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Originally published In Press as doi:10.1074/jbc.M307964200 on September 29, 2003

J. Biol. Chem., Vol. 278, Issue 50, 49954-49964, December 12, 2003
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Cyclooxygenase-2 Induction by Bradykinin in Human Pulmonary Artery Smooth Muscle Cells Is Mediated by the Cyclic AMP Response Element through a Novel Autocrine Loop Involving Endogenous Prostaglandin E2, E-prostanoid 2 (EP2), and EP4 Receptors*

Dawn A. Bradbury{ddagger}, Robert Newton§, Yong M. Zhu{ddagger}, Hala El-Haroun{ddagger}, Lisa Corbett{ddagger}, and Alan J Knox{ddagger}

From the {ddagger}Division of Respiratory Medicine, University of Nottingham, City Hospital, Nottingham NG5 1PB, United Kingdom and §Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College School of Medicine, London SW3 6LY, United Kingdom

Received for publication, July 22, 2003 , and in revised form, September 25, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bradykinin (BK) is an important mediator in several inflammatory and vascular diseases that acts in part via induction of cyclooxygenase-2 (COX-2). The mechanisms involved in BK-mediated COX-2 induction are unclear. Here we characterized the transcriptional mechanisms involved in human pulmonary artery smooth muscle cells. BK stimulated the activity of a transiently transfected 966-bp (–917 to + 49) COX-2 promoter luciferase reporter construct. There was no reduction in BK-induced luciferase activity in cells transfected with COX-2 promoter constructs of 674, 407, 239, and 135 bp or constructs with mutated CCAAT/enhancer-binding protein- or NF-{kappa}B-binding sites. In contrast luciferase activity was reduced in cells transfected with a 407-bp COX-2 promoter fragment containing a mutated cAMP response element (CRE)-binding site, suggesting that the CRE binding site is critical. Electrophoretic mobility shift assays using oligonucleotides specific for the CRE-binding region of the COX-2 promoter and consensus oligonucleotides showed strong specific binding. Furthermore BK increased consensus cAMP-responsive luciferase reporter (p6CRE/luc)-mediated luciferase expression. CRE activation occurred by BK inducing cytosolic phospholipase A2-mediated arachidonic acid release and rapid prostaglandin E2 (PGE2) production, thereby increasing cAMP. Indomethacin inhibited BK-induced PGE2 production, cAMP accumulation, and CRE/luc reporter and COX-2 promoter luciferase activity. Exogenous PGE2 and EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) PGE2 receptor agonists mimicked the effect of BK. Collectively these studies indicate that COX-2 induction by BK in human pulmonary artery smooth muscle cells is mediated by the CRE through a novel autocrine loop involving endogenous PGE2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The kinin peptide bradykinin (BK)1 is an important proinflammatory mediator associated with allergic and degenerative chronic inflammatory conditions such as asthma and rheumatoid arthritis (1, 2). BK together with Lys-BK or kallidin are also the principal kinins involved in the maintenance of cardiovascular homeostasis (3). BK binds to specific cell surface receptors, B1 and B2. The B1 receptor is induced in response to inflammatory challenge, whereas B2 receptor expression is constitutive. Both receptors are members of the seven-transmembrane G protein-coupled receptor family. Ligand binding to G protein-coupled receptors, which are expressed in many cell and tissue types, gives rise to diverse physiological responses including cytokine and growth factor production.

Cyclooxygenase (COX) catalyzes the production of prostaglandin, prostacyclin, and thromboxane from arachidonic acid. Arachidonic acid, which is released from membrane phospholipid by phospholipases, is converted to prostaglandin H2 by COX and then to the specific prostaglandin by terminal synthases. There are different isoforms of COX: COX-1, whose expression in constitutive, and COX-2, which is induced in response to pro-inflammatory peptides and mediators (4). There is also the recently identified COX-3, whose function has been less well characterized (5). We have recently shown that BK induces COX-2 in pulmonary artery smooth muscle cells (PASMC), but the transcriptional mechanisms used by BK to induce COX-2 have not been defined (6). The COX-2 promoter has binding sites for a number of transcription factors including nuclear factor-{kappa}B (NF-{kappa}B), C/EBP, AP-2, and cAMP response element (CRE), which are involved in COX-2 induction in response to several other stimuli. For example NF-{kappa}B is involved in IL-1{beta}- and hypoxia-induced COX-2 expression in lung epithelial cells and vascular endothelial cells (7, 8). In fibroblasts, COX-2 induction by both phorbol 12-myristate 13-acetate and IL-1{beta} utilizes the C/EBP-binding domain (9). There are a number of candidate transcription factors for the effect of BK on COX-2 because BK has been shown to activate transcription factors that have binding motifs on the COX-2 promoter. BK induced IL-1{beta} mRNA in a human lung fibroblast cell line by activation of NF-{kappa}B (10) and also activated NF-{kappa}B in HeLa cells transfected with BK B2 receptors (11). Our own studies of regulation of the IL-8 gene have shown that BK can activate C/EBP (12). A CRE is also present on the human COX-2 promoter, but its role has been less well defined. Signaling via the CRE- and AP-1-binding motifs has been reported in phorbol 12-myristate 13-acetate-stimulated COX-2 induction in murine osteoblastic cells (13).

Here we have determined the mechanisms involved in the transcriptional regulation of the COX-2 promoter by BK in HPASMC. Mutational and deletional analysis of the COX-2 promoter showed that NF-{kappa}B and C/EBP sites were not required for the increase in COX-2 promoter activity produced by BK. In contrast mutations of the CRE site abolished the effect, suggesting that the CRE was essential. Consistent with this, BK increased activity of a CRE luciferase construct and CREB binding to the COX-2 promoter by EMSA. Furthermore studies with exogenous PGE2, EP2 and EP4 PGE2 receptor subtype agonists, and the COX inhibitor indomethacin showed that BK-induced activation of the CRE was due to an autocrine loop involving the early release of PGE2 via cPLA2 and action on EP2 and EP4 prostanoid receptors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Proximal human HPASMC were purchased at passage 3 from Clonetics® (BioWhittaker UK Ltd., Wokingham, UK). The PASMC were cultured to passage 6 in smooth muscle cell growth medium-2 BulletKit® (Clonetics®, BioWhittaker UK Ltd.).

All of the experiments were set up in triplicate or quadruplicate using passage 6 cells cultured in 6- or 24-well plates at 37 °C in a 5% CO2, humidified incubator (Leec, Colwick, Nottingham, UK). The confluent cells were growth-arrested by serum withdrawal for 24 h. The medium was replaced with fresh serum-free medium with or without bradykinin (Sigma). For the inhibition experiments the cells were preincubated for 30 min with either 1 µM indomethacin (Sigma) or 0.1% Me2SO (Sigma) as the vehicle control. The highly selective EP2 and EP4 PGE2 receptor subtype agonists ONO-AE1 259 and ONO-AE1 329, which were a gift from ONO Pharmaceuticals (Osaka, Japan), were used in the PGE2 receptor studies (14, 15).

Assessment of Cell Viability—One well of the 24-well plate in each condition was detached using 0.025% trypsin and 0.01% EDTA. The cell number and percentage of viability were assessed using a hemocytometer and 0.4% Trypan blue (Sigma).

PGE2 Assay—The PGE2 levels in the culture supernatants were assessed by radioimmunoassay as described previously. The bound [3H]PGE2 (Amersham Biosciences) was measured using the Tri-Carb 2100TR liquid scintillation analyzer (Packard Bioscience Ltd., Pangbourne, UK). The PGE2 levels were calculated with Riasmart software (Packard Bioscience Ltd.). PGE2 was chosen because it is the main prostaglandin produced by pulmonary artery smooth muscle cells (16).

cAMP Assay—The cAMP was assayed after freon-amine (Sigma) extraction using [8-3H]adenosine 3',5'-cyclic phosphate, ammonium salt (Amersham Biosciences), and protein kinase A cAMP-dependent binding protein (Sigma) as described previously. This assay is based on the competitive binding between the unlabeled endogenous cAMP and a known, fixed quantity of 3H-labeled cAMP for an added known amount of the 3',5'-cAMP-dependent protein kinase (17).

[3H]Arachidonic Acid Release—Confluent PASMC were growth-arrested by withdrawal of serum for 24 h. The medium was then replaced with serum-free medium containing 18.5 KBq/ml of [3H]arachidonic acid (Amersham Biosciences), and the cells were incubated for 16 h. The culture supernatant was removed, and the cells were washed three times in sterile serum-free medium. The cells were then treated with or without 10 µM BK and for 0.25, 0.5, 1, 2, 3, and 4 h. The antibiotic and calcium ionophore calcimycin A23187 [GenBank] was used as a positive control. After the incubation times the supernatants were removed, and the released [3H]arachidonic acid was counted using Tri-Carb 2100TR liquid scintillation analyzer and Emulsifier Safe liquid scintillation mixture (Packard Bioscience Ltd.). The remaining cells were lysed with 0.1% Triton X-100 (Sigma) and counted as above. The percentage [3H]arachidonic acid release was calculated as shown in the following equation.

(Eq. 1)

Western Blot Analysis—Western blotting for COX-1 and COX-2 was performed as described previously (18). The goat anti-mouse horseradish peroxidase-conjugated secondary antibody was purchased from BD Biosciences (Cowley, Oxford, UK). The staining intensity of the bands was measured using a densitometer (Syngene, Braintree, UK) together with Genesnap and Genetools software (Syngene). The figures depicting Western blotting are representative of four blots.

RNA Isolation and Reverse Transcriptase-PCR—HPASMC were cultured to confluence in 6-well plates and growth-arrested for 24 h by serum withdrawal. The cells were treated with BK at a final concentration of 10 µM and collected at 0, 0.25, 0.5, 1, 2, and 4 h, respectively. Total RNA was isolated using the RNeasy mini kit (Qiagen) following the manufacturer's protocol. 1 µg of total RNA was reverse transcribed in a total volume of 25 µl including 200 units of Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI), 25 units of RNase inhibitor (Promega), 0.5 µg of oligo(dT)15 primer, 0.5 mM of each dNTPs, and 1x first strand buffer. (Promega). The reaction was incubated at 42 °C for 90 min.

PCR was carried out by adding 10 µl of cDNA to 40-µl reaction mixture giving final concentrations of 1 mM MgCl2, 0.12 mM of each dNTPs, 1 unit of Taq polymerase, 1x PCR buffer (Promega), and 0.5 µM of both the upstream and downstream PCR primers (Sigma). The primer sequences were as follows: COX-2 sense, 5'-TTC AAA TGA GAT TGT GGG AAA ATT GCT-3'; COX-2 antisense, 5'-AGA TCA TCT CTG CCT GAG TAT CTT-3'; GAPDH sense, 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3'; GAPDH antisense, 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3'; cPLA2 sense, 5'-CCA AAG TGA CAA AGG GGG CC-3'; cPLA2 antisense 5'-GCT ACC ACA GGC ACA TCA CG-3'; COX-1 sense, 5'-TGC CCA GCT CCT GGC CCG CCG CTT-3'; and COX-1 antisense 5'-GTG CAT CAA CAC AGG CGC CTC TTC-3'. Amplification was carried out with a PTC-100 programmable thermal controller (MJ Research Inc., Watertown, MA) after an initial denaturation at 94 °C for 3 min. This was followed by 30 cycles of PCR using the following temperatures and times: denaturation at 94 °C for 1 min, primer annealing at 58 °C for 2 min, primer extension at 72 °C for 1 min, and a final extension of 72 °C for 10 min. The PCR products were electrophoresed on 2% of agarose gel in 0.5x TBE buffer Tris-boric acid-EDTA (89 mM Tris borate, 2 mM EDTA, pH 8.3) containing 0.5 µg/ml ethidium bromide and visualized using ultraviolet illumination and the GeneGenius gel documentation and analysis system (Syngene).

Transfection of COX-2 Promoter with Deletions and Mutations—The PASMC were transiently transfected for 2 h using a liposomal transfection system (LipofectAMINE LF2000; Invitrogen). Transfections of the COX-2 promoter were performed using C2.2 (–2375/+49) 2424-bp or C2.1 (–917/+49) 966-bp fragments and C2.1 with a series of deletions or site-specific mutations ligated with a luciferase reporter plasmid pGL3 basic (Promega UK, Southampton, UK). Deletions consisted of Dra (–625/+49) 674-bp, Sty (–358/+49) 407-bp, Alu (–190/+49) 239-bp, and Rsa (–86/+49) 135-bp fragments. Mutations consisted of C/EBP (–132/–124), CRE (–59/–53), and NF-{kappa}B motifs {kappa}Bu (–447/–438) and {kappa}Bd (–224/–214) on the COX-2 promoter that bind p50/p65 NF-{kappa}B heterodimers.

The cells were cultured in 24-well plates to confluence, growth-arrested, and transfected using 1 µl of LF2000 and 0.8 µg of DNA/well according to the company instructions. The cells were co-transfected with 1 µg/well of the internal control plasmid pRL-SV40 (Promega UK) containing the Renilla luciferase gene (6). After 2 h of incubation with or without 10 µM bradykinin, the cells were harvested, and the firefly and Renilla luciferase activities were measured using a dual luciferase assay system kit (Promega UK) and a Microlumat Plus LB 96V luminometer (Berthold Technologies GmbH & Co. KG, Bad Wildbag, Germany). LipofectAMINE was shown to have a transfection efficiency of 10–20% under these conditions using HPASMC and green fluorescent protein (data not shown).

Transfection with p6CRE/luc Reporter—The p6CRE/luc construct contains the firefly luciferase gene under control of the minimal herpes simplex virus thymidine kinase promoter and six CREs and was obtained from S. Rees (Glaxo Wellcome, Stevenage, UK). This construct responds to agents that increase cAMP levels (19). Confluent, growth-arrested HPASMC in 24-well plates were transfected for 2 h using 2 µl of LF2000 and 1 µg of DNA/well. The medium was removed and incubated with serum-free medium containing either 1 µM indomethacin or 0.2% Me2SO for 30 min. BK was added to give a final concentration of 10 µM. Medium was added to the control wells. The cells were incubated for 2 h, after which the supernatants were removed, the cells were washed twice in phosphate-buffered saline and lysed with passive lysis buffer, and the luciferase levels were measured using a luciferase assay system kit (Promega UK) and the Microlumat Plus LB 96V luminometer (Berthold Technologies GmbH & Co. KG). In some of the wells 10 nM exogenous PGE2 was added.

Electrophoretic Mobility Shift Assay—The nuclear protein fractions for EMSA were prepared using Nu-Clear extraction kit (Sigma) following the manufacturer's protocol. The protein concentrations were determined using the Bio-Rad protein assay. Oligonucleotides containing the CREB sequences 5'-AAC AGT CAT TTC GTC ACA TGG GCT TG-3' (sense) and 5'-CA AGC CCA TGT GAC GAA ATG ACT GTT-3' (antisense) (Sigma) were labeled using [{gamma}-32P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (Promega UK). Fifteen micrograms of nuclear protein, 32P-labeled double-stranded probe (40,000 counts/min/ng), and 2 µl of 5x binding buffer (20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM dithiothreitol, 250 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.25 mg/ml poly(dI-dC)·poly(dI-dC)) were mixed in a total volume of 10 µl. In competition assays, 50x unlabeled competitors were added at the same time as probe addition. The mixture was incubated at room temperature for 30 min and then loaded on a 5% polyacrylamide gel in 0.5x TBE buffer and subjected to electrophoresis for 60 min. The gel was dried and exposed for autoradiography on Kodak XAR film at –70 °C for 19–48 h. Supershift was demonstrated using 400 ng of specific goat polyclonal and mouse monoclonal anti-human CREB-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Statistical Analysis—The results of the cAMP, PGE2, and luciferase levels were expressed as the means of the triplicate or quadruplicate wells for that experiment. The experiments were repeated at least three times, and the results shown represent the means ± S.E. Analysis of variance was used to determine significant differences. A p value of <0.05 (two-tailed) was regarded as statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
BK Induces COX-2 Protein and PGE2 Release—We have previously reported that BK induces COX-2 protein and stimulates increased PGE2 release in cultured human PASMC (6). Here we also found that BK increased PGE2 release. There was a significant difference in PGE2 levels in cells treated with 10 µM BK at 2, 4, 8, 16, and 24 h compared with unstimulated control cells (Fig. 1A). COX-2 was induced by BK with COX-2 protein detected at 2, 4, 8, 16, and 24 h, with the strongest bands at 4 and 8 h. COX-1 protein was constitutively expressed and remained constant at all the time points (Fig. 1B).



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  		FIG. 1.
A, time course of PGE2 production in PASMC treated with 10 µM BK for 0, 2, 4, 6, 8, 16, and 24 h and unstimulated cells. The PGE2 released into the culture medium was measured by radioimmunoassay. Each point represents the mean ± S.E. of quadruple determinations from three independent experiments. ***, p < 0.001 by analysis of variance. B, Western blotting showing time-dependent COX-2 protein induction by BK. PASMC were incubated for 0, 2, 4, 6, 8, 16, and 24 h with 10 µM BK. COX-2 protein expression was maximal at 4 h, whereas COX-1 protein expression was constitutive. The figure is representative of four blots from independent experiments.

 
BK Induction of COX-2 Is Transcriptional—Reverse transcriptase-PCR showed that BK increased COX-2 mRNA levels with time with a 2-fold increase at 60 min reducing to almost basal levels by 4 h (Fig. 2A). To confirm that this was due to COX-2 gene transcription rather than stabilization of mRNA, the cells were cultured for 1 h with 10 µM BK together with 5 and 10 µg/ml actinomycin D, an inhibitor of RNA polymerase II. The actinomycin D-treated cells showed a reduction in mRNA density (Fig. 2B). Western blotting demonstrated that incubation for 4 h with 10 µM BK together with 5 and 10 µg/ml actinomycin D inhibited COX-2 protein induction by BK (Fig. 2C). The cells were transfected with a 966-bp COX-2 promoter fragment (–917 to +49) ligated to firefly luciferase (C2.1). There was a 5.4 ± 0.27-fold increase in luciferase activity in the BK-treated cells compared with unstimulated cells (Fig. 2D). These results suggest that induction of COX-2 by BK is transcriptional and not mediated by post-transcriptional stabilization of COX-2 mRNA.



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  		FIG. 2.
A, time course of BK mediated COX-2 mRNA induction. PASMC were incubated with 10 µM BK for 0, 15, 30, 60, 120, and 240 min. COX-2 and internal control, GAPDH, mRNA were measured by reverse transcriptase-PCR. The relative band density was calculated by dividing the density of COX-2 band by the density of GAPDH band at the same time points. COX-1 mRNA was constitutive. The figure is representative of three experiments. B, Preincubation with 5 and 10 µg/ml actinomycin D (ACT D), an inhibitor of transcription, reduces COX-2 mRNA induced by 60 min of incubation with 10 µM BK. The figure is representative of three experiments. C, Western blotting for COX-2 showing that 5 and 10 µg/ml actinomycin D inhibit BK mediated COX-2 protein induction. D, luciferase activity in PASMC transiently transfected for 2 h with either a 966-bp fragment of the COX-2 promoter (–917 to +49) (C2.1) or control vector (pGL3 basic) ligated to a luciferase reporter construct. The cells were cultured to confluence, growth-arrested, and transfected with 1 µl of LF2000 and 0.8 µg of DNA/well. There was a significant increase in luciferase in cells transfected with the COX-2 promoter and stimulated for 2 h with 10 µM BK compared with unstimulated and control cells. ***, p < 0.001. The cells were co-transfected with the internal control plasmid pRL-SV40 containing the Renilla luciferase gene and corrected for any variations in transfection efficiency. The figure represents the mean and S.E. of three experiments performed in triplicate.

 
Mutations in the CRE but Not the NF-{kappa}B- or C/EBP-binding Sites in the COX-2 Promoter Reduce BK-stimulated Luciferase Activity—To determine which transcription factors are involved, the cells were transfected with 966 bp of the wild type COX-2 promoter (C2.1) and a series of deletion constructs ligated to a firefly luciferase reporter plasmid. A diagram representing the COX-2 promoter showing the key transcription sites and the positions where the restriction enzymes cleave the promoter to generate the series of deletions is shown in Fig. 3. BK increased the luciferase levels 5.4 ± 0.27 in the C2.1 construct. There was no reduction in luciferase activity with any of the deletions compared with the C2.1, suggesting that even the smallest Rsa fragment of the COX-2 promoter, which contains only the CRE transcription factor, was able to induce COX-2 (Fig. 4A).



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  		FIG. 3.
A schematic representation of the COX-2 promoter and constructs used in the transfection studies showing the positions of putative transcription factor-binding sites.

 



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  		FIG. 4.
A, increase in luciferase expression in PASMC transiently transfected with the deletion series of the COX-2 promoter luciferase constructs after 2 h of incubation with 10 µM BK. The cells were cultured in 24-well plates to confluence, growth-arrested, and transfected using 1 µl of LF2000 and 0.8 µg of DNA/well. The cells were co-transfected with the internal control plasmid pRL-SV40 containing the Renilla luciferase gene and corrected for any variations in transfection efficiency. The figure represents the mean and S.E. of three experiments performed in triplicate. There was no significant reduction in luciferase activity with the deletion series compared with the full-length construct, C2.1. B, the effect of mutations of the NF-{kappa}B-binding sites Kbu (–455/–428) and Kbd (–223/–214) on the COX-2 promoter activity in response to 10 µM BK. The graph shows the fold increase of luciferase activity in cells treated with BK compared with those with no BK treatment. The cells were cultured in 24-well plates to confluence, growth-arrested, and transfected using 1 µl of LF2000 and 0.8 µg of DNA/well. The figure represents the mean and S.E. of two experiments performed in quadruplicate. C, the effect of C/EBP and CRE-binding site mutations on the COX-2 promoter activity (Sty digest) in response to 10 µM BK. The cells were cultured in 24-well plates to confluence, growth-arrested, and transfected using 1 µl of LF2000 and 0.8 µg of DNA/well. The graph shows the fold increase of luciferase activity in cells treated with BK compared with those with no BK treatment. The figure represents the mean and S.E. of three experiments performed in triplicate and was analyzed by analysis of variance. **, p < 0.01.

 
Transfection studies using wild type and mutated C/EBP (–132/–124) and the NF-{kappa}B-binding sites from the COX-2 promoter {kappa}Bu (–447/–438) and {kappa}Bd (–224/–214) did not show reduction of the luciferase activity (Fig. 4, B and C). However, there was a significant inhibition (p < 0.01) with the mutated CRE construct (–59/–53), suggesting that this factor is necessary for COX-2 induction by BK (Fig. 4C).

BK Increases CREB Binding to the COX-2 Promoter—We used EMSA to determine whether BK treatment increased CREB binding to the COX-2 promoter. Incubation with 10 µM BK for 60 min induced CREB-1 binding activity with consensus and COX-2 promoter CRE oligonucleotides (Fig. 5, A and B). Supershift studies using a monoclonal antibody to CREB-1 produced gel retardation with the consensus sequence (Fig. 5A) and with the COX-2 promoter-specific primers (Fig. 5B). Competition with 50-fold excess unlabeled COX-2 promoter-specific CRE oligonucleotides blocked transcription factor binding, whereas excess AP-1 oligonucleotides did not block CRE transcription factor binding, demonstrating that the binding was specific (Fig. 5C).



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  		FIG. 5.
A, BK increases consensus CREB binding and the addition of 4 µg of anti-CREB-1 antibody to the nuclear extracts from cells treated with 10 µM BK for 60 min resulted in a supershift. The figures shown are representative of three experiments. B, 10 µM BK for 60 min increased CREB binding to the COX-2 promoter by EMSA. The addition of CREB-1 antibody to the nuclear extracts modified this CREB binding and induced a shift. The figures shown are representative of three experiments. C, for CRE binding competition, nuclear extracts from BK-treated cells were incubated with labeled CRE (Hot CRE) in the presence of a 50-fold excess of unlabeled CRE (Cold CRE) or unlabeled AP-1 (Cold AP-1). The figures shown are representative of three experiments.

 
BK Increases cAMP Production and Activates a 6CRE/luc Reporter Construct—To confirm that BK was increasing cAMP and thereby activating CRE-mediated transcription, we performed cAMP assays after BK stimulation and also measured activation of a 6CRE/luc reporter gene. BK rapidly increases intracellular cAMP levels (Fig. 6A) and increased the activity of the 6CRE/luc reporter construct (Fig. 6B), thereby demonstrating that BK was acting to increase cAMP and stimulate CRE-mediated transcription.



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  		FIG. 6.
A, confluent growth-arrested PASMC were incubated for 15, 30, 60, and 120 min with or without 10 µM BK. There was a significant increase in cAMP levels in the BK-treated cells compared with control cells. ***, p < 0.001. The cAMP levels in the unstimulated cells measured at the same times were below the level of detection of the assay. The figure shows the mean and S.E. of three experiments set up in triplicate. B, luciferase activity in PASMC cells transiently transfected for 2 h with pGL3.6CRE/luc containing six repeats of the CRE-binding site using 2 µl of LF2000 and 1 µg of DNA/well of a 24-well plate and incubated for 2 h with or without 10 µM BK. The figure represents the mean and S.E. of four experiments performed in triplicate. ***, p < 0.001.

 
BK-induced Release of PGE2 via cPLA2 Is Responsible for Generating the cAMP Signal—BK is known to produce a rapid increase in PGE2 by activating cPLA2 (20). Because PGE2 can increase cAMP via EP2 and EP4 receptors (21, 22), we postulated that cPLA2 induced early PGE2 production was the upstream signal to increase cAMP and stimulate cAMP-mediated CRE activation and gene transcription.

We found that BK induced an increase in cPLA2 mRNA that was maximal at 60 min. Furthermore we saw a significant increase in [3H]arachidonic acid release in the BK-treated cells at 30, 60, and 120 min (Fig. 7, A and B). This was accompanied by PGE2 generation (Fig. 7C). To test the role of this early PGE2 release, we determined the effect of its inhibition by indomethacin, a COX inhibitor. We found that PGE2 release was inhibited by preincubation with 1 µM indomethacin (Fig. 8A). Moreover indomethacin also inhibited BK-mediated increases in cAMP levels (Fig. 8B) and BK increases in 6CRE/luc reporter luciferase activity (Fig. 8C). Furthermore indomethacin inhibited CRE transcription factor binding by EMSA (Fig. 8D). The inhibitory effects of indomethacin on PGE2 release by cPLA2, cAMP generation, 6CRE/luc reporter activity, and EMSA CRE binding strongly suggest that early PGE2 release by cPLA2 is critical in the signal transduction cascade.



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  		FIG. 7.
A, reverse transcriptase-PCR of time-dependent increase in cPLA2 mRNA expression by 10 µM BK at 0, 15, 30, 60, 120, and 240 min time points. The figure is representative of three experiments. B, BK stimulation for 30, 60, and 120 min induces a significant increase in [3H]arachidonic acid release compared with unstimulated cells. Each point represents the mean ± S.E. of quadruple determinations from three independent experiments. **, p < 0.01; *, p < 0.05 by analysis of variance. C, BK stimulation for 30, 60, 90, and 120 min resulted in a significant increase in PGE2 levels in the BK-treated cells compared with control cells at all the time points. The figure shows the mean and S.E. of three experiments set up in quadruplicate. ***, p < 0.001.

 



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  		FIG. 8.
A, cells were cultured for 30 min with 10 µM BK after preincubation for 30 min with either 1 µM indomethacin or 0.1% Me2SO as a vehicle control. PGE2 release was measured and compared with unstimulated cells. BK increased PGE2 release. ***, p < 0.001. Pretreatment with indomethacin, a COX inhibitor, abolished the BK-associated increase in PGE2. +++, p < 0.001. B, cells were cultured for 30 min with 10 µM BK after preincubation for 30 min with either 1 µM indomethacin or 0.1% Me2SO. Intracellular cAMP levels were measured and compared with unstimulated control cells and time 0 levels. There was no detectable cAMP in the control cells. There was a significant increase in cAMP in the BK-treated cells after 30 min. ***, p < 0.001. Pretreatment with indomethacin abolished the BK-associated increase in cAMP. +++, p < 0.001. C, BK-mediated cAMP-driven luciferase activity is inhibited by indomethacin in PASMC transfected with 6CRE/luc luciferase reporter construct. Confluent, growth-arrested cells in 24-well plates were transfected for 2 h using 2 µl of LF2000 and 1 µg of DNA/well. BK increased luciferase relative light units (RLU). ***, p < 0.001, which was significantly reduced by indomethacin; +++, p < 0.001. The figure shows the mean and S.E. of three experiments set up in triplicate. D, indomethacin inhibits CREB binding to the COX-2 promoter by EMSA. The figure is representative of three experiments.

 
Exogenous PGE2 Mimics the Effect of BK—To confirm that the inhibitory effects of indomethacin are linked to the reduction in PGE2 levels, we performed additional studies to determine whether exogenous PGE2 would mimic the effect of BK. We found that exogenous PGE2 stimulated cAMP production in HPASMC in a concentration- and time-dependent manner (Fig. 9, A and B) and also increased 6CRE/luc reporter construct luciferase activity (Fig. 9C). Similarly exogenous PGE2 activated CRE transcription factor binding to the COX-2 promoter by EMSA (Fig. 9D) and increased luciferase levels in cells transfected with the COX-2 promoter construct C2.1 (Fig. 9E).



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  		FIG. 9.
A, concentration-dependent cAMP accumulation in response to exogenous PGE2. The confluent growth-arrested cells were incubated with 10 nM to 10 µM PGE2 for 20 min. There was a significant increase in intracellular cAMP in response to exogenous PGE2. ***, p < 0.001. B, time course showing increase in cAMP levels with 10 µM PGE2. ***, p < 0.001. The figure shows the mean and S.E. of three experiments set up in triplicate. C, incubation for 2 h with 10 nM PGE2 increases cAMP-driven luciferase activity compared with control cells in PASMC transfected for 2 h with 6CRE/luc reporter using 2 µl of LF2000 and 1 µg of DNA/well. ***, p < 0.001. D, treatment with 10 nM PGE2 for 1 h increases CRE binding to the COX-2 promoter by EMSA. E, 10 nM PGE2 for 2 h increases luciferase activity in cells transfected for 2 h with ~1 kb of the COX-2 promoter (C2.1). ***, p < 0.001. The cells were cultured in 24-well plates to confluence, growth-arrested, and transfected using 1 µl of LF2000 and 0.8 µg of DNA/well.

 
EP2 and EP4 Receptor Agonists Mimic the Effect of Exogenous PGE2—To determine which PGE2 receptors were important, we looked at the effect of PGE2 receptor agonists on cAMP production together with luciferase activity in cells transfected with 6CRE/luc reporter construct. Both EP2 and EP4 receptors are known to couple to adenylyl cyclase. We found that both EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) receptor agonists increased cAMP production and luciferase activity in a concentration-dependent manner (Fig. 10) in the same way as exogenous PGE2. This suggests that PGE2 is acting via both EP2 and EP4 receptors. The specificity of these PGE2 receptor agonists has been determined previously using Chinese hamster ovary cells (23). At the concentrations used here, the compounds are highly selective and do not cross-react with other prostanoid receptors.



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  		FIG. 10.
A, concentration dependent increase in cAMP generation after incubation for 20 min with EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) receptor agonists. B, fold increase in luciferase activity in cells transfected with 6CRE/luc reporter construct and stimulated with EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) receptor agonists. In both experiments 1 µM PGE2 was used as the positive control.

 
Collectively these results suggest that BK induces COX-2 gene transcription by a cAMP-dependent mechanism involving an autocrine prostanoid loop. BK up-regulates cPLA2, resulting in increased membrane-derived arachidonic acid release. Arachidonic acid is converted to PGE2. PGE2 binds to EP2 and EP4 receptors and increases intracellular cAMP production, which then phosphorylates CREB and induces transcription by binding to the CRE elements on the COX-2 promoter (Fig. 11).



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  		FIG. 11.
Mechanism of COX-2 induction by bradykinin in human pulmonary artery smooth muscle cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study shows that BK increases COX-2 expression through transcriptional mechanisms involving activation of the CRE region on the COX-2 promoter. Transfection experiments with ~1 kb of the COX-2 promoter construct and a series of deletions containing key transcription factor-binding sites showed that effective promoter activity was maintained with a construct having an isolated CRE region only. Mutational studies of the COX-2 promoter indicated that the NF-{kappa}B- and C/EBP-binding sites were not involved in BK induction of COX-2 but that the CRE binding site was essential. Furthermore, EMSA showed that after BK stimulation, CREB bound to the COX-2 promoter. BK increased intracellular cAMP levels and cAMP-driven p6CRE/luc luciferase expression. Additional studies with the COX inhibitor indomethacin showed that it inhibited the elevations in cAMP, 6CRE/luc activity, and EMSA CREB binding, suggesting that endogenous prostanoids were responsible for the effect of BK. Consistent with this, BK caused an early increase in cPLA2 mRNA expression, PGE2 and [3H]arachidonic acid release, and exogenous PGE2 increased CREB binding to the COX-2 promoter. Collectively these studies demonstrate that autocrine production of PGE2 and CRE activation is the major pathway involved in COX-2 induction by BK.

COX-2 is an important inducible gene in inflammatory and malignant diseases. A number of cytokines are known to induce COX-2 through both transcriptional and post-transcriptional mechanisms. Recently we have shown that the kinin bradykinin, which acts through seven-transmembrane G protein-coupled receptors is also capable of inducing COX-2 in airway smooth muscle, pulmonary artery smooth muscle, and lung fibroblasts, but the mechanisms involved have not been extensively studied. A greater understanding of these mechanisms may provide important information to aid our understanding of how BK acts in vascular and inflammatory diseases.

As in our previous studies in PASMC, we found that COX-2 protein was detected by Western blotting 1 h after BK treatment and peaked between 4 and 8 h. We extended these studies to show that BK-mediated COX-2 mRNA was also increased and was maximal at 1 h consistent with the lag between mRNA and protein synthesis. There was a reduction in COX-2 mRNA after 2 h, suggesting that post-transcriptional mechanisms were not involved in the effect of BK on COX-2 mRNA. The addition of actinomycin D prevented BK-mediated COX-2 mRNA induction, indicating that the mechanism was transcriptional. This lack of post-transcriptional effect of BK contrasts with the effects of IL-1{beta} and TNF-{alpha} in other studies. Both IL-1{beta} and TNF-{alpha} induced an increase in COX-2 mRNA in differentiated human macrophages that was stabilized by IL-1{beta} but destabilized by TNF-{alpha} (24). IL-1{beta} not only induced COX-2 mRNA in human endometrial stromal cells but significantly increased the mRNA stability (25).

In our experiments when cells were pretreated with actinomycin D prior to BK treatment COX-2, mRNA was reduced by actinomycin D, suggesting that COX-2 protein induction was transcriptional. This was confirmed in transfection experiments with a wild type COX-2 promoter ligated to a firefly luciferase reporter construct that had increased luciferase activity in BK-treated cells. We then proceeded to studies using a series of deletions of the 1-kb COX-2 promoter fragment. We found that deletions of the COX-2 promoter down to 0.135 kb all had maximal promoter activity, suggesting that a transcriptional binding site on the 0.135 kb was essential for promoter activity. Consistent with this, mutations of the NF-{kappa}B- or C/EBP-binding sites did not reduce activity. In contrast, when we used a COX-2 promoter construct with a mutated CRE site BK-induced luciferase activity was markedly reduced compared with the wild type construct, suggesting involvement of the CRE transcription factor-binding site in BK-mediated COX-2 transcription. This observation was supported by the EMSA experiments showing that BK induced CREB binding to CRE consensus sequences and also the COX-2 promoter CRE. We confirmed that binding was specific by studies with excess unlabeled CRE and AP-1 oligonucleotides and demonstrated supershift with a monoclonal antibody to CREB-1. Furthermore we showed that BK increased cAMP levels and activated a 6CRE/luc construct. The lack of involvement of NF-{kappa}B and C/EBP in BK-induced COX-2 expression contrasts with studies with IL-1{beta} and TNF-{alpha}. TNF-{alpha} enhanced COX-2 promoter activity in a mouse osteoblastic cell line, MC3T3-E1, by the activation of both NF-{kappa}B and C/EBP (26). COX-2 gene transcription by IL-1{beta} in the bronchial epithelial cell line A549 involved NF-{kappa}B (7) and C/EBP and NF-{kappa}B co-regulation in rabbit chondrocytes (27).

The lack of involvement of NF-{kappa}B and C/EBP in BK-induced regulation also contrasts with our own studies showing that BK can activate IL-8 in airway smooth muscle cells via NF-{kappa}B and C/EBP (12). Other studies have shown that BK induces IL-1{beta} gene expression by NF-{kappa}B activation in human epithelial cells and lung fibroblast cell lines (10, 28) and activates AP-1 in HEK 293 cells (29). Collectively these studies suggest that BK can activate several transcription factors that then regulate several genes in a manner that is gene- and transcription factor-specific.

CRE has been implicated in COX-2 induction by phorbol ester in murine osteoblastic cells, (13) and in phorbol 12-myristate 13-acetate-mediated differentiation of the human monocytoid U937 cells, (30), but none have looked at the involvement of the CRE in BK-mediated COX-2 induction. Our studies clearly indicate that the CRE is a critical part of BK signaling to the COX-2 promoter.

We then performed additional mechanistic studies to determine how BK was acting to increase cAMP and therefore CRE-mediated COX-2 transcription. BK is known to increase PGE2 production in smooth muscle cells in two stages. The first stage involves up-regulation of cPLA2-derived arachidonic acid providing the substrate for constitutively expressed COX-1 (20) with the second more sustained phase being due to COX-2 induction (18). In the first cPLA2-mediated phase the released PGE2 binds to cell surface receptors and stimulates intracellular cAMP synthesis. BK has also been shown to decrease the rate of cAMP degradation by inhibiting cellular phosphodiesterase activity, potentiating the effects of cAMP (31). Intracellular cAMP activates PKA resulting in phosphorylation of the CREB at Ser133, which binds to the CRE resulting in gene transcription. Because PGE2 can increase cAMP via EP2 and EP4 receptors, we hypothesized that early release of PGE2 via cPLA2 was responsible for the increase in cAMP, which then activated CRE-mediated transcription. To test this hypothesis, we performed studies using the broad spectrum COX inhibitor indomethacin and measured cPLA2 mRNA, arachidonic acid release, and PGE2 production. We found that BK induced cPLA2 mRNA expression, arachidonic acid mobilization, and PGE2 release at early time points. Both the BK-induced PGE2 release and cAMP production was markedly reduced by indomethacin. Indomethacin also prevented CREB binding to the COX-2 promoter CRE site in BK-treated cells. These studies strongly suggested that endogenous prostanoid produced via cPLA2 were responsible for generating the cAMP signal leading to CRE activation. To further test the hypothesis we determined whether exogenous PGE2 would mimic the effect of BK on CRE-mediated COX-2 expression. We found that exogenous PGE2-stimulated cAMP production increased 6CRE/luc luciferase activity and induced CREB binding to the CRE-binding site on the COX-2 promoter. Similarly the PGE2 receptor subtype agonists EP2 and EP4 stimulated both cAMP production and increased 6CRE/luc luciferase. PGE2-activated EP2/EP4 receptors are known to stimulate adenylyl cyclase, resulting in increased cAMP and activation of PKA (22).

This study and our previous studies with BK in airway smooth muscle where BK stimulation of ASM resulted in increased IL-8 and vascular endothelial growth factor production that was dependent on the generation of endogenous prostanoid (12, 32) suggest that generation of prostanoids by BK produces a strong signaling system that can affect the expression of a number of inflammatory response genes, which can contribute to inflammatory and vascular diseases.

In conclusion our studies provide strong evidence that BK induces COX-2 via the CRE-binding site on the COX-2 promoter. Furthermore autocrine PGE2 generation by cPLA2 with subsequent binding to the EP2 and EP4 receptors is responsible for generating the cAMP signal leading to CREB binding and CRE activation of the COX-2 promoter. These studies provide new insight into the mechanisms whereby transmembrane G protein-coupled receptor ligands such as BK cause COX-2 induction.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Div. of Respiratory Medicine, Clinical Science Bldg., City Hospital, Hucknall Rd., Nottingham NG5 1PB, UK. Tel.: 44-115-8404775; Fax: 44-115-8404771; E-mail: alan.knox{at}nottingham.ac.uk.

1 The abbreviations used are: BK, bradykinin; PASMC, pulmonary artery smooth muscle cell(s); HPASMC, human PASMC; PGE2, prostaglandin E2; NF-{kappa}B, nuclear factor {kappa}B; COX, cyclooxygenase; EMSA, electrophoretic mobility shift assay; CRE, cAMP response element; cPLA2, cytosolic phospholipase A2; IL, interleukin; TNF, tumor necrosis factor; CREB, cAMP-response element-binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EP, E-prostanoid; C/EBP, CCAAT/enhancer-binding protein. Back


   ACKNOWLEDGMENTS
 
We thank S. Rees (Glaxo Wellcome) for providing the 6CRE/luc reporter vectors.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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