Regulation of COX-2 expression in human intestinal myofibroblasts: mechanisms of IL-1-mediated induction

Randy C. Mifflin1, Jamal I. Saada1, John F. Di Mari1, Patrick A. Adegboyega3, John D. Valentich1, and Don W. Powell1,2

Departments of 1 Internal Medicine, 2 Physiology and Biophysics, and 3 Pathology, University of Texas Medical Branch, Galveston, Texas 77555


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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-1alpha 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-kappa B (NF-kappa 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-kappa 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-kappa 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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 beta -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-kappa B (NF-kappa 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.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials. Recombinant human IL-1alpha 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-kappa 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-beta -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-kappa 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 alpha -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-kappa 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 [alpha -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 beta  (cEBPbeta ) (NF-IL-6; -140 to -116 relative to human COX-2 transcription start site) 5'-CACCGGGCTTACGCAATTTTTTTAA-3'; NF-kappa B (-231 to -207 relative to human COX-2 transcription start site) 5'-GGAGAGTGGGGACTACCCCCTCTGC-3'; and consensus NF-kappa B (from kappa  light chain enhancer) 5'-AGTTGAGGGGACTTTCCCAGGC-3'.

Double-stranded oligonucleotides were end labeled using T4 polynucleotide kinase (GIBCO BRL, Bethesda, MD) and [gamma -32P]ATP. Probe (1 × 105 cpm; 30 fmol) was added to nuclear protein (10 µg) in a binding buffer containing 20% glycerol, 60 mM KCl, 5.0 mM MgSO4, 2.5 mM EDTA, 2.5 mM DTT, 50 mM HEPES, pH 7.9, 0.1 mg/ml poly(dI-dC) (Amersham-Pharmacia), 1 mM PMSF, and 5.0 mM NaF (20 µl final volume). Competition experiments contained a 200-fold molar excess of unlabeled specific or nonspecific competitor. Binding was carried out for 20 min at room temperature, and reactions were fractionated in 6.0% polyacrylamide gels in 0.5× TBE (89 mM Tris-borate, pH 8.3, and 2.0 mM EDTA) buffer. For supershift analyses, antibodies (2.0 µg) were added after the initial incubation period, and reactions were incubated for 45 min further before fractionation. Bound complexes were visualized following autoradiography of dried gels.

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 [gamma -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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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-1alpha (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-1beta (500 pg/ml) was used (data not shown).


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Fig. 1.   Time course of interleukin-1 (IL-1)-mediated cyclooxygenase-2 (COX-2) induction. Confluent 18Co monolayers were placed in fresh medium with or without IL-1alpha (500 pg/ml). At the indicated times, cultures were harvested for isolation of total RNA or protein. A: COX-2 and COX-1 mRNA Levels. Total RNA (20 µg) was analyzed for COX-2 message levels by Northern blotting as described in MATERIALS AND METHODS. The COX-2 transcript shown is the predominant 4.2-kb transcript. The COX-2 probe was removed and the blot was reprobed for COX-1 mRNA levels. Shown is the predominant 2.7-kb COX-1 transcript. B: COX-2 and COX-1 protein levels. Total protein (10 µg) was analyzed for COX-2 and COX-1 protein levels by Western blotting as described in MATERIALS AND METHODS. Shown are the 72-kDa COX-2 and COX-1.

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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Fig. 2.   COX-2 transcriptional activity following IL-1 treatment. Confluent 18Co monolayers were placed in fresh medium with or without IL-1alpha (500 pg/ml). After 6 h, nuclei were isolated and nuclear runoff analysis was performed as described in MATERIALS AND METHODS. Resultant phosphorimages from each hybridization (left) were quantitated and normalized to the beta -actin signal and are presented graphically at right. MCP-1, macrophage chemotactic protein-1; actin, beta -actin; vector, plasmid Bluescript SK- without insert, used to calculate background hybridization.

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-kappa B, cEBPbeta (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.

Specific binding to the Jun/ATF oligonucleotide was detected in untreated cells and this binding increased ~1.5-fold (1.55 ± 0.07, P < 0.005) in response to IL-1 (Fig. 3A). A single specific complex was observed bound to the cEBPbeta (NF-IL6) oligonucleotide, and this binding was unaffected by IL-1 treatment (Fig. 3B). Binding activity to the cEBPbeta (NF-IL6) oligonucleotide in control and untreated cells was generally low, requiring extended exposure times for detection.


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Fig. 3.   IL-1-mediated induction of DNA binding activities. Electrophoretic mobility shift assay (EMSA) analysis of binding to the COX-2 Jun/ATF (A), COX-2 cEBPbeta (NF-IL6) (B), consensus NF-kappa B (C), and COX-2 NF-kappa B (D) oligonucleotides. Confluent 18Co monolayers were placed in fresh medium with or without IL-1alpha (500 pg/ml) for 6 h. Nuclear extracts were then prepared, and EMSA was performed as described in MATERIALS AND METHODS. Representative autoradiograms are shown that demonstrate binding activities in extracts prepared from untreated (control) and IL-1-treated (IL-1) cells. Spec Comp, specific competitor: binding reactions that contained a 200-fold molar excess of specific unlabeled oligonucleotide; NS Comp, nonspecific competitor: binding reactions that contained a 200-fold molar excess of an unlabeled nonspecific oligonucleotide. For the nuclear factor-kappa B (NF-kappa B) and cellular enhancer binding protein (cEBP) reactions, the nonspecific competitor was the Jun/activating transcription factor (ATF) oligonucleotide. For the Jun/ATF reactions, the nonspecific competitor was the NF-kappa B oligonucleotide. In D, C1, C2, C3, and C4 denote specific complexes binding the COX-2 NF-kappa B oligonucleotide. In D, specific antibodies for NF-kappa B family members (p50, RelB, p65, cRel, and p52) were included in binding reactions that contained the labeled COX-2 NF-kappa B oligonucleotide and extracts prepared from cells treated with IL-1 for 6 h. The composition of specific complexes that could be identified is denoted at right.

NF-kappa B activation in response to IL-1 was investigated by using oligonucleotides representing the consensus NF-kappa B binding sequence and the proximal NF-kappa B site of the human COX-2 promoter. Virtually undetectable in untreated cells, binding activity to the consensus oligonucleotide was increased dramatically following IL-1 treatment (Fig. 3C). Two complexes that bound the COX-2 NF-kappa B site were observed in control cells (Fig. 3D, C1 and C3). IL-1 treatment resulted in the appearance of new complexes, denoted C2 and C4 in Fig. 3D. Inclusion of antibodies to members of the NF-kappa B family in the binding reactions (Fig. 3D) demonstrated that C4 is composed of p50 homodimers and that the C2 band is composed of p50/p65 heterodimers. Nuclear p50 and p65 were not detected in untreated cells with the use of antibody shift analysis (data not shown). C1 and C3 probably represent specific binding of factors, unrelated to NF-kappa B, to sequences within the oligonucleotide, since they did not interact with any of the NF-kappa B antibodies (Fig. 3D).

In summary, IL-1 treatment resulted in the induction of nuclear p50/p65 binding to a cognate NF-kappa B element of the human COX-2 promoter. Inducible binding to the cEBPbeta (NF-IL6) element was not observed and Jun/ATF binding was modestly increased 6 h after IL-1 treatment. Similar results were observed in extracts prepared from cells 30 min or 2 h after IL-1 treatment (data not shown).

Effect of NF-kappa B on IL-1-mediated COX-2 induction. The effect of IL-1-mediated NF-kappa B activation on COX-2 expression was studied by using inhibitors of NF-kappa B. PG-490 (400 ng/ml), which inhibits transcriptional transactivation of bound NF-kappa 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-kappa B activation by preventing degradation of inhibitor of kappa B (Ikappa 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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Fig. 4.   Effect of NF-kappa B inhibition on IL-1-mediated COX-2 induction. A: Western analysis of COX-2 expression. Confluent 18Co monolayers were pretreated for 1 h with either MG-132 (50 µM), PG-490 (400 ng/ml), or solvent (DMSO, 0.5%) and then incubated for 24 h in the presence or absence of IL-1alpha (500 pg/ml). Cells were then harvested and COX-2 protein levels were assessed by Western blotting. Shown is the 72-kDa COX-2. The membrane was then stripped and reprobed using an antibody to alpha -smooth muscle actin (SMA), ensuring that equal loading of lanes had occurred. B: EMSA analysis of NF-kappa B binding. Cells treated as in A were harvested 6 h following IL-1 addition, and nuclear extracts were prepared as described in MATERIALS AND METHODS. Binding activities to the consensus NF-kappa B oligonucleotide are shown.

EMSA analysis was performed by using nuclear extracts prepared from cells treated with MG-132 or PG-490 to verify their effect on NF-kappa B mobilization. Although MG-132 potently suppressed the bulk of IL-1-induced binding activity to the consensus oligonucleotide, a single complex was observed in cells treated with IL-1 and MG-132 (Fig. 4B). Antibody shift experiments showed that this complex consisted of p50/p65 heterodimers (data not shown). The residual NF-kappa B mobilization seen in the presence of MG-132 probably results from incomplete inhibition of proteosome activity, although proteasome-independent means of NF-kappa B translocation have been described (46, 58). In contrast, PG-490 treatment enhanced nuclear NF-kappa B binding activity in control and IL-1-treated cells (Fig. 4B). Because PG-490 inhibits the transactivation activity of bound NF-kappa B complexes and not the mobilization (translocation) of NF-kappa B subunits, enhanced binding in the presence of PG-490 is expected, since NF-kappa B positively regulates synthesis of Ikappa Balpha (17). The data presented in Fig. 4 demonstrate that NF-kappa B mobilization and transactivation are necessary for IL-1-mediated induction of COX-2 expression in human intestinal myofibroblasts.

MAPK/SAPK and PKC modulation of COX-2 expression. The data presented above demonstrate that NF-kappa 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.

MAPK/SAPK activation following IL-1 treatment was assessed by Western blotting using antibodies specific for phosphorylated active p38 (Thr-180/Tyr-182) and ERK-1/2 (Thr-202/Tyr-204). A phospho-c-Jun (Ser-73) specific antibody was used as a measure of JNK activation. Substantial activation of all three types was observed 30 min after IL-1 treatment, and increased p38 and c-Jun phosphorylation persisted for at least 4 h (Fig. 5A). As an additional indicator of p38 or JNK activation, phosphorylation of the transcription factor ATF-2 (Thr-71) was also observed in response to IL-1 (Fig. 5A). This observation is significant, since phosphorylation of the ATF transactivation domain is required for bound ATF/Jun heterodimers to activate transcription (25).


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Fig. 5.   Mitogen-activated protein kinase (MAPK)/stress-activated protein kinase (SAPK) modulation of COX-2 expression. A: IL-1-mediated stimulation of MAPK/SAPK phosphorylation. 18Co monolayers were incubated for 0.5 or 4 h in the presence or absence of IL-1alpha (500 pg/ml, delivered in serum-free medium). Cells were harvested, and Western blotting was performed to determine relative levels of total (T) and phosphorylated (P) p38, extracellular signal-regulated protein kinase (ERK-1/2), and cJun as described in MATERIALS AND METHODS. Levels of phosphorylated ATF-2 were also determined. B: effect of p38 and mitogen-activated/extracellular response kinase (MEK) inhibition on IL-1-mediated COX-2 induction. Confluent 18Co monolayers were incubated for 24 h in the presence or absence of IL-1, SB-203580 (p38 inhibitor, 20 µM), PD-98059 (MEK1/2 inhibitor, 20 µM), and various combinations thereof as indicated. Cells were then harvested and COX-2 protein levels were determined by Western analysis as described in MATERIALS AND METHODS. Shown is the 72-kDa COX-2. The membrane was then stripped and reprobed using an antibody to SMA, ensuring that equal loading of lanes had occurred.

The contribution of MAPK activation to IL-1-mediated COX-2 induction was assessed by Northern blotting for COX-2 mRNA levels in cells treated with the combination of IL-1 and a specific p38 (SB-203580, 20 µM) or MEK1 (ERK-1/2 kinase, PD-98059, 20 µM) inhibitor. p38 and MEK inhibition each profoundly suppressed COX-2 protein induction by 93% (± 4.7%, P < 0.001) and 76% (± 7.8%, P < 0.001), respectively (Fig. 5B). The combination of SB-203580 and PD-98059 resulted in 98% inhibition (± 1.5%, P < 0.001; Fig. 5B). Thus ERK and p38 activation are likewise required for maximal IL-1-mediated induction of COX-2 expression in human intestinal myofibroblasts.

The role played by PKC in IL-1-mediated COX-2 induction was next examined. Initially, PKC activity was measured by an in vitro kinase assay. Cells were incubated for 20 min in the presence or absence of IL-1 or the PKC inhibitor BIS (10 µM) at a concentration that is inhibitory to all PKC isoforms. PKC activity was elevated approximately fivefold following IL-1 treatment, while inclusion of BIS completely suppressed IL-1-induced PKC activity (Fig. 6A). The observed activation of PKC was necessary for COX-2 induction by IL-1, since the inclusion of BIS significantly suppressed (59% inhibition, P < 0.01) resultant COX-2 protein levels (Fig. 6B). Thus IL-1 treatment resulted in rapid induction of PKC activity, and PKC activation was necessary for maximal COX-2 induction by IL-1.


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Fig. 6.   Modulation of COX-2 expression by protein kinase C (PKC). A: PKC enzymatic activity following IL-1 addition. Confluent 18Co monolayers were incubated for 20 min in the presence or absence of IL-1alpha (500 pg/ml) or the PKC inhibitor bisindolylmaleimide I (BIS; 10 µM). Cells were then harvested and PKC activity was determined as described in MATERIALS AND METHODS. The data are presented as %PKC activity, relative to control cells, ±SE from 3 separate determinations. B: effect of PKC inhibition on COX-2 expression. Confluent 18Co monolayers were incubated 24 h in the presence or absence of IL-1alpha (500 pg/ml) or BIS (10 µM). COX-2 protein levels were then assessed by Western blotting as described in MATERIALS AND METHODS. Shown is the 72-kDa COX-2. The membrane was then stripped and reprobed using an antibody to SMA, ensuring that equal loading of lanes had occurred. C: effect of PKC inhibition on MAPK/SAPK activation. Confluent 18Co monolayers were incubated 30 min in the presence or absence of IL-1alpha (500 pg/ml) or BIS (10 µM). ERK, p38, and JNK activation was assessed by Western blotting using phosphorylation-specific antibodies as described in MATERIALS AND METHODS. D: effect of PKC inhibition on NF-kappa B activation. Confluent 18Co monolayers were incubated 2 h in the presence or absence of IL-1alpha (500 pg/ml) or BIS (10 µM). Nuclear extracts were prepared, and NF-kappa B binding activity to the consensus NF-kappa B oligonucleotide was determined by EMSA as described in MATERIALS AND METHODS.

The effect of PKC inhibition on ERK, p38, JNK, and NF-kappa B mobilization was next investigated to determine whether PKC is a critical upstream mediator required for activation of these pathways or whether PKC activation operates in parallel. PKC inhibition (BIS, 10 µM) had no inhibitory effect on ERK, p38, or JNK activation by IL-1 (Fig. 6C). In fact, BIS treatment enhanced phospho-ERK, phospho-p38, and phospho-JNK levels to varying degrees (Fig. 6C). NF-kappa B mobilization, assessed by EMSA analysis, was not affected by the PKC inhibitor BIS (Fig. 6D). In summary, although PKC inhibition did not abrogate activation of MAPK, SAPK, and NF-kappa B pathways, absolute levels of ERK-1/2, p38, and JNK activation were enhanced to varying degrees by the pan-PKC inhibitor BIS.

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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Fig. 7.   Influence of MAPK/SAPK and PKC activation on COX-2 message stability. Confluent 18Co cultures were incubated 4 h in the presence of IL-1alpha (500 pg/ml). Cells were 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 5,6-dichloro-1-beta -D- ribofuranosylbenzimidazole (DRB; 50 µM). Cultures were then frozen 1, 2, and 3 h later for determination of COX-2 mRNA levels by Northern blotting. A: phosphorimages from a representative experiment. Also shown are digitized photographs of the membranes showing equivalent levels of 28S ribosomal RNA (28) prior to hybridization. B: graph of relative COX-2 mRNA levels (normalized to 28S rRNA) as a function of the time spent in the presence of each inhibitor. Data compiled from 3 separate experiments indicates that the COX-2 message half-life in the presence of DRB is 1.1 ± 0.06 h. The half-lives of the COX-2 message in the presence of PD-98059 or BIS are 0.88 ± 0.04 and 0.83 ± 0.04 h, respectively. The COX-2 message half-life in the presence of the P38 inhibitor SB-203580 is 0.35 ± 0.03 h. P < 0.003 compared with DRB, and P < 0.01 compared with PD-98059 or BIS.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa 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-kappa B, MAPK/SAPK), since PKC inhibition had little inhibitory effect on NF-kappa 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-kappa B complexes to a cognate site within the human COX-2 promoter, suggesting that NF-kappa B is an important component of the observed transcriptional induction (Fig. 3). The importance of NF-kappa B activation was demonstrated when NF-kappa 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 cEBPbeta /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-kappa 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-kappa 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-kappa 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-kappa B activation. One consequence of p38 activation would be stabilization of the COX-2 message. Mobilized NF-kappa 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-kappa B, MAPK/SAPK, and PKC signaling.


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Fig. 8.   IL-1-mediated COX-2 induction in intestinal myofibroblasts: a working model. Simplified cartoon illustrating pathways activated by IL-1, which culminate in induction of COX-2 gene expression. See text for discussion. IL-1RACP, IL-1 receptor accessory protein.

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.


    ACKNOWLEDGEMENTS

We are grateful to Drs. Timothy Hla, Dan Dixon, and Stephen Prescott for providing human COX-2 cDNA plasmids.


    FOOTNOTES

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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Am J Physiol Cell Physiol 282(4):C824-C834
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