total RNA was isolated using the Ultraspec RNA Isolation Reagent (Biotecx Laboratories, Houston, TX). Northern blotting and hybridizations were as described (21). Gene-specific mRNA levels were detected and quantified using a Packard Phosphorimager and OptiQuant software (Packard, Downer's Grove, IL). Slight variations in signal strength resulting from differences in the amount of total RNA loaded in each well were corrected by normalization to 28S rRNA levels using digitized images of the membranes after transfer.
Nuclear runoff analysis.
Nuclei were isolated using the protocol described by Bender
(6). Briefly, confluent monolayers were washed and
collected in ice-cold phosphate-buffered saline (PBS). Cell pellets
were resuspended in 4 ml of ice-cold sucrose buffer I [0.32
M sucrose, 3.0 mM CaCl2, 2.0 mM magnesium acetate, 0.1 mM
EDTA, 10 mM Tris · HCl, pH 8.0, 1.0 mM dithiothreitol (DTT),
and 0.5% Nonidet P-40]. Resuspended cells were broken by Dounce
homogenization using a loose pestle for 10 strokes. The lysed mixture
was mixed with an equal volume of sucrose buffer II (2.0 M
sucrose, 5.0 mM magnesium acetate, 0.1 mM EDTA, 10 mM
Tris · HCl, pH 8.0, and 1.0 mM DTT) and layered over a 4.4-ml
cushion of sucrose buffer II in a polyallomer SW 40.1 (Beckman) ultracentrifuge tube. After centrifugation for 45 min at
30,000 g (15,500 rpm) and 4°C, the nuclear pellet was suspended in 200 µl of ice-cold glycerol storage buffer (50 mM Tris · HCl, pH 8.3, 40% glycerol, 5.0 mM MgCl2,
and 0.1 mM EDTA) and frozen at 86°C until extension reactions were
carried out. Nascent transcripts were extended using the protocol
described by Greenberg (19). Nuclei (1 × 107) were mixed with an equal volume of 2× reaction buffer
[10 mM Tris · HCl, pH 8.0, 0.3 M KCl, 5.0 mM DTT, 5.0 mM
MgCl2, 1.0 mM GTP, 1.0 mM CTP, 1.0 mM ATP, and 100 µCi
[
-32P]UTP (800 Ci/mmol)] and incubated for 30 min at
30°C. After the labeling reaction, 50 µg carrier tRNA was added and
samples were subjected to sequential DNase I and proteinase K
digestion. Labeled transcripts were then purified by TCA precipitation.
Hybridizations and washes were carried out according to Greenberg
(19), and transcription levels were determined by using
phosphorimager analysis of the hybridized membranes.
EMSA.
Nuclear extracts were prepared by using the method of Schreiber et al.
(47). Confluent monolayers were washed and collected in
ice-cold Tris-buffered saline, pH 7.4. Cellular pellets were resuspended in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.0 mM DTT, 0.1 mM EDTA, 0.1 mM EGTA, 2.0 µg/ml aprotinin, 2.0 µg/ml leupeptin, 1.0 mM PMSF, 1.0 mM sodium fluoride, and 1.0 mM sodium orthovanadate) and allowed to swell on ice for 15 min. While vortexing, Nonidet P-40 was added to 0.6%, and vortexing was continued for 30 s to lyse cells. Nuclei were collected by centrifugation at 12,000 g for 30 s and were resuspended in buffer
B (20 mM HEPES, pH 7.9, 0.4 M KCl, 1.0 mM DTT, 0.1 mM EDTA, 0.1 mM
EGTA, 2.0 µg/ml aprotinin, 2.0 µg/ml leupeptin, 1.0 mM PMSF, 1.0 mM
sodium fluoride, and 1.0 mM sodium orthovanadate) and placed on a
shaking platform at 4°C for 15 min. After centrifugation at 12,000 g for 5 min at 4°C, the soluble extracts were frozen in
aliquots at 86°C. Protein concentrations were determined with the
Pierce bichinchoninic acid assay reagent by using the microplate
determination protocol as directed by the supplier (Pierce, Rockford
IL). The double-stranded oligonucleotides used for EMSA analysis were
as follows (complementary strands not shown): Jun/ATF (
68 to
44 relative to human COX-2 transcription start site)
5'-AACAGTCATTTCGTCACATGGGCTT-3'; cellular enhancer binding protein
(cEBP
) (NF-IL-6;
140 to
116 relative to human COX-2
transcription start site) 5'-CACCGGGCTTACGCAATTTTTTTAA-3'; NF-
B
(
231 to
207 relative to human COX-2 transcription start site)
5'-GGAGAGTGGGGACTACCCCCTCTGC-3'; and consensus NF-
B (from
light
chain enhancer) 5'-AGTTGAGGGGACTTTCCCAGGC-3'.
Western analysis. Cells were washed with ice-cold PBS and lysed in Laemmli sample buffer (10% glycerol, 5% 2-mercaptoethanol, 2% SDS, 0.002% bromphenol blue, and 62.5 mM Tris · HCl, pH 6.8). Ten micrograms of protein were run on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). The membranes were saturated with 5% fat-free dry milk in Tris-buffered saline (50 mM Tris, pH 7.5, 150 mM NaCl) with 0.05% Tween 20 (TBS-T) for 1 h at room temperature. Blots were then incubated overnight with the appropriate primary antibody, diluted in 5% BSA TBS-T. After washing with TBS-T solution, blots were further incubated for 1 h at room temperature with the appropriate peroxidase-conjugated secondary antibody (Amersham) for chemiluminescent detection. Blots were then washed three times in TBS-T before visualization. Chemiluminescent detection was performed using the Enhanced Chemiluminescence Detection Kit (Amersham) according to the supplier's recommendations.
PKC activity.
PKC activity was measured using a commercially available kit following
the supplier's instructions (Calbiochem). After treatment, cells were
washed and collected in ice-cold PBS. Cellular pellets were homogenized
by Dounce homogenization in ice-cold 25 mM Tris · HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM 2-mercaptoethanol,
1 µg/ml leupeptin, and 1 µg/ml aprotinin. The lysate was
centrifuged for 5 min at 14,000 g and 4.0°C, and the
supernatant was saved and assayed for PKC activity at 30°C for 10 min
in a buffer containing 0.5 mM CaCl2, 10 mM
MgCl2, 20 mM Tris · HCl, pH 7.5, 15 µM ATP, 25 µCi [-32P]ATP, 0.3 mg/ml phosphatidylserine, 30 µg/ml diacylglycerol, 0.3% Triton X-100, and 25 µM biotinylated
PKC pseudosubstrate (RFARKGSLRQKNV). Reactions were terminated by the
addition of TCA to 5%. After centrifugation, TCA-soluble material was
neutralized and biotinylated substrate was purified using affinity
ultrafiltration as recommended by the supplier. The amount of label
incorporated into the substrate was determined by scintillation
counting, and PKC activity was calculated as picomoles of phosphate
incorporated per minute per microgram of protein. Each assay was
performed in triplicate and repeated three times.
Data analysis. All experiments were repeated a minimum of three times with similar results. Where appropriate, data were expressed as means ± SE. Statistical analysis was performed by using Student's t-test with a P value of 0.05 as statistically significant. In all cases where comparative data are presented, the autoradiograpic images originated from the same exposure of the same gel: in some cases, for clarity of presentation, lanes containing samples not germane to this study were removed.
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RESULTS |
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Time course of IL-1-mediated COX-2 induction.
COX-1 expression is constitutive in 18Co cells, whereas COX-2 is highly
inducible by a variety of stimuli (21). Increased COX-2
mRNA levels, assessed by Northern blotting, were observed in 18Co
intestinal myofibroblasts within 1 h of IL-1 (500 pg/ml) treatment. These levels peaked after 8 h (38.5 ± 6-fold, P < 0.001), yet were still elevated 16 h
following IL-1 treatment (Fig.
1A). Increased COX-2 mRNA
levels were also observed in control cells at the 1 h time
point, which reflects the transient induction observed when cells were
placed in fresh serum-containing medium (Fig. 1A).
Serum-induced COX-2 mRNA levels in control cells returned to baseline
within 4 h. As previously reported by our laboratory, COX-1 mRNA
levels (but not protein) were elevated approximately threefold
16-24 h following stimulation with IL-1 (Fig. 1A)
(21). COX-2 protein expression, assessed by Western
blotting, was seen as early as 4 h and was maximal 16-24 h
following IL-1 treatment (Fig. 1B). COX-1 protein levels did
not change in response to IL-1 (Fig. 1B). The magnitude and
kinetics of COX-2 mRNA and protein induction were similar to those
shown when IL-1
(500 pg/ml) was used (data not shown).
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COX-2 transcriptional activity following IL-1 treatment.
Nuclear runoff analysis performed upon nuclei isolated 4 h
following IL-1 treatment demonstrated a marked (17.6 ± 2.8-fold, P < 0.01) transcriptional induction of the COX-2 gene
relative to control cells (Fig. 2). A
similar level of transcriptional activation was seen for the chemokine
IL-8, while the induction of macrophage chemotactic protein-1 was not
as pronounced.
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IL-1-mediated induction of DNA binding activities.
The human COX-2 promoter contains numerous potential binding sites for
transcription factors whose activities can be modulated during
inflammatory episodes. Proximal binding sites for the factors NF-B,
cEBP
(NF-IL6), and Jun/ATF are important for COX-2 induction by
various agents in other cell types (23). We designed
synthetic double-stranded oligonucleotides corresponding to these sites in the human COX-2 promoter and assessed binding activities in nuclear
extracts prepared from control and IL-1-treated cells using EMSA.
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Effect of NF-B on IL-1-mediated COX-2 induction.
The effect of IL-1-mediated NF-
B activation on COX-2 expression was
studied by using inhibitors of NF-
B. PG-490 (400 ng/ml), which
inhibits transcriptional transactivation of bound NF-
B complexes
(28), completely suppressed IL-1-mediated COX-2 induction (98 ± 0.16% inhibition, P < 0.001) as
assessed by Western blotting (Fig.
4A). The proteasome inhibitor
MG-132 (50 µM), which inhibits NF-
B activation by preventing
degradation of inhibitor of
B (I
B) (26),
also potently inhibited COX-2 induction (83 ± 6% inhibition,
P < 0.003) by IL-1 as assessed by Western blotting (Fig. 4A). The trace levels of COX-2 protein seen in cells
treated with MG-132 alone probably represent COX-2 accumulation as the result of proteasome inhibition (Fig. 4A).
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MAPK/SAPK and PKC modulation of COX-2 expression.
The data presented above demonstrate that NF-B activation plays an
important role in IL-1-mediated induction of COX-2 expression in
intestinal myofibroblasts. However, COX-2 expression is subject to
regulation by other signaling pathways as well. Activation of PKC and
the mitogen- and stress-activated protein kinases (SAPKs, MAPKs) is
frequently associated with increased COX-2 expression (20), and these pathways are likewise stimulated by IL-1
(37). Therefore, we next determined the role played by
MAPKs, SAPKs, and PKC during IL-1-mediated induction of COX-2
expression in intestinal myofibroblasts.
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Influence of MAPK/SAPK and PKC activation on COX-2 message
stability.
p38 or ERK activation results in COX-2 mRNA stabilization in other cell
types (42, 50). Therefore, we determined the effect of
MAPK/SAPK and PKC activation on COX-2 message stability. Cultures were
pretreated 4 h with IL-1 and then placed in serum-free medium containing SB-203580 (20 µM), PD-98059 (20 µM), BIS (10 µM), or the RNA polymerase II transcriptional inhibitor DRB (50 µM). Cultures were then frozen 1, 2, and 3 h later for determination of COX-2 mRNA levels by Northern blotting. In these experiments, the COX-2 message half-life in IL-1-treated cells, as judged by decay in the
presence of DRB, was ~1 h (Fig. 7). MEK
or PKC inhibition had no significant effect on COX-2 mRNA stability
(Fig. 7). However, the COX-2 message rapidly disappeared in the
presence of the p38 inhibitor SB-203580, having a half-life of ~20
min (Fig. 7). Thus IL-1-mediated p38 activation results in
stabilization of the COX-2 message.
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DISCUSSION |
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Intestinal myofibroblasts have recently been implicated as
significant sources of inducible COX-2 during colonic inflammation and
colorectal carcinogenesis and thus represent a potential site for
intervention during these processes. We have not only identified signal
transduction pathways necessary for optimal COX-2 induction, but we
provide data on how these pathways interact with each other in this
novel cell type. In this report we demonstrated that IL-1-mediated COX-2 induction in human intestinal myofibroblasts involves regulation at transcriptional and posttranscriptional levels. IL-1-mediated NF-B, p38, and MEK1 activation were necessary for maximal induction, since inhibition of each significantly curtailed resultant COX-2 levels. The p38 inhibition destabilized the COX-2 message, although an
additional positive effect of p38 activation at the transcriptional level cannot be ruled out. Significant JNK activation was also observed
following IL-1 treatment, although in the absence of a specific JNK
inhibitor, we can only speculate on its role in COX-2 regulation.
Activation of PKC signaling was also essential for maximal induction,
since PKC inhibition severely blunted resultant COX-2 levels. In
contrast to what has been reported in other systems, including renal
mesangial cells that are also myofibroblasts (43, 44), PKC
signaling apparently acts in parallel and in relative autonomy from the
other pathways (NF-
B, MAPK/SAPK), since PKC inhibition had little
inhibitory effect on NF-
B mobilization or MAPK/SAPK activation.
These results suggest a model of COX-2 regulation in intestinal
myofibroblasts involving multiple independent paths that are each
necessary for optimal levels of expression analogous to what has been
proposed in macrophages and monocytic cells (34).
We observed a robust transcriptional induction of the COX-2 gene
following IL-1 treatment (Fig. 2). IL-1 treatment mobilized binding of
NF-B complexes to a cognate site within the human COX-2 promoter,
suggesting that NF-
B is an important component of the observed
transcriptional induction (Fig. 3). The importance of NF-
B
activation was demonstrated when NF-
B inhibition by either PG-490 or
MG-132 suppressed COX-2 expression in response to IL-1 (Fig. 4). No
change in binding activity for cEBP
/NF-IL6 was observed, while
Jun/ATF binding increased modestly (1.5-fold) following IL-1 addition
(Fig. 3). The EMSA analyses employed in our studies assay binding
activity of nuclear proteins to cognate sites within the COX-2
promoter. Such analyses do not assay the transactivation potential of
bound complexes, which represents an additional level of regulation.
For example, phosphorylation events within the Rel homology or
transactivation domains of the NF-
B p65 subunit are required for
optimal transactivation of bound complexes (57). Likewise,
prebound Jun/ATF or CREB/ATF heterodimers require phosphorylation
within their respective transactivation domains for maximal
transactivation activity (29, 36). Consequently, the
slight changes in binding activity we observed do not rule out the
possible involvement of Jun/ATF in IL-1-mediated induction of COX-2
expression, especially since we observed increased ATF-2 phosphorylation following IL-1 administration (Fig. 5).
The sites at which each PKC isoform exerts its regulatory effect are
unknown. Unlike other systems, PKC inhibition had little inhibitory
effect on NF-B mobilization or MAPK/SAPK activation (Fig. 6).
Because atypical PKC isoforms are recruited to the nucleus on
activation (33), it is possible that atypical PKC
activation enhances COX-2 expression by enhancing the transactivation
capability of bound transcription factors such as NF-
B and Jun/ATF.
In our studies, BIS treatment, alone or in combination with IL-1,
enhanced the phosphorylation of JNK, ERK-1/2, and p38 to varying
degrees (Fig. 6C). If certain PKC isoforms exert negative
feedback on IL-1-mediated ERK and p38 activation, we would expect to
see enhanced ERK and p38 activation in the presence of BIS. We are
currently employing detailed biochemical and genetic approaches to test this hypothesis.
Another difference between intestinal myofibroblasts and mesangial cells is that IL-1 has not been reported to activate ERKs in mesangial cells (55). ERK activation by IL-1 has recently been shown to depend on establishment of focal adhesions (30), and it is possible that, in the mesangial cell study mentioned, culture conditions were suboptimal for formation of focal adhesion complexes. In this regard it is interesting that we observe maximal COX-2 induction by IL-1 in cells that are 2-3 wk postconfluent. Perhaps this period reflects the time needed for establishment and organization of focal adhesions and other cell-matrix interactions necessary for optimal ERK signaling.
We found that p38 inhibition destabilized the COX-2 message in IL-1-treated cells, changing the half-life from ~1 h to 20 min. IL-1-mediated stabilization of the COX-2 message has been reported (18, 53), and in some cases p38 activation has been associated with increased COX-2 mRNA stability (27, 42), even at low levels of p38 activity (42). Although not as high as the initial burst seen within minutes of treatment, elevated p38 phosphorylation and activity can be detected as long as 24 h following IL-1 treatment. Thus the timing of p38 activation agrees well with the kinetics of COX-2 mRNA accumulation seen in IL-1-treated 18Co cells. The mechanism by which p38 regulates COX-2 message stability is not known. One possibility is that p38 modulates the activity of proteins that bind target sequences identified in the 3'-untranslated region of the COX-2 message (11, 27, 53). ERK activation likewise enhances COX-2 message stability in rat aortic smooth muscle and gastric and intestinal epithelial cells (50, 60). We saw no such effect in human intestinal myofibroblasts.
A working model for how IL-1 induces COX-2 gene expression in
intestinal myofibroblasts is presented in Fig.
8. Upon binding its receptor, IL-1
triggers recruitment and activation of receptor-proximal factors, which
activate MAPK kinase kinases (MAPKKKs). MAPKKK activation results in
ERK, JNK, P38, and possibly NF-B activation. One consequence of p38
activation would be stabilization of the COX-2 message. Mobilized
NF-
B would act via binding its cognate sites within the COX-2
promoter. We have shown a requirement for ERK activation during this
process, and we hypothesize that ERK, and possibly JNK, may act via
phosphorylation of necessary transcription factors such as Jun or ATF
family members, which may also bind to the COX-2 promoter. As mentioned
above, we do not know how the PKC activation observed in our studies
augments COX-2 expression. One possible pathway, shown in Fig. 8, is
via enhancement of transactivation functions of bound transcription
factors. Future experiments will address upstream activators and
targets of NF-
B, MAPK/SAPK, and PKC signaling.
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A thorough understanding of COX-2 gene regulation in human intestinal myofibroblasts offers the potential to develop novel therapeutics that target this particular cell type. Such an approach is appealing, given recent reports that document significant myofibroblast COX-2 expression during acute and chronic inflammation (9, 24, 45, 49, 54) and during the early phases of colonic polyp formation and cancer progression (38, 49, 59). It should be noted, however, that while inhibition of COX-2 expression and activity is desirable in the context of colorectal carcinogenesis, its absence can have a negative impact on the course and resolution of colitis in experimental models (3, 35, 41). Furthermore, myofibroblasts represent significant sources of other proinflammatory and angiogenic factors, as well as epithelial trophic factors (14, 15, 32, 39, 40, 59). Knowledge of signal transduction within these cells is thus important to a complete understanding of colonic inflammation and cancer.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Timothy Hla, Dan Dixon, and Stephen Prescott for providing human COX-2 cDNA plasmids.
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FOOTNOTES |
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This work was funded by grants from the Crohn's and Colitis Foundation of America and the National Institute of Diabetes and Digestive and Kidney Diseases (DK-55783).
Present address of J. D. Valentich: MetaMatrix, 100 Denniston #41, Pittsburgh, PA 15206.
Address for reprint requests and other correspondence: D. W. Powell, Dept. of Internal Medicine, 4.124 John Sealy Annex, Mail Route 0567, Univ. of Texas Medical Branch, 301 Univ. Blvd., Galveston, TX 77555-0567 (E-mail: dpowell{at}utmb.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00388.2001
Received 10 August 2001; accepted in final form 8 November 2001.
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REFERENCES |
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1.
Adegboyega, PA,
Mifflin RC,
Saada JI,
DiMari JF,
and
Powell DW.
Cox-2 is localized to stromal cells in human colorectal adenomas (Abstract).
Gastroenterology
120:
A162,
2001[ISI].
2.
Adegboyega, PA,
Mifflin RC,
Saada JI,
DiMari JF,
and
Powell DW.
Transdifferentiation of mesenchymal stromal cells to myofibroblasts in colorectal polyps (Abstract).
Gastroenterology
120:
A5,
2001.
3.
Ajuebor, MN,
Singh A,
and
Wallace JL.
Cyclooxygenase-2-derived prostaglandin D2 is an early anti-inflammatory signal in experimental colitis.
Am J Physiol Gastrointest Liver Physiol
279:
G238-G244,
2000
4.
Arai, Y,
Takanashi H,
Kitagawa H,
and
Okayasu I.
Involvement of interleukin-1 in the development of ulcerative colitis induced by dextran sulfate sodium in mice.
Cytokine
10:
890-896,
1998[ISI][Medline].
5.
Beltinger, J,
McKaig BC,
Makh S,
Stack WA,
Hawkey CJ,
and
Mahida YR.
Human colonic subepithelial myofibroblasts modulate transepithelial resistance and secretory response.
Am J Physiol Cell Physiol
277:
C271-C279,
1999
6.
Bender, TP.
Isolation of nuclei by sucrose gradient centrifugation.
In: Current Protocols in Molecular Biology (Suppl. 26), edited by Ausubel FM,
Brent R,
Kingston RE,
Moore DD,
Seidman JG,
Smith JA,
and Struhl K.. New York: Wiley, 1995, p. 4.10.16-14.10.11.
7.
Casini-Raggi, V,
Kam L,
Chong YJ,
Fiocchi C,
Pizarro TT,
and
Cominelli F.
Mucosal imbalance of IL-1 and IL-1 receptor antagonist in inflammatory bowel disease. A novel mechanism of chronic intestinal inflammation.
J Immunol
154:
2434-2440,
1995
8.
Chulada, PC,
Thompson MB,
Mahler JF,
Doyle CM,
Gaul BW,
Lee C,
Tiano HF,
Morham SG,
Smithies O,
and
Langenbach R.
Genetic disruption of PTGS-1, as well as of PTGS-2, reduces intestinal tumorigenesis in Min mice.
Cancer Res
60:
4705-4708,
2000
9.
Davies, NM,
Sharkey KA,
Asfaha S,
MacNaughton WK,
and
Wallace JL.
Aspirin causes rapid up-regulation of cyclooxygenase-2 expression in the stomach of rats.
Aliment Pharmacol Ther
11:
1101-1108,
1998[ISI].
10.
Dinarello, CA.
Biologic basis for interleukin-1 in disease.
Blood
87:
2095-2147,
1996
11.
Dixon, DA,
Kaplan CD,
McIntyre TM,
Zimmerman GA,
and
Prescott SM.
Post-transcriptional control of cyclooxygenase-2 gene expression. The role of the 3'-untranslated region.
J Biol Chem
275:
11750-11757,
2000
12.
Dubois, RN.
Cyclooxygenase: a target for colon cancer prevention.
Aliment Pharmacol Ther
14, Suppl1:
64-67,
2000[ISI][Medline].
13.
Eckmann, L,
Stenson WF,
Savidge TC,
Lowe DC,
Barrett KE,
Fierer J,
Smith JR,
and
Kagnoff MF.
Role of intestinal epithelial cells in the host secretory response to infection by invasive bacteria. Bacterial entry induces epithelial prostaglandin H synthase-2 expression and prostaglandin E2 and F2 production.
J Clin Invest
100:
296-309,
1997
14.
Elenbaas, B,
and
Weinberg RA.
Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation.
Exp Cell Res
264:
169-184,
2001[ISI][Medline].
15.
Fiocchi, C.
Intestinal inflammation: a complex interplay of immune and nonimmune cell interactions.
Am J Physiol Gastrointest Liver Physiol
273:
G769-G775,
1997
16.
Fiocchi, C.
Inflammatory bowel disease: etiology and pathogenesis.
Gastroenterology
115:
182-205,
1998[ISI][Medline].
17.
Ghosh, S,
May MJ,
and
Kopp EB.
NF-B and Rel proteins: evolutionarily conserved mediators of immune responses.
Annu Rev Immunol
16:
225-260,
1998[ISI][Medline].
18.
Gou, Q,
Liu CH,
Ben-Av P,
and
Hla T.
Dissociation of basal turnover and cytokine-induced transcript stabilization of the human cyclooxygenase-2 mRNA by mutagenesis of the 3'-untranslated region.
Biochem Biophys Res Commun
242:
508-512,
1998[ISI][Medline].
19.
Greenberg, ME.
Nuclear runoff transcription in mammalian cells.
In: Current Protocols in Molecular Biology (Suppl. 26), edited by Ausubel FM,
Brent R,
Kingston RE,
Moore DD,
Seidman JG,
Smith JA,
and Struhl K.. New York: Wiley, 1995, p. 4.10.11-14.10.14.
20.
Guan, Z,
Buckman SY,
Springer LD,
and
Morrison AR.
Regulation of cyclooxygenase-2 by the activated p38 MAPK signaling pathway.
Adv Exp Med Biol
469:
9-15,
1999[ISI][Medline].
21.
Hinterleitner, TA,
Saada JI,
Berschneider HM,
Powell DW,
and
Valentich JD.
IL-1 stimulates intestinal myofibroblast cox gene expression and augments activation of Cl secretion in T84 cells.
Am J Physiol Cell Physiol
271:
C1262-C1268,
1996
22.
Hla, T,
and
Neilson K.
Human cyclooxygenase-2 cDNA.
Proc Natl Acad Sci USA
89:
7384-7388,
1992[Abstract].
23.
Inoue, H,
and
Tanabe T.
Transcriptional role of the nuclear factor B site in the induction by lipopolysaccharide and suppression by dexamethasone of cyclooxygenase-2 in U937 cells.
Biochem Biophys Res Commun
244:
143-148,
1998[ISI][Medline].
24.
Jackson, LM,
Wu KC,
Mahida YR,
Jenkins D,
and
Hawkey CJ.
Cyclooxygenase (COX) 1 and 2 in normal, inflamed, and ulcerated human gastric mucosa.
Gut
47:
762-770,
2000
25.
Karin, M,
Liu Z,
and
Zandi E.
AP-1 function and regulation.
Curr Opin Cell Biol
9:
240-246,
1997[ISI][Medline].
26.
Kothny-Wilkes, G,
Kulms D,
Poppelmann B,
Luger TA,
Kubin M,
and
Schwarz T.
Interleukin-1 protects transformed keratinocytes from tumor necrosis factor-related apoptosis-inducing ligand.
J Biol Chem
273:
29247-29253,
1998
27.
Lasa, M,
Mahtani KR,
Finch A,
Brewer G,
Saklatvala J,
and
Clark AR.
Regulation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade.
Mol Cell Biol
20:
4265-4274,
2000
28.
Lee, KY,
Chang W,
Qiu D,
Kao PN,
and
Rosen GD.
PG490 (triptolide) cooperates with tumor necrosis factor- to induce apoptosis in tumor cells.
J Biol Chem
274:
13451-13455,
1999
29.
Livingstone, C,
Patel G,
and
Jones N.
ATF-2 contains a phosphorylation-dependent transcriptional activation domain.
EMBO J
14:
1785-1797,
1995[Abstract].
30.
MacGillivray, MK,
Cruz TF,
and
McCulloch CA.
The recruitment of the interleukin-1 (IL-1) receptor-associated kinase (IRAK) into focal adhesion complexes is required for IL-1 -induced ERK activation.
J Biol Chem
275:
23509-23515,
2000
31.
McCall, RD,
Haskill S,
Zimmermann EM,
Lund PK,
Thompson RC,
and
Sartor RB.
Tissue interleukin-1 and interleukin-1 receptor antagonist expression in enterocolitis in resistant and susceptible rats.
Gastroenterology
106:
960-972,
1994[ISI][Medline].
32.
McKaig, BC,
Makh SS,
Hawkey CJ,
Podolsky DK,
and
Mahida YR.
Normal human colonic subepithelial myofibroblasts enhance epithelial migration (restitution) viaTGF-3.
Am J Physiol Gastrointest Liver Physiol
276:
G1087-G1093,
1999
33.
Mellor, H,
and
Parker PJ.
The extended protein kinase C superfamily.
Biochem J
332:
281-292,
1998[ISI][Medline].
34.
Mestre, JR,
Mackrell PJ,
Rivadeneira DE,
Stapleton PP,
Tanabe T,
and
Daly JM.
Redundancy in the signaling pathways and promoter elements regulating cyclooxygenase-2 gene expression in endotoxin-treated macrophage/monocytic cells.
J Biol Chem
276:
3977-3982,
2001
35.
Morteau, O,
Morham SG,
Sellon R,
Dieleman LA,
Langenbach R,
Smithies O,
and
Sartor RB.
Impaired mucosal defense to acute colonic injury in mice lacking cyclooxygenase-1 or cyclooxygenase-2.
J Clin Invest
105:
469-478,
2000
36.
Nakajima, T,
Uchida C,
Anderson SF,
Parvin JD,
and
Montminy M.
Analysis of a cAMP-responsive activator reveals a two-component mechanism for transcriptional induction via signal-dependent factors.
Genes Dev
11:
738-747,
1997[Abstract].
37.
O'Neill, LA,
and
Greene C.
Signal transduction pathways activated by the IL-1 receptor family: ancient signaling machinery in mammals, insects, and plants.
J Leukoc Biol
63:
650-657,
1998[Abstract].
38.
Oshima, M,
Dinchuk JE,
Kargman SL,
Oshima H,
Hancock B,
Kwong E,
Trzaskos JM,
Evans JF,
and
Taketo MM.
Suppression of intestinal polyposis in APC 716 knockout mice by inhibition of cyclooxygenase 2 (COX-2).
Cell
87:
803-809,
1996[ISI][Medline].
39.
Powell, DW,
Mifflin RC,
Valentich JD,
Crowe SE,
Saada JI,
and
West AB.
Myofibroblasts. I. Paracrine cells important in health and disease.
Am J Physiol Cell Physiol
277:
C1-C19,
1999
40.
Powell, DW,
Mifflin RC,
Valentich JD,
Crowe SE,
Saada JI,
and
West AB.
Myofibroblasts. II. Intestinal subepithelial myofibroblasts.
Am J Physiol Cell Physiol
277:
C183-C201,
1999
41.
Reuter, BK,
Asfaha S,
Buret A,
Sharkey KA,
and
Wallace JL.
Exacerbation of inflammation-associated colonic injury in rat through inhibition of cyclooxygenase-2.
J Clin Invest
98:
2076-2085,
1996
42.
Ridley, SH,
Dean JL,
Sarsfield SJ,
Brook M,
Clark AR,
and
Saklatvala J.
A p38 map kinase inhibitor regulates stability of interleukin-1-induced cyclooxygenase-2 mRNA.
FEBS Lett
439:
75-80,
1998[ISI][Medline].
43.
Rzymkiewicz, DM,
Tetsuka T,
Daphna-Iken D,
Srivastava S,
and
Morrison AR.
Interleukin-1 activates protein kinase C
in renal mesangial cells. Potential role in prostaglandin E2 up-regulation.
J Biol Chem
271:
17241-17246,
1996
44.
Sanz, L,
Diaz-Meco MT,
Nakano H,
and
Moscat J.
The atypical PKC-interacting protein p62 channels NF-B activation by the IL-1-TRAF6 pathway.
EMBO J
19:
1576-1586,
2000
45.
Schmassmann, A,
Peskar BM,
Stettler C,
Netzer P,
Stroff T,
Flogerzi B,
and
Halter F.
Effects of inhibition of prostaglandin endoperoxide synthase-2 in chronic gastrointestinal ulcer models in rats.
Br J Pharmacol
123:
795-804,
1998[Abstract].
46.
Schmitz, ML,
Bacher S,
and
Kracht M.
IB-independent control of NF-
B activity by modulatory phosphorylations.
Trends Biochem Sci
26:
186-190,
2001[ISI][Medline].
47.
Schreiber, E,
Matthias P,
Muller MM,
and
Schaffner W.
Rapid detection of octamer binding proteins with "mini-extracts," prepared from a small number of cells.
Nucleic Acids Res
17:
6419,
1989[ISI][Medline].
48.
Shao, J,
Sheng H,
Inoue H,
Morrow JD,
and
DuBois RN.
Regulation of constitutive cyclooxygenase-2 expression in colon carcinoma cells.
J Biol Chem
275:
33951-33956,
2000
49.
Shattuck-Brandt, RL,
Varilek GW,
Radhika A,
Yang F,
Washington MK,
and
DuBois RN.
Cyclooxygenase 2 expression is increased in the stroma of colon carcinomas from IL-10(/
) mice.
Gastroenterology
118:
337-345,
2000[ISI][Medline].
50.
Sheng, H,
Williams CS,
Shao J,
Liang P,
DuBois RN,
and
Beauchamp RD.
Induction of cyclooxygenase-2 by activated Ha-ras oncogene in rat-1 fibroblasts and the role of mitogen-activated protein kinase pathway.
J Biol Chem
273:
22120-22127,
1998
51.
Singer, II,
Kawka DW,
Schloemann S,
Tessner T,
Riehl T,
and
Stenson WF.
Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease.
Gastroenterology
115:
297-306,
1998[ISI][Medline].
52.
Smith, WL,
and
DeWitt DL.
Prostaglandin endoperoxide H synthases-1 and -2.
Adv Immunol
62:
167-215,
1996[ISI][Medline].
53.
Srivastava, SK,
Tetsuka T,
Daphna-Iken D,
and
Morrison AR.
IL-1 stabilizes COX II mRNA in renal mesangial cells: role of 3'-untranslated region.
Am J Physiol Renal Fluid Electrolyte Physiol
267:
F504-F508,
1994
54.
Takahashi, S,
Shigeta J,
Inoue H,
Tanabe T,
and
Okabe S.
Localization of cyclooxygenase-2 and regulation of its mRNA expression in gastric ulcers in rats.
Am J Physiol Gastrointest Liver Physiol
275:
G1137-G1145,
1998
55.
Uciechowski, P,
Saklatvala J,
Vonderohe J,
Resch K,
Szamel M,
and
Kracht M.
Interleukin 1 activates Jun-N-terminal kinases Jnk1 and Jnk2 but not extracellular regulated map kinase (ERK) in human glomerular mesangial cells.
FEBS Lett
394:
273-278,
1996[ISI][Medline].
56.
Valentich, JD,
Popov V,
Saada JI,
and
Powell DW.
Phenotypic characterization of an intestinal subepithelial myofibroblast cell line.
Am J Physiol Cell Physiol
272:
C1513-C1524,
1997
57.
Wang, D,
and
Baldwin AS, Jr.
Activation of nuclear factor-b-dependent transcription by tumor necrosis factor-
is mediated through phosphorylation of RelA/p65 on serine 529.
J Biol Chem
273:
29411-29416,
1998
58.
Wang, Q,
Kim S,
Wang X,
and
Evers BM.
Activation of NF-b binding in HT-29 colon cancer cells by inhibition of phosphatidylinositol 3-kinase.
Biochem Biophys Res Commun
273:
853-858,
2000[ISI][Medline].
59.
Williams, CS,
Tsujii M,
Reese J,
Dey SK,
and
DuBois RN.
Host cyclooxygenase-2 modulates carcinoma growth.
J Clin Invest
105:
1589-1594,
2000
60.
Xu, K,
Robida AM,
and
Murphy TJ.
Immediate-early MEK-1-dependent stabilization of rat smooth muscle cell cyclooxygenase-2 mRNA by Gq-coupled receptor signaling.
J Biol Chem
275:
23012-23019,
2000
61.
Youngman, KR,
Simon PL,
West GA,
Cominelli F,
Rachmilewitz D,
Klein JS,
and
Fiocchi C.
Localization of intestinal interleukin 1 activity and protein and gene expression to lamina propria cells.
Gastroenterology
104:
749-758,
1993[ISI][Medline].