p38 MAPK and NF-{kappa}B mediate COX-2 expression in human airway myocytes

Cherie A. Singer,1 Kimberly J. Baker,2 Alan McCaffrey,1 David P. AuCoin,2 Melissa A. Dechert,2 and William T. Gerthoffer1,2

1Department of Pharmacology and 2Cell and Molecular Biology Program, University of Nevada School of Medicine, Reno, Nevada 89557-0046

Submitted 27 November 2002 ; accepted in final form 17 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
We have previously demonstrated that p38 and extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinases (MAPK) are components of proinflammatory induced cytokine expression in human airway myocytes. The experiments described here further these studies by examining p38 MAPK and NF-{kappa}B regulation of cyclooxygenase-2 (COX-2) expression in response to a complex inflammatory stimulus consisting of 10 ng/ml interleukin (IL)-1{beta}, tumor necrosis factor-{alpha} (TNF-{alpha}), and interferon (IFN)-{gamma}. COX-2 expression was induced with this stimulus in a time-dependent manner, with maximal expression seen 12-20 h after treatment. Semiquantitative RT-PCR and immunoblotting experiments demonstrate decreased COX-2 expression following treatment with the p38 MAPK inhibitor SB-203580 (25 µM) or the proteosome inhibitor MG-132 (1 µM). SB-203580 did not affect cytokine-stimulated I{kappa}B{alpha} degradation, NF-{kappa}B nuclear binding activity, or NF-{kappa}B-dependent signaling from the COX-2 promoter, indicating that p38 MAPK and NF-{kappa}B may affect COX-2 expression via separate signaling pathways. SB-203580, but not MG-132, also increased the initial rate of COX-2 mRNA decay, indicating p38 MAPK, but not NF-{kappa}B, participates in the regulation of COX-2 mRNA stability. These findings suggest that although p38 MAPK and NF-{kappa}B signaling regulate steady-state levels of COX-2 expression, p38 MAPK additionally affects stability of COX-2 mRNA in cytokine-stimulated human airway myocytes.

inflammation; mitogen-activated protein kinase; mRNA stability; cytokines


INFLAMMATION OF THE AIRWAYS is a complex process involving the recruitment and infiltration of immune cells into the airway, increases in epithelial mucosal secretions, and smooth muscle hyperreactivity and hyperplasia. These events are coordinated by chemical mediators of inflammation released from leukocytes such as eosinophils and macrophages, as well as lung fibroblasts and airway myocytes. In particular, the synthesis and release of inflammatory mediators by airway myocytes have become increasingly well documented (reviewed in 7, 14), and investigation of mechanisms regulating this synthetic function of airway smooth muscle is of active interest. Our laboratory has undertaken studies to address the role of mitogen-activated protein kinases (MAPK) in the expression and synthesis of inflammatory mediators in airway smooth muscle. Previous work using a cDNA expression array demonstrated that a complex proinflammatory stimulus consisting of interleukin (IL)-1{beta}, tumor necrosis factor (TNF)-{alpha}, and interferon (IFN)-{gamma} induces the expression of multiple inflammatory mediators in human bronchial smooth muscle cells (BSMC) (13). In these experiments, both p38 MAPK and extracellular signal-regulated protein kinase (ERK) MAPK were found to be components regulating cytokine-stimulated expression of IL-6 and IL-8, whereas only ERK MAPK was involved in mediating IL-1{beta} expression.

Prostanoids, such as prostaglandin (PG) E2, prostacyclin, and thromboxanes, are produced by all cell types in the lung (9, 34) and are known to modulate cytokine release, cell proliferation, inflammatory cell recruitment, and contractile tone of both airway and vascular smooth muscle (21, 37). Two isoforms of cyclooxygenase (COX) are responsible for prostanoid synthesis from arachidonic acid. COX-1 is constitutively expressed in most cell types to produce prostanoids involved in maintaining cell homeostasis. COX-2 expression is induced by mitogenic and proinflammatory stimuli (11, 20, 28) and is regulated by p38 MAPK in many different cell types (16, 17, 42). Analysis of the COX-2 gene, however, reveals several transcriptional regulatory sequences in the promoter including a site for CCAAT/enhancer-binding protein and cAMP response element motifs, three SP1 sites, two NF-{kappa}B sites, two activator protein-2 sites, and an Ets-1 site (2), suggesting that multiple signaling pathways participate in the induction of COX-2 expression.

Regulation of gene expression by MAPK or other signaling pathways can occur at both the transcriptional and posttranscriptional levels. Transcriptional regulation occurs through activation of transcription factors and control of transcriptional initiation (36), whereas posttranscriptional regulation can occur through mRNA processing, modification of mRNA stability (4, 8), and control of translation initiation (31, 39). Stabilization of mRNA affects the abundance and increases the half-life of transcripts within the cell and is a mechanism important for the rapid and sustained induction of early-response genes, such as those induced by proinflammatory mediators. Induction of TNF-{alpha}, IL-6, and macrophage inflammatory protein-1{alpha} by lipopolysaccharide (LPS) is dependent on p38 MAPK regulation of mRNA stability in human blood monocytes (38), whereas TNF-{alpha} induction of granulocyte-macrophage colony-stimulating factor is dependent on mRNA stability regulated by ERK MAPK in blood eosinophils (10).

The experiments described here examine p38 MAPK and NF-{kappa}B regulation of COX-2 in human BSMC stimulated with 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma}. The data provide evidence suggesting that IFN-{gamma} is an important addition to the cytokine stimulus when used in combination with IL-1{beta} and TNF-{alpha}. Further experiments demonstrate that both p38 MAPK and NF-{kappa}B participate in the regulation of steady-state levels of COX-2 expression by this cytokine stimulus and that p38 MAPK additionally affects the decay of COX-2 mRNA transcripts from these cultures. The results from these studies describe p38 MAPK-mediated mRNA stability for the first time in airway myocytes and suggest that while multiple signaling pathways participate in regulating COX-2, posttranscriptional modification of COX-2 gene expression is an important mechanism affecting the inflammatory response in airway smooth muscle.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Materials. Normal human BSMC and smooth muscle cell growth medium (SmGm) were obtained from Clonetics (San Diego, CA). IFN-{gamma}, IL-1{beta}, TNF-{alpha}, and all other tissue culture reagents were purchased from Sigma (St. Louis, MO). SB-203580, PD-98059, and MG-132 were purchased from Calbiochem (La Jolla, CA). TRIzol, Superscript II, and PCR reagents were purchased from Invitrogen (Carlsbad, CA). Thermus aquaticus (Taq) polymerase, RNAse H, reagents for probe labeling, gel shift assays, in vitro site-directed mutagenesis, and luciferase assays were purchased from Promega (Madison, WI). Reagents for transient transfections were purchased from Stratagene (La Jolla, CA). Antibodies for COX-1, COX-2, p38 MAPK and inhibitor of {kappa}B{alpha} (I{kappa}B{alpha}) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). An antibody recognizing phosphorylated p38 MAPK was purchased from Cell Signaling Technology (Beverly, MA). Reagents for Northern analysis were purchased from Ambion (Austin, TX). The STAT-PGE2 enzyme immunoassay was purchased from Cayman Chemical (Ann Arbor, MI).

Cell culture and drug treatments. BSMC were maintained in a humidified 5% CO2 atmosphere at 37°C in SmGm or M199 media supplemented with 5% fetal bovine serum (FBS), 0.5 ng/ml epidermal growth factor, 5 µg/ml insulin, 2 ng/ml fibroblast growth factor, 50 µg/ml gentamicin, and 50 ng/ml amphotericin B. These cells have been screened by Clonetics for human pathogens and nonsmooth muscle cell types. Further characterization of these cultures by our laboratory has been previously described (13), and cultures from passages 4-8 were used for these experiments. When the cultures reached confluence, they were growth arrested for 24 h in media supplemented with 0.1% FBS, growth factors, gentamicin, and amphotericin B before further treatments. Cultures were then stimulated with the cytokine cocktail consisting of 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma}, or 10 ng/ml of individual cytokines at the indicated time points. Selected cultures were pretreated for 15 min with the 0.1% DMSO vehicle, 25 µM SB-203580, 50 µM PD-98059, 1 or 10 µM MG-132 before and during cytokine stimulation.

RNA isolation and relative RT-PCR. Total RNA was extracted with TRIzol reagent at 1 ml/10 cm2 according to the manufacturer's instructions. After TRIzol extraction, RNA samples were dissolved in nuclease-free water and treated with DNase I (5 units) for 10 min at 37°C. The reaction was stopped by the addition of 25 mM EDTA and incubation at 65°C for 15 min. We quantitated RNA concentrations by measuring absorbance at 260 nm. First-strand cDNA synthesis was performed at 42°C from 2 µg of total RNA using 250 ng random hexamers, 50 mM Tris·HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.125 mM dATP, dTTP, dGTP, and cCTP, and 1 unit SuperScript II RT. After cDNA synthesis, 20 units of RNAse H were then added to remove RNA complementary to the cDNA.

Semiquantitative relative PCR was performed using QuantumRNA 18S internal standards according to the manufacturer's protocol (Ambion). COX-2-specific oligonucleotides were synthesized by Bio-Synthesis (Lewisville, TX) from the following sequence obtained from the human COX-2 (M90100 [GenBank] ): 5'-CAATCTGGCTGAGGGAACACAACA-'3 and 5'-ATCTGCCTGCTCTGGTCAATGGA-'3. The reaction mixture contained 60 mM Tris·HCl (pH 8.5); 15 mM (NH4)SO4; 1.5 mM MgCl2; 0.25 mM dATP, dCTP, dGTP, and dTTP; 10% DMSO; 50 µM of each primer; 5 µl template cDNA; and 2.5 units Taq polymerase. Amplification within the linear range took place at 94°C for 30 s and 68°C for 2 min followed by a final 72°C extension for 5 min. An optimized ratio of 18S rRNA primers was added to the reaction as an endogenous standard along with 18S competimers allowing modulation of 18S amplification without affecting the performance of the gene-specific PCR targets in the reaction. PCR products were quantified by ethidium bromide staining and analyzed by densitometry. Results were normalized to the amount of 18S rRNA present in each sample.

Cytosolic and nuclear protein extraction. Cytosolic and nuclear protein extractions were performed according to standard protocols (1). Briefly, treated cells were rinsed twice and then scraped in 100 µl of ice-cold PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4) containing 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, and 1 µg/ml pepstatin A. The cells were pelleted by centrifugation at 3,000 rpm for 5 min at 4°C. The pellet was washed twice with 1 ml of buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF] and centrifuged as just described. The supernatant was discarded, and the cellular proteins remaining in the pellet were released by hypotonic lysis in 100 µl of buffer A containing 0.1% Nonidet P-40 and centrifuging at 14,000 rpm for 10 min at 4°C. The supernatant containing the cytosolic fraction was transferred to a new tube, while the nuclear proteins remaining in the pellet were obtained by extraction in a high-salt buffer B [20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM Na2 EDTA, pH 8.0, 25% glycerol, 0.5 mM DTT, and 0.5 mM PMSF] and centrifugation at 14,000 rpm for 10 min at 4°C. The supernatant containing nuclear proteins was then diluted with an equal volume of buffer C [20 mM HEPES (pH 7.9), 50 mM KCl, 25% glycerol, 0.5 mM DTT, and 0.5 mM PMSF]. Protein concentrations for all extracts were determined by the bicinchoninic acid (BCA) method using bovine serum albumin as the standard.

Northern and slot blot analysis. Total RNA was isolated with TRIzol reagent. Northern analysis was done with the formaldehyde-based NorthernMax system (Ambion). Five micrograms of RNA were denatured, size fractionated by gel electrophoresis, immobilized to Nytran membranes, and UV cross-linked. Prehybridization took place for 30 min at 42°C in Ultrahyb hybridization buffer. Radiolabeled probes for COX-2 were added to the membranes at 1 x 106 cpm/ml and hybridized overnight at 42°C. Membranes were then washed in 2x SSC (300 mM NaCl, 30 mM sodium citrate) at room temperature and in 0.1x SSC (15 mM NaCl, 1.5 mM sodium citrate)/0.1% SDS at 60°C, exposed to a phosphorimager screen, and analyzed by densitometry. After hybridization for COX-2, the membranes were stripped by boiling in 0.05x SSC, 0.01 M EDTA, and 0.1% SDS and hybridized for 18S rRNA using the same conditions described above. Densitometric data for COX-2 expression were then normalized to 18S rRNA in the samples.

For slot blot analysis of transcript decay, cultures were treated with 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma} for 20 h. The cytokine stimulus was then removed, and fresh media were added containing 0.1% DMSO, 25 µM SB-203580, or 10 µM MG-132. Total RNA was isolated as described at the times indicated after removal of cytokine stimulus. RNA was then denatured in a solution of 5x SSC containing 6.8% formalin at 65°C. Serial dilutions were prepared from 2.5 µg of the RNA samples, and 25 µg/ml of tRNA were added to those with <1 µg of RNA. RNA was blotted onto duplicate Nytran membranes using the Minifold II Slot-Blot System (Schleicher and Schuell, Keene, NH) at 0.25, 0.125, 0.0625, and 0.03125 µg per well to determine hybridization signal within the linear range. Membranes were hybridized, washed, and analyzed as described above for COX-2 or 18S rRNA.

Random priming. Probes for Northern and slot blot analysis were generated by PCR amplifying COX-2 or 18S rRNA with the primers above. PCR products were separated by agarose gel electrophoresis and purified using spin-column filtration chromatography (Qiagen, Valencia, CA). The Prime-A-Gene labeling system (Promega) was used to generate labeled probes by random priming. We diluted DNA in water and denatured it at 100°C before assembling the reaction. The reaction proceeded for 60 min at room temperature and contained 50 mM Tris·HCl (pH 8.0); 5 mM MgCl2; 2 mM DTT; 200 mM HEPES (pH 6.6); random hexadeoxyribonucleotides; 20 µM dATP, dTTP, and dGTP; 25 ng denatured DNA template; 50 µCi [{alpha}-32P]dCTP; and 5 units Klenow. The reaction was terminated by heating at 100°C and adding 20 mM EDTA. Unincorporated label was removed by size exclusion chromatography with Sephadex G-50 spin columns (Amersham Pharmacia Biotech, Piscataway, NJ).

Western analysis. For immunoblotting experiments, cultures were washed twice in phosphate-buffered saline following cytokine stimulation. Whole cell lysates for analysis of COX-1 and COX-2 were prepared by lysis in an extraction buffer containing 20 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1 mM Na3VO4, 1 mM NaF, 10 mM sodium {beta}-glycerophosphate, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 10 µg/ml trypsin inhibitor, and 10 µg/ml aprotinin. For p38 MAPK, whole cell extracts were prepared in an extraction buffer containing 60 mM Tris·HCl (pH 6.8), 2% SDS, 10% glycerol, 0.1 mM EGTA, 1 mM EDTA, 5 mM NaF, 1 µM leupeptin, and 1 mM AEBSF. In both cases, lysates were clarified by centrifugation at 10,000 g for 10 min at 4°C, and protein concentrations were determined by the BCA method. Cytosolic extracts for I{kappa}B{alpha} analysis were prepared as described above. Fifteen micrograms of protein were separated by 10-12% SDS-PAGE, and proteins were transferred to nitrocellulose paper in 25 mM Tris, 192 mM glycine, and 10% methanol at 24 V for 2 h under continuous cooling. Membranes were blocked for at least 1 h with 0.5% gelatin in TNT [100 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% (wt/vol) Tween 20] or 5% milk. Blots were probed with COX-1- or COX-2-specific antibodies at a dilution of 1:500 in 0.1% gelatin in TNT. For p38 MAPK, membranes were probed with anti-p38 MAPK (1:1,000) or a dual anti-phosphotyrosinethreonine-p38 MAP kinase antibody (1:5,000). I{kappa}B{alpha} was visualized with anti-I{kappa}B{alpha} monoclonal antibody at a 1:1,000 dilution. All secondary antibodies were conjugated to alkaline phosphatase and used at a dilution of 1:10,000. Images of immunoblots were scanned with a UMAX Powerlook flatbed scanner and analyzed by densitometry.

PGE2 assay. An enzyme immunoassay was used to measure the secretion of PGE2 in media from cytokine-stimulated human BSMC. The assay was performed according the manufacturer's instructions. The minimum detectable levels of PGE2 with this assay were ~95 pg/ml.

Site-directed mutagenesis of the COX-2 promoter. The human COX-2 promoter containing -1840 bases upstream and +123 downstream of the transcriptional start site was kindly provided by Dr. Stephen Prescott (15). Restriction digests with SacI and HindIII generated a 1.1-kb fragment (-1,024 to +118) that was ligated into pGEM11. This portion of the promoter contains the two consensus sites for NF-{kappa}B binding located at -455 to -428 (5'-NF-{kappa}B: 5'-GGCGGGAGAGGGGATTCCC-TGCGCCCCC-'3) and -232 to -205 (3'-NF-{kappa}B: 5'-CAGGAGAGTGGGGACTACCCCCTCT-GCT-3') with the binding element underlined. To generate promoter reporter constructs containing mutations in NF-{kappa}B binding, we synthesized the following oligonucleotides from previously published work (33) with the mutated bases in boldface and italics: 5'-NF-{kappa}B-MU (5'-CGGCGGCGGGA-GAGCTCATTCCCTGCGCCC-3'), which introduces a SacI site (GAGCTC), and 3'-NF-{kappa}B-MU (5'-AGACAGGAGAGTGGCCAC-TACCCCCTCTGC-3'), which introduces an EaeI site (TGGCCA). In vitro site-directed mutagenesis took place using the Gene Editor system (Promega) according to the manufacturer's instructions by annealing the mutagenic oligonucleotides to alkaline-denatured template DNA followed by synthesis of the mutant strand by T4 DNA polymerase and T4 DNA ligase. Positive clones were screened by restriction digests with SacI and EaeI following transformation into BMH 71-18 mutS and JM109. Introduction of the mutations was verified by sequencing at the Nevada Genomics Center. The wild-type and mutated promoters were cut out of pGEM11 with SfiI and HindIII for blunt-end ligation into the luciferase construct pGL2 at SmaI and HindIII.

Transient transfections and promoter-reporter assays. Human BSMC were transiently transfected with luciferase constructs using a modified CaPO4 protocol found in the modified bovine serum (MBS) Mammalian Transfection kit from Stratagene. Twenty-four hours before transfection, cells were dispersed in 12-well plates at a density of 40,000 cells/well. The following day, the normal growth medium was removed and replaced with M199 containing 6% MBS. The DNA precipitate was prepared by adding 5 µg of the pGL2 firefly luciferase construct DNA and 100 ng of pRL Renilla luciferase DNA to 2x N,N-bis (2-hydroxylethyl)-2-aminoethanesulfonic acid-buffered saline, pH 6.95, and 0.125 M CaCl2. After careful addition of the DNA suspension, cultures were maintained for 4 h in a humidified 3% CO2 atmosphere at 37°C, washed thoroughly in PBS, and maintained in M199 containing 0.1% FBS, growth factors, gentamicin, and amphotericin B for 36 h before the described cytokine treatments. The Dual-Luciferase Reporter Assay system (Promega) was used to evaluate both firefly and Renilla luciferase activity simultaneously from the same sample. Background luminescence was subtracted from nontransfected control cultures, and transfection efficiency was determined by normalizing firefly luciferase activity to Renilla activity from the same sample in a Zylux Sirius model luminometer (Zylux, Oak Ridge, TN).

Electrophoretic mobility shift assays. Gel shift assays were performed according to the manufacturer's instructions for the Gel Shift Assay Core System using double stranded NF-{kappa}B consensus oligonucleotides (Promega). Briefly, 3.5 pmol of NF-{kappa}B consensus oligonucleotides were end labeled with 1 µCi [{gamma}-32]ATP using 5 units of T4 polynucleotide kinase in 70 mM Tris·HCl (pH 7.6), 10 mM MgCl2,and 5 mM DTT at 37°C for 10 min. The reaction was stopped by the addition of 50 mM EDTA and diluted with 10 mM Tris·HCl (pH 8.0) and 1 mM EDTA. Unincorporated label was removed by size exclusion chromatography with Sephadex G-50 spin columns. DNA binding reactions (15 µl) were performed using 1 µl of labeled NF-{kappa}B consensus oligo and 4 µg of nuclear extract in binding buffer containing 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris·HCl (pH 7.5), and 0.05 mg/ml poly(dI-dC). HeLa nuclear extracts supplied with the kit were used as positive controls. The reactions were incubated at room temperature for 20 min and stopped with an appropriate volume of 10x gel loading buffer containing 250 mM Tris·HCl (pH 7.5), 0.2% bromphenol blue, and 40% glycerol. Samples were loaded onto 1.0-mm, 10 x 12-cm nondenaturing 4% acrylamide gels and electrophoresed at 100 V. Gels were dried overnight at room temperature between cellophane sheets and exposed to a phosphorimager screen.

Densitometry and statistical analysis. Densitometry was performed using a Bio-Rad model 525 Molecular Imager and the Volume Analyze feature of Molecular Analyst software (Bio-Rad, Hercules, CA). Results are expressed as percent control from cultures treated with cytokines for 20 h (100%). Statistical analysis was performed by one-way ANOVA followed by post hoc testing with the Student-Newman-Keuls method or a paired t-test using SigmaStat software (Jandel Scientific, San Rafael, CA).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cytokine-stimulated COX expression. Human BSMC were stimulated with a cytokine cocktail containing 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma} over a period of 20 h to examine the effect of a proinflammatory stimulus on COX expression. Northern analysis identified a prolonged increase in the expression of a 4.6-kb COX-2 transcript within 4-8 h of cytokine stimulation that peaks within 12-20 h of exposure. A graphical summary of these results is depicted in Fig. 1A following densitometric analysis of COX-2 expression and normalization to the amount of 18S rRNA present in the samples. This proinflammatory stimulus did not appear to have any effect on the expression of COX-1 and was correlated with an increase in COX-2 protein expression (Fig. 1B). In these experiments, isoform-specific antibodies were used to identify COX-1 at ~70-72 kDa and COX-2 at 74 kDa, although immunoreactivity for COX-1 always appeared as a doublet. The appearance of a doublet for COX-1 has been previously observed by others using similar antibodies (11). These results establish that the proinflammatory stimulus consisting of IL-1{beta}, TNF-{alpha}, and IFN-{gamma} significantly increases the expression of COX-2, but not COX-1, in human BSMC.



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Fig. 1. Effect of cytokine stimulation on cyclooxygenase expression. Human bronchial smooth muscle cells (BSMC) were treated at the indicated times with 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma}. A: 5 µg of total RNA were prepared and fractionated by gel electrophoresis under denaturing conditions. Northern analysis depicting cyclooxygenase (COX)-2 transcript at 4.6 kb (top) or 18S rRNA (bottom) from the same blot is shown from a representative experiment. A graphical summary of COX-2 expression normalized to 18S rRNA is also shown (n = 3) ± SE. B: 15 µg of total protein were prepared from whole cell lysates and resolved by SDS-PAGE. Western analysis depicting immunoreactivity for COX-1 (top) and COX-2 (bottom) is shown from a representative experiment.

 

Effect of individual cytokines on COX-2 expression and activity. To determine the individual contributions of IL-1{beta}, TNF-{alpha}, and IFN-{gamma} to COX-2 expression, human BSMC cultures were treated for 20 h with 10 ng/ml of IL-1{beta}, TNF-{alpha}, or IFN-{gamma}. Western analysis of COX-2 expression from whole cell lysates (Fig. 2A) demonstrates that IL-1{beta} produced 58% and TNF-{alpha} produced 20% of the COX-2 expression seen with the cytokine cocktail, whereas IFN-{gamma} did not significantly increase COX-2 expression above basal levels. Secretion of PGE2 was also examined in response to changes in COX-2 expression as a measure of COX-2 activity (Fig. 2B). IL-1{beta} or TNF-{alpha} alone increased PGE2 secretion from ~297 pg/ml at basal levels to 1,485 pg/ml with IL-1{beta} or 722 pg/ml with TNF-{alpha} stimulation, whereas IFN-{gamma} alone (270 pg/ml) was not as effective in stimulating PGE2 synthesis. In comparison, the cytokine cocktail containing IL-1{beta}, TNF-{alpha}, and IFN-{gamma} synergistically stimulated PGE2 secretion compared with that seen with the individual components with concentrations rising to 10,367 pg/ml. This indicates that the addition of IFN-{gamma} to the cytokine stimulus significantly contributes to COX-2 expression and, more dramatically, PGE2 synthesis in these cultures when combined with IL-1{beta} and TNF-{alpha}, even though stimulation with IFN-{gamma} alone has little or no effect.



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Fig. 2. Cytokine stimulation of COX-2 activity and expression. Human BSMC were treated for 20 h with 10 ng/ml of the indicated cytokines. A: Western blots depicting COX-2 immunoreactivity are shown with densitometric analysis of the results (n = 4) ± SE. Data are expressed as the % change from control cultures treated with IL-1{beta}, TNF-{alpha}, and IFN-{gamma} (100%). B: PGE2 enzyme immunoassay from the media of treated cultures (n = 3) ± SE. *Significant difference from cultures left untreated or treated with individual cytokines, P < 0.05. {dagger}Significant difference from cultures treated with IL-1{beta} or TNF-{alpha}.

 

Effect of MAPK inhibition on COX-2 expression. The regulation of cytokine-stimulated COX-2 expression by MAPK signaling pathways was examined in the presence of chemical inhibitors of p38 MAPK and ERK MAPK activation. Cultures were treated for 20 h with the cytokine cocktail described above following a 15-min pretreatment with 0.1% DMSO vehicle, 25 µM SB-203580, or 25 µM PD-98059 (Fig. 3). Although there are multiple chemical inhibitors of p38 MAPK and ERK MAPK available, SB-203580 and PD-98059 have been extensively used in our laboratory and by others to study various aspects of MAPK regulation in smooth muscle. Additionally, previous work has confirmed that the concentrations used in these experiments inhibit p38 MAPK and ERK MAPK activity in human BSMC following stimulation with the cytokine cocktail (13), and the effect of the individual components in the cytokine cocktail on p38 MAPK phosphorylation is shown in Fig. 4A. Semiquantitative relative RT-PCR (Fig. 3A) and Western analysis (Fig. 3B) were used to compare the expression of COX-2 between the treatment groups. In RT-PCR experiments, treatment with SB-203580 decreased cytokine-stimulated COX-2 expression by 63% from control cultures treated with DMSO, whereas in immunoblotting experiments COX-2 expression was decreased by 38% from control cultures. PD-98059 did not have a significant or a consistent effect on COX-2 expression in either case, although the use of PD-98059 in previous experiments demonstrated a consistent downregulation of IL-1{beta}, IL-6, and IL-8 expression (13). It is interesting to note that SB-20380 did not return COX-2 expression levels back to that seen in unstimulated cultures. Furthermore, SB-203580 treatment in combination with PD-98059 did not have any additional effect on decreasing COX-2 expression (data not shown). Individually, it appears that IL-1{beta} and TNF-{alpha} have effects similar to the cytokine cocktail on p38 MAPK phosphorylation when used at 10 ng/ml, whereas IFN-{gamma} does not stimulate p38 MAPK phosphorylation.



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Fig. 3. SB-203580 inhibits cytokine-stimulated COX-2 expression. Human BSMC were treated for 20 h with 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma} in the presence of the 0.1% DMSO vehicle, 25 µM SB-203580, or 25 µM PD-98059. Data are expressed as the % change from cytokine treated control cultures (100%). A: relative multiplex RT-PCR amplifies products of the predicted sizes for COX-2 (466 bp) and 18S rRNA (324 bp). A graphical summary of COX-2 expression normalized to 18S rRNA is shown (n = 4 ± SE). B: a representative COX-2 Western blot is shown with densitometric analysis of the results (n = 6-8) ± SE. *Significant difference from cytokine treated control cultures, P < 0.05.

 


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Fig. 4. Cytokine stimulation of p38 MAPK phosphorylation Human BSMC were treated for 15 min with 10 ng/ml of the indicated cytokines. A: Western blots of phosphorylated and nonphosphorylated p38 MAPK are shown from a representative experiment along with densitometric analysis of phosphorylated p38 MAPK. Results are expressed as the fold change from unstimulated cultures (n = 6-11) ± SE. B: I{kappa}B{alpha} immunoreactivity was visualized by Western analysis in 15 µg of cytosolic protein. Densitometric data are expressed relative to the I{kappa}B{alpha} immunoreactivity seen in unstimulated cultures (100%) (n = 2-6) ± SE.

 

Cytokine-stimulated activation of NF-{kappa}B. Although it is clear that p38 MAPK regulates proinflammatory gene expression in airway myocytes, we sought to determine whether other mediators of transcription, namely NF-{kappa}B, are activated in response to the complex cytokine stimulus used in these studies. In the cytosol, NF-{kappa}B exists as an inactive hetero- or homodimer complexed with regulatory I{kappa}B, of which there are several isoforms including I{kappa}B{alpha}, I{kappa}B{beta}, and I{kappa}B{epsilon}. Phosphorylation of I{kappa}B proteins by IKK causes ubiquitination of I{kappa}B and targets it for degradation by the 26S proteosome. This releases NF-{kappa}B dimers from the complex and allows for nuclear translocation of NF-{kappa}B (reviewed in 21, 35). In Fig. 5A, the disappearance of I{kappa}B{alpha} from cytosolic extracts of human BSMC is shown following stimulation with the cytokine cocktail. In these experiments, I{kappa}B{alpha} immunoreactivity was abundant in unstimulated cultures, but following 15-30 min of exposure, I{kappa}B{alpha} decreased 82% and returned to within 20-30% of basal levels within 1-2 h. An examination of the effect of the individual cytokines demonstrated that the only components of the cytokine cocktail that appear to contribute to I{kappa}B{alpha} degradation are IL-1{beta} and TNF-{alpha} (Fig. 4B). The addition of 10 µM MG-132, a proteosome inhibitor that would be expected to inhibit I{kappa}B{alpha} degradation, blocked the disappearance of I{kappa}B{alpha} from cytosolic extracts (Fig. 5B) with 91% of I{kappa}B{alpha} immunoreactivity remaining after treatment. These results indicate that I{kappa}B{alpha} is targeted for degradation following this cytokine stimulus, presumably releasing NF-{kappa}B dimers for nuclear translocation.



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Fig. 5. Cytokine-stimulated I{kappa}B{alpha} immunoreactivity. I{kappa}B{alpha} immunoreactivity was visualized in 15 µg of cytosolic protein. A: human BSMC were treated at the indicated times with 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma}. Densitometric data are expressed relative I{kappa}B{alpha} immunoreactivity seen in unstimulated cultures at time = 0, (n = 2-4) ± SE. B: human BSMC were treated with 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma} for 15 min in the presence of the 0.1% DMSO vehicle, 10 µM MG-132, or 25 µM SB-203580. Densitometric data are expressed relative the I{kappa}B{alpha} immunoreactivity seen in DMSO-treated control cultures (n = 3-4) ± SE.

 

To determine whether degradation of I{kappa}B{alpha} results in an increase in NF-{kappa}B-dependent DNA binding, we performed gel shift assays with a representative gel shown in Fig. 6. In the absence of stimulus, very little binding of nuclear proteins was seen in the presence of an oligonucleotide encoding the DNA consensus sequence for NF-{kappa}B. After 15 min of cytokine stimulation, an increase in binding is observed. This binding is ablated in the presence of excess unlabeled NF-{kappa}B oligonucleotide, indicating that the increase in binding seen is specific for NF-{kappa}B. Moreover, in the presence of an unlabeled, nonspecific competitor encoding the consensus sequence for SP-2, there was no change in NF-{kappa}B-dependent binding (data not shown). In the presence of 10 µM MG-132, NF-{kappa}B-specific binding was diminished, although not completely abolished. The effect of MG-132 on NF-{kappa}B binding was variable, and treatment with MG-132 always caused a shift in the pattern of binding. These results correlate with other reports in vascular smooth muscle cells demonstrating that MG-132 decreases but does not completely block NF-{kappa}B binding (41) and demonstrates that this cytokine stimulus causes an increase in NF-{kappa}B-dependent DNA binding in human BSMC.



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Fig. 6. NF-{kappa}B-dependent nuclear binding in cytokine stimulated human BSMC. Human BSMC were treated with 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma} for 15 min in the presence of 0.1% DMSO, 10 µM MG-132, or 25 µM SB-203580. Electrophoretic mobility shift assays were performed using 4 µg of nuclear extracts in the presence of 32P-labeled NF-{kappa}B consensus oligonucleotides. Excess unlabeled NF-{kappa}B oligonucleotide was added to the reaction in lane 3 as a specific binding competitor. A representative experiment is shown from n = 5.

 

p38 MAPK does not participate in NF-{kappa}B activation. In airway smooth muscle cells stimulated with IL-1{beta}, Laporte et al. (16) have reported that 30 µM SB-203580 reduces NF-{kappa}B-dependent nuclear binding by ~30%, whereas a lower concentration of SB-203580 (3 µM) has little effect. Other reports in vascular smooth muscle have indicated that 10 µM SB-203580 has no effect on LPS-stimulated NF-{kappa}B activity (41), whereas still others have reported that 30 µM SB-203580 has no effect on IL-1{beta}-stimulated NF-{kappa}B activity (42). Because we find IL-1{beta} combined with TNF-{alpha} and IFN-{gamma} synergistically increases PGE2 synthesis and cytokine synthesis in BSMC, it is plausible that the cytokine mixture may recruit different signaling mechanisms than that seen with IL-1{beta} alone. To determine whether p38 MAPK activity is necessary for NF-{kappa}B signaling in human BSMC stimulated with the cytokine cocktail, several aspects of NF-{kappa}B activity were examined in the presence of 25 µM SB-203580. This inhibitor had little effect on cytokine-stimulated degradation of I{kappa}B{alpha} (Fig. 5B) or nuclear binding activity of NF-{kappa}B (Fig. 6). These results indicate p38 MAPK does not affect activation of NF-{kappa}B in human BSMC treated with IL-1{beta}, TNF-{alpha}, and IFN-{gamma}, even though IL-1{beta} and TNF-{alpha} alone stimulate p38 MAPK phosphorylation and I{kappa}B{alpha} degradation.

Role of NF-{kappa}B in cytokine-stimulated COX-2 expression. It is clear that the complex inflammatory stimulus of IL-1{beta}, TNF-{alpha}, and IFN-{gamma} activates NF-{kappa}B in human BSMC, and subsequent experiments were performed to determine whether NF-{kappa}B mediates COX-2 expression with this stimulus. In these experiments, cultures were again treated for 20 h with the cytokine cocktail described above in the presence of 0.1% DMSO vehicle or 1 µM MG-132. This concentration of MG-132 reduced COX-2 expression by 57%, as measured by semiquantitative RT-PCR in Fig. 7A, and by 71%, as measured by Western blots in Fig. 7B, from control cultures receiving the cytokine stimulus in the presence of DMSO vehicle. Greater decreases in expression were seen in cultures treated with 10 µM MG-132 (data not shown). However, higher concentrations of MG-132 were toxic when cultures were treated for 20 h, even though concentrations of MG-132 up to 25 µM have been shown to be effective at inhibiting NF-{kappa}B transcription from a model promoter-reporter construct (16).



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Fig. 7. MG-132 inhibits cytokine-stimulated COX-2 expression. Human BSMC were treated for 20 h with 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma} in the presence of the 0.1% DMSO vehicle or 1 µM MG-132. Data are expressed as the % change from cytokine treated control cultures (100%). A: relative multiplex RT-PCR shows the PCR products for COX-2 and 18S rRNA. A graphical summary of COX-2 expression normalized to 18S rRNA (n = 4) ± SE. B: a representative COX-2 Western blot is shown with densitometric analysis of the results (n = 3) ± SE. *Significant difference from cytokine-treated control cultures, P < 0.05.

 

A further analysis of the regulation of COX-2 expression by NF-{kappa}B signaling in response to IL-1{beta}, TNF-{alpha}, and IFN-{gamma} was completed by examining NF-{kappa}B-dependent activation of the human COX-2 promoter -1,024 bases upstream and +123 downstream of the transcriptional start site, as diagrammed in Fig. 8A. Human BSMC were transiently transfected with pGL2 luciferase constructs containing the wild-type COX-2 promoter (COX2WT) or a promoter in which the two NF-{kappa}B-binding elements at -455 to -428 (5'-NF-{kappa}B) and 232 to -205 (3'-NF-{kappa}B) have been mutated (COX2MU) as described above and in previous publications (33). Transfection of COX2WT increased basal luciferase activity in DMSO-treated control cultures compared with cultures transfected with pGL2 alone and also resulted in a significant increase in luciferase activity upon stimulation with the cytokine cocktail for 1 h (Fig. 8B). In contrast, transfection with the COX2MU construct resulted in a 55% decrease in basal luciferase activity and a 70% decrease in cytokine-stimulated luciferase activity. This indicates that this region of the promoter contributes to NF-{kappa}B-dependent stimulation of COX-2 transcription. The contribution of p38 MAPK signaling pathways to activation of this COX-2 promoter-reporter construct was examined by the addition of 25 µM SB-203580 to cytokine-stimulated cultures. SB-203580 decreased luciferase activity by 30% in COX2WT-transfected cells and did not further stimulate promoter-reporter activity in COX2MU-transfected cultures. These results support the idea that NF-{kappa}B, along with p38 MAPK and probably other transcription factors, is a component regulating cytokine-stimulated expression of COX-2 in airway myocytes, although the role of p38 MAPK in the region of the COX-2 promoter examined in these studies appears to be minimal. This suggests that p38 MAPK may be involved in other processes regulating COX-2 expression.



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Fig. 8. NF-{kappa}B-dependent stimulation of COX-2 promoter reporter activity. A: schematic diagram of the human COX-2 promoter used for reporter assays with the NF-{kappa}B sites noted. B: human BSMC were transiently transfected with 5 µg of empty pGL2, pGL2-COX2WT containing the COX-2 promoter from -1024 to +118 with intact NF-{kappa}B sites, or pGL2-COX2MU containing mutations in both the 5'- and 3'-NF-{kappa}B sites. Cultures were pretreated for 15 min with the 0.1% DMSO vehicle or 25 µM SB-203580 and then exposed to 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma} for 1 h. Firefly luciferase activity from cultures transfected with the pGL2 constructs was normalized to Renilla luciferase activity from cotransfected pRL (n = 10-12) ± SE. *Significant difference from untreated pGL2-COX2WT cultures, P < 0.05. {dagger}Significant difference from cytokine-stimulated pGL2-COX2MU cultures, P < 0.05.

 

Effect of p38 MAPK inhibition of COX-2 mRNA stability. In addition to the transcriptional elements found in the 5'-untranslated region (UTR) of the COX-2 gene, several elements found in the 3'-UTR are of regulatory importance. The 4.6-kb transcript of COX-2 contains 22 copies of adenosine and uridine (AU) repeats common in unstable RNA transcripts (2). In human monocytes stimulated with LPS and in HeLa cells stimulated with IL-1, it appears that the stability of COX-2 mRNA is mediated by p38 MAPK (8, 32). This led us to examine the participation of p38 MAPK in COX-2 mRNA stability during a cytokine stimulus in airway myocytes (Fig. 9A). In these experiments, human BSMC were treated for 20 h with 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma} to induce the expression of COX-2. The media were then removed and replaced with media containing 0.1% DMSO vehicle or 25 µM SB-203580. The decay of COX-2 mRNA was followed by isolating RNA from the cultures over a period of 2 h and examining COX-2 expression by slot blot analysis. Duplicate membranes were also hybridized for 18S rRNA to control for RNA loading, and the probes used to examine COX-2 and 18S rRNA are the same used in the Northern blots described above. At the beginning of the experiment, the amount of COX-2 expression seen with 20 h of cytokine stimulus is deemed 100%. After removal of the cytokine stimulus, COX-2 expression increased slightly in the cultures that received the DMSO control and then remained relatively stable for the duration of the experiment. This increase in COX-2 expression may be due to a transient activation of p38 MAPK following removal of cytokine stimulus (Fig. 10). However, in the cultures that received SB-203580, COX-2 expression rapidly decreased 48% within the first 30 min of the experiment and remained 30% below that seen in the control cells. Similar results were seen in the presence of 5 µg/ml actinomycin D (data not shown) and correlate with previous studies in macrophage cell lines and monocytes, suggesting that degradation of TNF-{alpha} mRNA (4) and COX-2 mRNA (8) is sensitive to SB-203580 in the presence and absence of actinomycin D. Interestingly, COX-2 expression remained relatively stable in the control cells and did not appear to decay rapidly, even 4 h after removal of the cytokine stimulus (data not shown). The results demonstrate that the initial rate of COX-2 mRNA decay is regulated by activation of p38 MAPK. The results also suggest additional undefined pathways contribute to long-term stability of the transcript after stimulation with the cytokine mixture. We repeated the same experiments in the presence of 10 µM MG-132 to determine whether NF-{kappa}B signaling is necessary for COX-2 mRNA stability. As shown in Fig. 9B, MG-132 did not affect the stability of COX-2 mRNA, since COX-2 expression levels were similar to that seen in the presence of the DMSO vehicle. Therefore, it appears even though p38 MAPK and NF-{kappa}B participate in regulating the steady-state levels of COX-2 mRNA, p38 MAPK additionally affects the stability of COX-2 transcripts in cytokine-stimulated BSMC.



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Fig. 9. Cytokine-stimulated COX-2 mRNA stability. After 20 h of treatment with 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma}, media were removed and replaced with fresh media containing the 0.1% DMSO vehicle, 25 µM SB-203580 (A), or 10 µM MG-132 (B). Total RNA was prepared for slot blot analysis of COX-2 transcript and 18S rRNA decay following removal of cytokine stimulus at the times indicated. COX-2 densitometric data are normalized to that seen with 18S within the linear range of hybridization and expressed as the % change from control cultures at time = 0 (n = 3-5) ± SE.

 


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Fig. 10. p38 MAPK phosphorylation following removal of cytokine stimulus. After 20 h of treatment with 10 ng/ml IL-1{beta}, TNF-{alpha}, and IFN-{gamma}, media were removed and replaced with fresh media containing 0.1% DMSO. Whole cell extracts were prepared at the times indicated following removal of cytokine stimulus and phosphorylated p38 MAPK evaluated by Western analysis. Densitometric data are expressed as the fold change in immunoreactivity (n = 5) ± SE.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
COX-2 expression has been described in many different cell types in the lung during airway inflammation, including eosinophils, macrophages, neutrophils, mast cells, epithelial cells, fibroblasts, and vascular and airway smooth muscle (9, 29, 34). The data presented here further describe expression of COX-2 in cultured BSMC exposed to a complex proinflammatory stimulus of IL-1{beta}, TNF-{alpha}, and IFN-{gamma}, which has been previously shown to induce COX-2 expression in cells isolated from human tracheal smooth muscle (3). Although several reports in the literature have examined COX-2 expression in response to IL-1{beta} or a combination of IL-1{beta} and TNF-{alpha} in airway smooth muscle (22, 26, 42), our data suggest that the addition of IFN-{gamma} to the cytokine stimulus synergistically stimulates PGE2 synthesis from endogenous arachidonic acid and also contributes to increases in COX-2 expression. Therefore, the use of a stimulus that includes IFN-{gamma} may yield important information about the regulation of inflammatory mediators. Previous work in our laboratory demonstrated that IFN-{gamma} potentiates IL-6 release from airway smooth muscle cells when used in combination with IL-1{beta} and TNF-{alpha} but has only an additive effect on the synthesis of IL-1{beta} and no effect on the release of IL-8 (13). This is in contrast to other reports in the literature where the addition of IFN-{gamma} affected PGE2 synthesis in the presence of exogenous but not endogenous arachidonic acid (3), whereas others have demonstrated that IL-1{beta}, but not TNF-{alpha} or IFN-{gamma}, contributes to PGE2 synthesis in airway myocytes (27).

The data we present here demonstrate that inhibition of p38 MAPK with SB-203580 decreases the steady-state levels of cytokine-stimulated COX-2 expression. This work concurs with other reports examining p38 MAPK-mediated regulation of COX-2 expression in airway smooth muscle cells stimulated with IL-1{beta} and TNF-{alpha} (16, 22, 42). The experiments presented here more clearly delineate that p38 MAPK, and not ERK MAPK, contributes to cytokine-stimulated COX-2 expression in response to a more complex inflammatory stimulus. This may be particularly important when examining expression in the presence of multiple inflammatory mediators, which have the potential to activate multiple signaling pathways. We also provide data supporting a role for NF-{kappa}B regulation of COX-2 expression in airway smooth muscle cells that has not been previously reported using this cytokine stimulus. In human lung alveolar epithelial cells and epithelial cell lines, COX-2 expression was found to be mediated by NF-{kappa}B-dependent signaling pathways (5, 18). However, in other studies of airway smooth muscle, lung epithelial cells, and vascular smooth muscle, a role for NF-{kappa}B in COX-2 expression was not found (6, 16, 24). From the experiments presented here, it is clear that IL-1{beta} and TNF-{alpha} stimulate p38 MAPK and NF-{kappa}B signaling pathways and that the combination of IL-1{beta}, TNF-{alpha}, and IFN-{gamma} regulates steady-state levels of COX-2 expression in a p38 MAPK- and NF-{kappa}B-dependent manner. However, the p38 MAPK inhibitor SB-203580 does not affect NF-{kappa}B activity or NF-{kappa}B-dependent activation when NF-{kappa}B binding sites in the COX-2 promoter are mutated, but other binding sites are left intact. These sites may include Ets family members phosphorylated by the p38 MAPK substrate MAPK-activating protein kinase-2 (MK2), and in fact, the addition of SB-203580 does appear to inhibit wild-type COX-2 promoter activity, possibly through the aforementioned Ets family members or other transcription factors. This indicates that p38 MAPK and NF-{kappa}B signaling pathways may regulate COX-2 expression through parallel pathways with no apparent cross talk, as seen in the regulation of TNF-{alpha} expression in vascular smooth muscle cells (41).

In human monocytes, LPS upregulates COX-2 mRNA in a manner that can be inhibited by the presence of SB-203580 (8). In these cells, p38 MAPK induces COX-2 expression by regulating transcription of the COX-2 gene and by stabilizing COX-2 mRNA transcripts. In the presence of actinomycin D, COX-2 mRNA decays slowly, but the addition of SB-203580 causes rapid disappearance of COX-2 transcript. We describe similar results for the first time in airway smooth muscle, where the addition of SB-203580 caused a rapid decay of cytokine-stimulated COX-2 mRNA. The data presented here demonstrate that p38 MAPK affects the initial rate of COX-2 mRNA decay but does not appear to have any additional effects on mRNA stability over the time period examined. These results could be explained by the 20-h treatment used to upregulate COX-2 expression. It is likely that the levels of COX-2 expression we are seeing are in fact due to induction of COX-2 transcription followed by stabilization of the mRNA. These two events acting together could substantially prolong the half-life of the transcript. It has been proposed that p38 MAPK mediates mRNA stability through activation of MK2. Expression of dominant-negative forms of MK2 in HeLa cells blocks stabilization of a tetracycline-inducible chimeric transcript containing the 3'-UTR of the COX-2 gene (17). AU-rich sequences in transcripts targeted for regulation by mRNA stability have been identified as important cis-acting elements. These sequences can bind to AU-rich element (ARE) binding proteins regulating mRNA stability. The presence of ARE is necessary for MK-2-mediated mRNA stability of TNF, IL-6, IL-8, and COX-2 (17, 23, 40), and there is now evidence suggesting that MK2 phosphorylates the RNA-binding protein tristetraprolin to mediate its effects on mRNA stability (19).

The importance of COX-2 expression in airway inflammation is becoming clear in vivo in animal models and in patients with asthmatic inflammation. When both mice and guinea pigs are sensitized to ovalbumin in a model of allergic inflammation, COX-2 expression and PGE2 synthesis are upregulated (12, 25). In airway smooth muscle, p38 MAPK mediates the responses of IL-1{beta} involved in regulation of COX-2 expression, PGE2 synthesis, and {beta}-adrenergic and histaminergic smooth muscle responsiveness (16, 30). The data presented here support a role for COX-2 in mediating the inflammatory response of airway smooth muscle and provide evidence demonstrating that the regulation of mRNA stability by p38 MAPK may be an important mechanism affecting COX-2 gene expression in these cells. These data further demonstrate that the use of a complex inflammatory stimulus in evaluating regulation of inflammatory gene expression in human airway myocytes will be helpful in determining the role of proinflammatory cytokines in situ.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
C. A. Singer is a recipient of a National Institutes of Health National Research Service Award Postdoctoral Fellowship (HL-10072). This study was supported by National Heart, Lung, and Blood Institute Grant HL-48183 (to W. T. Gerthoffer).


    ACKNOWLEDGMENTS
 
The authors acknowledge the members of W. T. Gerthoffer's laboratory for providing helpful discussion of the manuscript and express their gratitude to Stephen Prescott at the University of Utah for the human COX-2 promoter.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. A. Singer, Dept. of Pharmacology/318, Univ. of Nevada School of Medicine, Reno, NV 89557-0046 (E-mail: cas{at}med.unr.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.


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 MATERIALS AND METHODS
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 REFERENCES
 

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