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Am J Physiol Lung Cell Mol Physiol 288: L238-L250, 2005; doi:10.1152/ajplung.00313.2004
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Prostanoid receptor expression by human airway smooth muscle cells and regulation of the secretion of granulocyte colony-stimulating factor

Deborah L. Clarke,1 Maria G. Belvisi,2 Susan J. Smith,1 Elizabeth Hardaker,2 Magdi H. Yacoub,2 Koremu K. Meja,1 Robert Newton,3 Donna M. Slater,4 and Mark A. Giembycz3

1Thoracic Medicine and 2Cardiothoracic Surgery (Respiratory Pharmacology Group), National Heart and Lung Institute, Imperial College London, London; 4Department of Biological Sciences, Biomedical Research Institute, University of Warwick, Coventry, United Kingdom; and 3Departments of Pharmacology & Therapeutics, and Cell Biology & Anatomy, Respiratory Research Group, University of Calgary, Calgary, Alberta, Canada

Submitted 20 August 2004 ; accepted in final form 21 September 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The prostanoid receptors on human airway smooth muscle cells (HASMC) that augment the release by IL-1{beta} of granulocyte colony-stimulating factor (G-CSF) have been characterized and the signaling pathway elucidated. PCR of HASM cDNA identified products corresponding to EP2, EP3, and EP4 receptor subtypes. These findings were corroborated at the protein level by immunocytochemistry. IL-1{beta} promoted the elaboration of G-CSF, which was augmented by PGE2. Cicaprost (IP receptor agonist) was approximately equiactive with PGE2, whereas PGD2, PGF2{alpha}, and U-46619 (TP receptor agonist) were over 10-fold less potent. Neither SQ 29,548 nor BW A868C (TP and DP1 receptor antagonists, respectively) attenuated the enhancement of G-CSF release evoking any of the prostanoids studied. With respect to PGE2, the EP receptor agonists 16,16-dimethyl PGE2 (nonselective), misoprostol (EP2/EP3 selective), 17-phenyl-{omega}-trinor PGE2 (EP1 selective), ONO-AE1-259, and butaprost (both EP2 selective) were full agonists at enhancing G-CSF release. AH 6809 (10 µM) and L-161,982 (2 µM), which can be used in HASMC as selective EP2 and EP4 receptor antagonists, respectively, failed to displace to the right the PGE2 concentration-response curve that described the augmented G-CSF release. In contrast, AH 6809 and L-161,982 in combination competitively antagonized PGE2-induced G-CSF release. Augmentation of G-CSF release by PGE2 was mimicked by 8-BrcAMP and abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA). These data demonstrate that PGE2 facilitates G-CSF secretion from HASMC through a PKA-dependent mechanism by acting through EP2 and EP4 prostanoid receptors and that effective antagonism is realized only when both subtypes are blocked concurrently.

cAMP-dependent protein kinase; human airway smooth muscle; granulocyte colony-stimulating factor; prostanoid receptors; airway inflammation

COLONY-STIMULATING FACTORS (CSFs) were discovered in the 1960s as a family of glycoproteins that are essential for the differentiation, proliferation and survival of bone marrow-derived hematopoietic stem cells (7, 36). Three human CSFs have been unequivocally identified as granulocyte/macrophage colony-stimulating factor (GM-CSF) (65), granulocyte colony-stimulating factor (G-CSF) (52), and macrophage colony-stimulating factor (M-CSF) (39). In addition to acting upon immature myeloid cells, CSFs promote the activation and survival of mature leukocytes including neutrophils, monocytes, and eosinophils (6, 45). GM-CSF has effects on many leukocytes, whereas G-CSF and M-CSF have a more restricted action modifying the function of neutrophils and cells of the monocytes/macrophage lineage, respectively (50).

The ability of CSFs to increase the longevity and activity of proinflammatory cells implies that their inappropriate secretion may perpetuate airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease, where eosinophils, neutrophils, and monocytes/macrophages play a pathogenic role. CSFs can be synthesized by a variety of motile and structural cells. These include human airway smooth muscle (HASM) cells, which have a substantial synthetic capacity and may contribute to inflammatory processes through the generation of a plethora of mediators including cytokines, chemokines, and bioactive lipids (37). We have reported previously that HASM cells can produce G-CSF and GM-CSF (but not M-CSF) in response to proinflammatory cytokines, such as interleukin (IL)-1{beta}, that are secreted in to the asthmatic, emphysematous, and bronchitic airways (11, 44). Of relevance to the present study is that PGE2 and other agents that elevate cAMP attenuate the elaboration of GM-CSF (10, 44), whereas the release of G-CSF is augmented under identical experimental conditions (11). The latter finding is particularly intriguing as it implies that cAMP (through the production of G-CSF) will only further enhance the survival and activity of neutrophils, monocytes, and macrophages during ongoing inflammation. Thus agonism of specific prostaglandin receptors on HASM and other pulmonary cells could theoretically exacerbate or suppress an inflammatory response by altering the balance of cytokines secreted into the airways. Ultimately, this effect could have an impact on the progression of respiratory diseases by influencing the lifespan and functional activity of pulmonary leukocytes.

Studies performed over the last 15 years have provided pharmacological evidence for five main classes of G protein-coupled receptor for the naturally occurring prostanoid agonists, and these have been given the prefix DP, EP, FP, IP, and TP (8, 15, 41, 53, 64). Due to a lack of selective antagonists, this taxonomy was formulated from rank orders of agonist potency obtained in various pharmacological preparations where the first letter denotes the agonist most selective at that receptor and is at least one order of magnitude more potent than the other natural ligands. Molecular biological techniques subsequently confirmed this pharmacological classification with the cloning and expression of cDNAs for representatives of the five prostanoid receptors in a number of species including humans (53).

The finding that the release of G-CSF from cytokine-treated HASM cells is augmented by PGE2 implicates prostanoid receptors of the EP subtype. Currently, pharmacological evidence and primary sequence information of partial and full-length cDNA clones have identified four EP receptor variants that mediate diverse biological responses (8, 15, 42, 53, 64). EP1 receptors generally mediate contraction of smooth muscle and are coupled to the hydrolysis of inositol phospholipids with subsequent Ca2+ mobilization via the Gq/G11 class of pertussis toxin-insensitive G proteins. In contrast, prostanoid receptors of the EP2 subtype are positively coupled to adenylyl cyclase via Gs and mediate relaxation of a variety of smooth muscle preparations. The EP3 receptors comprise a family of similar but structurally distinct proteins of which eight splice variants in humans can theoretically be derived from the same gene. This heterogeneity presumably explains the fact that EP3 subtypes subserve a variety of functions including inhibition of autonomic neurotransmission, smooth muscle contraction, inhibition of lipolysis, and water reabsorption. Indeed, transfection experiments of the cDNAs encoding these EP3 variants into Chinese hamster ovary cells revealed that they can couple to at least three second messenger systems including phospholipase C, via Gq/G11, and adenylyl cyclase, through both Gs and Gi/Go. In 1993, a fourth EP receptor subtype (denoted EP4) was originally identified from studies performed with piglet saphenous vein where the pharmacological behavior of prostanoid agonists and antagonists did not resemble an interaction with the other defined EP receptor subtypes (12). The most salient characteristics of the EP4 receptor are the findings that PGE2 can exhibit subnanomolar potency, that the EP2-selective agonists butaprost and AH 13205 have relatively low affinity, and that AH 23848B is a weak but competitive antagonist (5, 12, 35, 47, 55).

Although much is known about the expression of GM-CSF in HASM cells and its regulation by cAMP-elevating agent (e.g., Refs. 31, 32, 44), only a paucity of information is available on G-CSF. Thus the objective of the present study was to classify the prostanoid receptor(s) expressed by HASM cells through which PGE2 enhances the release of G-CSF and probe the molecular basis of this effect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Drugs and analytical reagents. IL-1{beta} was from R & D Systems (Abingdon, Oxon, UK), and DMEM and HBSS were from Flow Laboratories (Rickmansworth, Hertfordshire, UK). Nonessential amino acids were purchased from Life Technologies (Paisley, UK); indomethacin, diclofenac, heat-inactivated FCS, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), PGD2, PGE2, PGF2{alpha}, and 9,11-dideoxy-11{alpha},9{alpha}-methanoepoxy-PGF2{alpha} (U-46619) were from Sigma-Aldrich (Poole, Dorset, UK); and 16,16-dimethyl PGE2, 17-phenyl-{omega}-trinor PGE2, 6-isopropoxy-9-oxoxanthine-2-carboxylic acid (AH 6809), [1S-[1{alpha},2{alpha}(Z),3{alpha},4{alpha}]]-7-[3-[[2-[(phenyl-amino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-y1]-5-heptenoic acid (SQ-29,548), [1{alpha}(Z),2{beta},5{alpha}]-(±)-7-[5-[[(1,1'-biphenyl)-4-yl-methoxy]-3-hydroxy-2-(1-piperidinyl) cyclopentyl]-4-heptanoic acid] (AH 23848B), 3-[(2-cyclohexyl-2-hydroxyethyl)amino]-2,5-dioxo-1-(phenylmethyl)-4-imidazolidine-heptanoic acid hydantoin (BW A868C), fluprostenol, PGE1-OH, sulprostone, misoprostol, and butaprost were obtained from Cayman Chemicals (Ann Arbor, MI). All other synthetic prostanoid reagents were donated as follows: cicaprost by Schering (Berlin, Germany), (16S)-9-deoxy-9{beta}-chloro-15-deoxy-16-hydroxy-17,17-trimethyl-ene-19,20-didehydro-PGE2-sodium salt (ONO-AE1-259) by Ono Pharmaceuticals (Osaka, Japan), and 4'-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide (L-161,982) by Merck Frosst (Montreal, Quebec, Canada). Human EP2 and IP receptor primary antibodies were purchased from Université de Sherbrooke (Sherbrooke, Quebec, Canada). The human EP4 receptor primary antibody was from Cayman Chemicals. Goat anti-human PKI{alpha} (code sc no. 1944) and goat anti-human {beta}-actin (code sc no. 1615) were from Autogen Bioclear (Calne, Wiltshire, UK). Goat anti-human phosphorylated cAMP response element binding protein (pCREB) (code 9191S) was purchased from New England Biolabs (Hitchin, Hertfordshire, UK).

Isolation of HASM cells. Tracheal rings from either lung or heart and lung transplantation donors (7 female, 17 male; age range, 17–57 yr; median age, 36.5 yr) were dissected under sterile conditions in Hanks' balanced salt solution (HBSS, in mM): 136.8 NaCl, 5.4 KCl, 0.8 MgSO4, 0.4 Na2HPO4·7H2O, 1.3 CaCl2·2H2O, 4.2 NaHCO3, and 5.6 glucose supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (2.5 µg/ml). The smooth muscle layer was dissected free of adherent connective tissue and cartilage, and the epithelium was removed with a rounded scalpel blade. The smooth muscle was incubated for 30 min at 37°C in 5% CO2/air in HBSS containing BSA (10 mg/ml), collagenase (type XI, 1 mg/ml), and elastase (type I, 3.3 U/ml). After the removal of any remaining connective tissue, the smooth muscle was chopped finely and incubated for a further 150 min in the enzyme solution described above with the elastase concentration increased to 15 U/ml. Dissociated cells were centrifuged (100 g, 5 min, 4°C) and resuspended in DMEM containing heat-inactivated FCS (10% vol/vol), sodium pyruvate (1 mM), L-glutamine (2 mM), nonessential amino acids (1x), and antimicrobial agents as detailed above.

Primary culture of HASM cells. HASM cells in suspension were placed in a tissue culture flask (75 cm2) containing 6 ml of supplemented DMEM and allowed to adhere (~12 h) at 37°C in 5% CO2/air. The culture medium was replaced after 4–5 days (12 ml) and thereafter every 3–4 days. When the cells reached confluence (~10–14 days) and demonstrated a typical "hill and valley" appearance and positive immunostaining for {alpha}-actin, they were placed onto either 96-well plates (2,000 cells/well; Costar UK, High Wycombe, UK) or eight-well Labtek slides (4,000 cells/well, Nunc) for cytokine release and PCR experiments, respectively. At subconfluence the cells were growth arrested by being placed in DMEM containing apotransferin (5 µg/ml), insulin (1 µM), ascorbate (100 µM), and BSA (0.1% wt/vol) for 24 h. The medium was replaced with DMEM containing 3% FCS and drugs or appropriate vehicles as indicated.

Infection of HASM cells with Ad5.CMV.PKI{alpha}. In some experiments subconfluent, growth-arrested HASM cells were infected [multiplicity of infection (MOI) = 100] with an E1–/E3– replication-deficient adenovirus vector (Ad5.CMV.PKI{alpha}) containing a 251-bp DNA fragment encoding the complete amino acid sequence of the {alpha}-isoform of cAMP-dependent protein kinase inhibitor (PKI{alpha}) (18) downstream of the constitutively active CMV immediate-early promoter (30, 46). After 48 h, when >90% of cells expressed the transgene, cells were processed for G-CSF release and Western blotting as described below. To control for biological effects of the virus per se, the vector Ad5.CMV.Null, expressing no transgene, was used in parallel. Preliminary experiments established that infection with Ad5.CMV.PKI{alpha} resulted in the expression of a completely functional transgene and that neither vector at an MOI of 100 had any effect on HASM cell viability (data not shown).

Measurement of G-CSF. HASM cells (naïve and virus infected) were pretreated for 30 min with indomethacin (10 µM), diclofenac (10 µM), and, where indicated, receptor antagonist(s) before being exposed for a further 5 min or 30 min to prostanoid agonists or 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), respectively. IL-1{beta} (10 pg/ml) was then added, and the cells were incubated at 37°C in a thermostatically controlled incubator under a 5% CO2 atmosphere. At 24 h the amount of G-CSF released into the culture supernatant was quantified by a sandwich ELISA (human DuoSet development system, R & D Systems Europe) according to the manufacturer's instructions. The detection limit of this assay is 16 pg/ml.

RNA extraction. Total RNA was prepared from untreated cells in 3% FCS on six-well plates by guanidine isothiocyanate and phenol-chloroform extraction. The concentration of RNA was measured spectrophotometrically at 260 nm, and the samples were stored at –70°C until required.

Reverse transcription and PCR. Total RNA was heated at 70°C for 5 min, placed on ice, and incubated at 25°C for a further 5 min before the addition of a reverse transcription (RT) mix. Each sample (0.5 µg) was reverse transcribed in a 20-µl reaction volume containing 200 units of Superscript II (GIBCO, Paisley, UK), 10 mM DTT (GIBCO), 30 units of RNase inhibitor (Promega, Southampton, UK), and 0.25 µg of random hexanucleotides (Promega). The reaction was carried out at 37°C for 1 h and was stopped by heating to 90°C for 5 min. The mix was diluted to 100 µl in distilled H2O and kept at 4°C until used or –70°C if longer storage was required.

DNAs encoding the prostanoid receptors were amplified by PCR using primers designed from the reported primary sequences deposited with the GenBank database (Table 1). PCR amplification was carried out with a Hybaid OmniGene Thermocycler (Hybaid, Teddington, UK) in a total volume of 25 µl containing 2 µl of cDNA, 0.2 mM dNTPs (Promega), 1x NH4 buffer [16 mM (NH4)2SO4, 67 mM Tris·HCl (pH 8.8), 0.1% vol/vol Tween 20], 1.5 mM MgCl2, 1 unit BioTaq polymerase (Bioline), and 0.125 µg of both forward and reverse primers. Cycling parameters were set for 35–36 cycles (see Table 1) at an initial denaturing temperature of 94°C for 4 min, specific annealing temperature (see Table 1) for 30 s, and an extension temperature of 72°C for 5 min. The number of cycles chosen for the PCR reactions was determined empirically from cDNA pooled from a mixture of HASM cells from different donors. PCR products were resolved on 1% wt/vol agarose gels, stained with ethidium bromide, and visualized under UV light. After amplification, each PCR product was analyzed by agarose gel electrophoresis, subcloned into pGEM-T Easy vectors (Promega), and sequenced.


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  		Table 1. Primers and conditions for RT-PCR

 
Immunocytochemistry. HASM cells, grown on LabTek slides, were treated with 3% vol/vol FCS for 24 h, washed in PBS (150 mM, pH 7.4; 3x 30 s), and fixed (10 min) in methanol. The cells were washed again and blocked for 2 h at room temperature with human serum (10% vol/vol in PBS) to inhibit nonspecific binding. Cells were incubated with either the primary antibody (immunized rabbit serum) directed against the human EP2, EP4, or IP receptor subtype (diluted 1:200, 1:770, and 1:2,500, respectively, in 10% vol/vol human serum/PBS) or rabbit control serum for 25 min (EP2) overnight (EP4) and 1 h (IP) at 4°C. For the detection of EP2 and IP receptors, slides were washed in PBS/1% vol/vol Triton X-100 (5 min) and PBS (5 min) and then blocked again for 20 min. For the EP4 receptors, slides were washed (3 x 5 min) in PBS only. All slides were then incubated with a biotinylated anti-rabbit IgG (diluted 1:200; Vector Laboratories, Peterborough, UK) for 30 min at room temperature and washed again as described above. Immunopositive cells were subsequently detected using an avidin-biotin-alkaline phosphatase detection system (Vector Laboratories). The reaction was visualized using fast red in the presence of levamisole (Vector Laboratories), and the cells were counterstained with hematoxylin (VWR International, Lutterworth, UK) and mounted with glycergel (Dako, Ely, UK).

For the staining of PKI{alpha}, HASM cells were infected with Ad5.CMV.PKI{alpha} and 48 h later fixed in formaldehyde (4% vol/vol in PBS). The fixative was decanted, and cells were permeabilized with Nonidet P-40 (0.5% vol/vol in PBS). After rehydration in glycine (100 mM in PBS), cells were "blocked" by immersion in 0.5% vol/vol BSA in PBS containing 0.1% wt/vol gelatin followed by overnight incubation at 4°C with a goat anti-human antibody directed against PKI{alpha}. Cells were washed again and exposed (60 min) at room temperature to a biotinylated swine anti-rabbit IgG secondary antibody (diluted 1:200, Vector Laboratories) and for a further 60 min with fluorescein-conjugated streptavidin (diluted 1:100). Cells were counterstained with the nuclear marker 4',6'-diamidino-2-phenylindole (DAPI; 1 µg/ml, 5 min), and the subcellular localization of PKI{alpha} was visualized by laser-scanning confocal microscopy.

Western blot analysis. HASM cells were treated with 3% vol/vol FCS for 24 h and, in some experiments, exposed to 8-BrcAMP (1 mM, 30 min) or PGE2 (10 µM, 5 min). The medium was removed, the cells were washed with HBSS and lysed, and proteins were extracted in 20 mM Tris·HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.1% vol/vol Nonidet P-40, 0.05% wt/vol sodium deoxycholate, 0.025% wt/vol SDS, and 0.1% vol/vol Triton X-100 supplemented with PMSF (0.1 mg/ml), leupeptin (10 µg/ml), and aprotinin (25 µg/ml). Insoluble protein was removed by centrifugation (10,000 g, 3 min), and aliquots of the resulting supernatant were diluted 1:4 in Laemmli buffer and boiled for 5 min. Denatured proteins (25 µg) were separated by SDS-PAGE using a 4–20% gradient gel (Bio-Rad, Hemel Hempstead, UK) and transferred to Hybond-enhanced chemiluminescence (ECL) membranes (Amersham Pharmacia Biotech, Little Chalfont, UK) in Tris buffer (50 mM Tris base, pH 8.3, 192 mM glycine, 20% vol/vol methanol). The nitrocellulose was incubated overnight in TBS-T (25 mM Tris-base, pH 7.4, 150 mM NaCl, 0.1% vol/vol Tween 20) containing 5% wt/vol nonfat dry milk. The filters were incubated for 1 h at room temperature in TBS-T containing 5% wt/vol BSA plus an anti-human pCREB or anti-human PKI{alpha} polyclonal antibody (diluted 1:1,000 and 1:500, respectively) as indicated. Membranes were washed with TBS-T and incubated with horseradish peroxide-conjugated sheep, anti-rabbit IgG (diluted 1:4,000, Dako) in TBS-T/5% wt/vol nonfat dry milk for 1 h at room temperature. The nitrocellulose was washed in TBS-T and developed using ECL Western blotting detection reagents on Kodak X-OMAT-S film (Amersham Pharmacia Biotech).

Cell viability. At the end of each experiment, we determined cell viability colorimetrically by measuring the reduction of the tetrazolium salt MTT to formazan by mitochrondial dehydrogenases.

Data and statistical analyses. Data points and values in the text and figure legends represent the means ± SE of n independent determinations using tissue from different donors. Concentration-response curves were analyzed by least-squares, nonlinear iterative regression with the PRISM curve fitting program (GraphPad software, San Diego, CA), and –log EC50 values were subsequently interpolated from curves of best fit. Equieffective molar concentration ratios (e.c.r.) were calculated with the formula: EC50 prostanoid/EC50 PGE2.

Estimates of antagonist affinity were calculated using the equation pKB = log (CR-1) – log [B] as described by Schild (60), where CR is the concentration ratio calculated from the EC50 of agonist in the presence of the antagonist divided by the EC50 of the agonist alone, KB is the equilibrium dissociation constant, and [B] is the concentration of antagonist. In the experiments described herein the term pA2 is substituted for pKB, as antagonists were used at one concentration only, which precludes assumptions being made about the nature of the antagonism.

Where appropriate, data were analyzed statistically by Student's paired t-test or by one-way ANOVA/Newman-Keuls multiple-comparison test. The null hypothesis was rejected when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We have reported previously that IL-1{beta} promotes a time- and concentration-dependent release of G-CSF from HASM cells with a time to achieve 50% of the maximum response (t1/2) and EC50 of 16 h and 62 pg/ml, respectively (11). In the experiments described herein, IL-1{beta} was used at a concentration of 10 pg/ml (~EC40), and G-CSF was measured in the culture supernatant 24 h after addition of the stimulus. None of the compounds or their vehicles used in these experiments affected cell viability as determined by the reduction of MTT to formazan. None of the vehicles used had any significant effect on G-CSF release (data not shown).

Effect of naturally occurring prostaglandins, cicaprost, and U-46619 on G-CSF release. PGE2, PGD2, PGF2{alpha}, and the thromboxane and prostacyclin mimetics U-46619 and cicaprost, respectively, failed to stimulate the release of G-CSF from indomethacin-treated HASM cells. In contrast, all of the prostanoids tested potentiated, in a concentration-dependent manner, the elaboration of G-CSF evoked by the proinflammatory cytokine IL-1{beta}. Significant variation in the maximum amount of G-CSF released was observed between tissue from different donors (150–700% of IL-1{beta} control, 0.43–2.1 ng/ml) that was unrelated to passage number or the amount of G-CSF secreted by IL-1{beta} alone. Consequently, an analysis of data across experiments did not give an accurate measure of the intrinsic activity ({alpha}) of each prostanoid relative to PGE2. This problem was circumvented in a separate study where all prostanoid agonists were tested at the same time on the same cells at a concentration (10 µM) that approximates the EC100 of each ligand. Indeed, the positions of the log concentration-response curves (using untransformed data within experiments) were parallel to one another, implying that similar asymptotes would be achieved (data not shown). As shown in Fig. 1A, there was no statistical difference in the ability of any of the prostanoids to augment IL-1{beta}-induced G-CSF release (Table 2). Accordingly, all data were normalized, where 0 and 100% represent G-CSF release effected by IL-1{beta} alone and IL-1{beta} in the presence of 10 µM prostanoid agonist, respectively (Fig. 1B). Analyzing the results in this way yielded a rank order of agonist potency of: PGE2 = cicaprost > PGF2{alpha} = U-46619 = PGD2. –Log EC50 (M) values are shown in Table 2.



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  		Fig. 1. Effect of prostanoids on IL-1{beta}-induced granulocyte colony-stimulating factor (G-CSF) release. Adherent human airway smooth muscle (HASM) cells were pretreated for 5 min with PGD2, PGE2, PGF2{alpha}, cicaprost, and U-46619 before being exposed to IL-1{beta} (10 pg/ml). Cells were maintained at 37°C in a thermostatically controlled incubator under a 5% CO2 atmosphere, and the amount of G-CSF released into the culture supernatant was quantified at 24 h by a sandwich ELISA. A and B illustrate the effect of 10 µM of each prostanoid and concentration-response curves, respectively. In the latter case data are expressed as a percentage of the response elicited by 10 µM prostanoid. Each data point represents the mean ± SE of 4–9 determinations (see Table 2) using tissue from different donors. Indomethacin (10 µM) was present in the culture medium throughout the experiment. See MATERIALS AND METHODS for further details.

 

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  		Table 2. Potency and intrinsic activity values of prostanoid and EP-selective agonists at potentiating G-CSF release

 
Effect of fluprostenol on IL-1{beta}-induced G-CSF release. A selective FP receptor agonist, fluprostenol (1 and 10 µM) (1), failed to augment IL-1{beta}-induced G-CSF release from HASM cells (IL-1{beta}: 2.51 ± 0.8 ng/ml, n = 3; + 1 µM fluprostenol: 2.76 ± 0.86 ng/ml, n = 3; + 10 µM fluprostenol, n = 3; 2.79 ± 0.82 ng/ml).

Effect of SQ-29,548 and BW A868C on prostanoid-induced G-CSF release. Pretreatment (30 min) of HASM cells with SQ 29,548 (1 µM) or BW A868C (1 µM), at concentrations that are ~500 and 2,000 times greater than their affinity at TP and DP receptors, respectively (28, 62), failed to antagonize the stimulatory effect on G-CSF secretion of any of the prostanoids studied (Table 3).


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  		Table 3. Effect of a TP- and a DP1-receptor antagonist on the augmentation of IL-1{beta}-stimulated G-CSF release evoked by prostanoid agonists

 
Expression of DP, EP, and IP receptor subtype mRNA. The expression of mRNA transcripts by HASM cells for the DP, EP, and IP receptor subtypes was determined by RT-PCR using primer pairs (Table 1) that recognize unique sequences in the relevant human genes. In these studies generic primers were designed to detect all subtypes of the EP3 receptor. Figure 2 shows ethidium bromide-stained agarose gels of representative experiments; cDNA from human myometrium was used as a positive control for all receptors except EP1 receptor subtype. In three independent determinations the PCR amplified products from HASM cDNA correspond to the predicted size of the DP1 (634 bp), DP2 (CRTH2; GPR44) (593 bp), EP2 (1,056 bp), EP3 (526 bp), EP4 receptor (713 bp), and IP (258 bp) receptor subtypes (Fig. 2). A product corresponding to the predicted size (361 bp) for the EP1 receptor was never detected in any cDNA sample following 36 cycles of amplification (Fig. 2A). As the primers for EP1 receptor were designed not to span an intron, the detection of a product of the correct size (361 bp) obtained from genomic DNA confirms the ability of the PCR to amplify this fragment and suggests that the absence of a band in the cDNA samples (HASM and myometrium) is a genuine reflection of low or nonexistent mRNA expression. In all cases the amplified products were cloned into pGEM-T Easy vectors and sequenced to confirm their identity.



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  		Fig. 2. Qualitative RT-PCR analysis of DP, EP, and IP receptor subtype mRNA. Adherent HASM cells were cultured with 3% vol/vol FCS for 24 h. RNA was extracted, reverse-transcribed into cDNA, amplified using specific primers (Table 1), and subjected to electrophoresis on 1% agarose gels. Shown are ethidium bromide-stained agarose gels representative of 3 experiments using tissue from different donors. Product sizes for DP1-R (A), DP2-R (B), EP1-R (C), EP2-R (D), EP3-R (E), EP4-R (F), and IP-R (G) are 634, 593, 361, 1,065, 520, 713, and 258 bp, respectively. Note: primers for the DP1-R and IP-R subtypes span a very large intron, which is why no product is detected in lane 5. Lane 1, 1-kb ladder molecular-weight markers (GIBCO); lane 2, pool of HASM cDNA; lane 3, negative RT sample; lane 4, cDNA from human myometrial smooth muscle (positive control); lane 5, genomic DNA; lane 6, PCR blank.

 
Effect of PGE2 on IL-1{beta}-induced G-CSF release from diclofenac-treated HASM cells. In 2002 Hirai et al. (33) reported that indomethacin is a potent agonist at the human DP2 receptor subtype (aka CRTH2; GPR44). The detection of DP2 receptor mRNA in HASM cells and the inability of BW A868C to act as an antagonist at this subtype (34) prompted us to determine whether PGE2 would also potentiate G-CSF release in the presence of a diclofenac, a cyclooxygenase (COX) inhibitor that has no agonist activity at the DP2 receptor. In diclofenac (10 µM)-treated HASM cells, PGE2 (10 µM) augmented G-CSF release by 321.3 ± 90.2% (n = 6) over the effect of IL-1{beta} alone, which was not significantly different (P > 0.05) from the augmentation evoked by indomethacin (275.1 ± 45.8%, n = 6) under identical experimental conditions. Moreover, indomethacin (10 µM) did not promote G-CSF in the absence of IL-1{beta} and neither inhibited nor augmented IL-1{beta}-induced G-CSF release in diclofenac-treated HASM cells (IL-1{beta}: 668.2 ± 44.7 pg/ml; IL-1{beta} + indomethacin: 599.5 ± 48.5 pg/ml, n = 3).

Expression of prostanoid receptors at the protein level. Immunocytochemistry was used to determine whether HASM cells expressed the EP2, EP4, and IP receptors at the protein level. In all experiments, receptor-positive HASM cells stained red with the fast red substrate, whereas hematoxylin stained cell nuclei and cytoplasm dark blue and light blue, respectively. As shown in Fig. 3, HASM cells stained positive for the EP2 (Fig. 3A), EP4 (Fig. 3C), and IP receptor (Fig. 3E). There was negligible staining of HASM cells by rabbit Ig control antibodies confirming the specificity of the immunoreactivity (Fig. 3, B, D, and F).



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  		Fig. 3. Identification by immunocytochemistry of EP2, EP4, and IP receptors on HASM cells. Adherent cells were cultured in 3% vol/vol FCS for 24 h, washed in PBS, and fixed in methanol. HASM cells were incubated with either the EP2 polyclonal antiserum (1:200 for 25 min at 4°C, A), the EP4 polyclonal antiserum (1:770 overnight at 4°C, C), or the IP polyclonal antiserum (1:2,500 for 1 h at 4°C, E). Cells that were positive for the relevant receptors were stained red. Cell nuclei and cytoplasm were stained dark blue and light blue, respectively. B, D, and F: cells incubated with rabbit serum only, which represent negative controls. The data are representative of 3 experiments of tissue from different donors.

 
Effect of EP-selective prostanoid agonists on G-CSF release. The role of EP receptors in regulating G-CSF release was determined with a variety of PGE analogs that have different degrees of selectivity for the EP1, EP2, and EP3 receptor subtypes (Table 2). These included 16,16-dimethyl PGE2, 17-phenyl-{omega}-trinor PGE2, butaprost, misoprostol, ONO-AE1-259, and sulprostone. None of the EP agonists had any effect on G-CSF release by themselves, whereas in the presence of IL-1{beta}, G-CSF release was augmented above the effect of IL-1{beta} alone. At the maximally effective concentration, all prostanoids except sulprostone, which enhanced G-CSF release by ~22% (from 2.82 ± 1.26 to 3.44 ± 1.98 ng/ml, n = 3), were apparently full agonists in this system relative to PGE2 (P > 0.05) augmenting G-CSF release by ≥100% (Fig. 4A, Table 2). Experiments involving ONO-AE1-259 were not performed at the same time as the other agonists, and, hence, the results with this prostanoid are graphed separately with PGE2 as the internal control (Fig. 5B). As shown in Fig. 4, there was no statistical difference (P > 0.05, one-way ANOVA) between PGE2, ONO-AE1-259, 16,16-dimethyl PGE2, misoprostol, 17-phenyl-{omega}-trinor PGE2, and butaprost in their ability to augment G-CSF release. Accordingly, the data were normalized where 0 and 100% represent G-CSF release effected by IL-1{beta} alone and IL-1{beta} in the presence of 10 µM prostanoid agonist, respectively. Analyzing the results in this way yielded a rank order of agonist potency of ONO-AE1-259 > 16,16-dimethyl PGE2 = PGE2 > misoprostol > 17-phenyl-{omega}-trinor PGE2 = butaprost (Fig. 4C, Table 2). –Log EC50 (M) values are shown in Table 2.



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  		Fig. 4. Effect of EP-selective prostanoid agonists on IL-1{beta}-induced G-CSF release. Adherent HASM cells were pretreated for 5 min with PGE2 and 6 synthetic PGE analogs before being exposed to IL-1{beta} (10 pg/ml). Cells were maintained at 37°C in a thermostatically controlled incubator under a 5% CO2 atmosphere, and the amount of G-CSF released into the culture supernatant was quantified at 24 h by a sandwich ELISA. A, B, and C: effect of 10 µM of each prostanoid and concentration-response curves, respectively. In the latter case, data are expressed as a percentage of the response elicited by 10 µM prostanoid. Each data point represents the mean ± SE of 3–11 determinations (see Table 2) using tissue from different donors. Indomethacin (10 µM) was present throughout the experiment. See MATERIALS AND METHODS for further details. *Significantly weaker agonist compared with PGE2 (P < 0.05).

 


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  		Fig. 5. Effect of an EP2 and EP4 receptor antagonist on the augmentation of IL-1{beta}-induced G-CSF release evoked by PGE2. Adherent HASM cells were pretreated (30 min) with either AH 6809 (10 µM, A) or L-161,982 (2 µM, B) and then for a further 5 min with PGE2. IL-1{beta} (10 pg/ml) was then added to the cells, and the G-CSF released in to the culture medium was quantified at 24 h by a sandwich ELISA. Each data point represents the mean ± SE of 5 determinations using tissue from different donors. Indomethacin (10 µM) was present throughout the experiment. See MATERIALS AND METHODS for further details.

 
Effect of an EP2 and EP4 receptor antagonist on PGE2-induced G-CSF release. As EP1 receptor mRNA was not detected by HASM cells (Fig. 2) and PGD2 did not positively or negatively regulate G-CSF release (Table 3), AH 6809, which is an antagonist at human EP1, EP2, and DP1 receptors (14, 40, 66), was used as a selective EP2 receptor antagonist. Pretreatment (30 min) of HASM cells with AH 6809 at a concentration (10 µM) that should displace EP2 receptor-mediated response curves ~10-fold to the right did not antagonize the stimulatory effect of PGE2 on IL-1{beta}-induced G-CSF release (Fig. 5A). L-161,982 (2 µM), a potent and highly selective antagonist at the EP4 receptor subtype (48), also failed to block the ability of PGE2 to enhance G-CSF release from HASM cells (Fig. 5B). Confirmation that AH 6809 and L-161,982 were active in these experiments was the respective antagonism of butaprost- and PGE1-OH (selective EP4 receptor agonist)-induced G-CSF release from which corresponding pA2 values of 5.91 and 7.13 were derived (Fig. 6). In the latter case, the affinity was derived assuming the concentration-response curve constructed in the presence of L-161,982 reached the same maximum as that achieved in the absence of antagonist.



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  		Fig. 6. Effect of AH 6809 and L-161,982 on the respective augmentation of IL-1{beta}-induced G-CSF release evoked by butaprost and PGE1-OH. Adherent HASM cells were pretreated (30 min) with either AH 6809 (10 µM, A) or L-161,982 (10 µM, B) and then for a further 5 min with butaprost and PGE1-OH, respectively. IL-1{beta} (10 pg/ml) was then added to the cells, and the G-CSF released in to the culture medium was quantified at 24 h by a sandwich ELISA. Each data point represents the mean ± SE of 3 determinations using tissue from different donors. Indomethacin (10 µM) was present throughout the experiment. See MATERIALS AND METHODS for further details.

 
Effect of concurrent antagonism of EP2 and EP4 receptors on PGE2-induced G-CSF release. Consistent with the data presented in Fig. 6, pretreatment (30 min) of HASM cells with AH 6809 (10 µM) or L-161,982 (2 µM) failed to antagonize the enhancement by PGE2 of G-CSF release (data not shown). In contrast, in AH 6809-treated cells, L-161,982 produced a parallel 9.7 ± 3.5-fold rightward shift of the PGE2 concentration-response curves that described the augmentation of G-CSF release relative to the effect of AH 6809 alone (Fig. 7A). Conversely, the PGE2 concentration-response curve constructed in the presence of L-161,982 was displaced 8.5 ± 4.2-fold to the right when AH 6809 was used concurrently (Fig. 7B). There was no significant difference in the fold displacement between these two experiments (P > 0.05).



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  		Fig. 7. Effect of dual blockade of EP2 and EP4 receptors on the augmentation of IL-1{beta}-induced G-CSF evoked by PGE2. Adherent HASM cells were pretreated (30 min) with AH 6809 (10 µM, A) or L-191,982 (2 µM, B) alone and in combination and then for a further 5 min with PGE2. IL-1{beta} (10 pg/ml) was then added to the cells, and the G-CSF released in to the culture medium was quantified at 24 h by a sandwich ELISA. Each data point represents the mean ± SE of 4 determinations using tissue from different donors. Indomethacin (10 µM) was present throughout the experiment. See MATERIALS AND METHODS for further details.

 
Effect of 8-BrcAMP on G-CSF release. Pretreatment (30 min) of HASM cells with 8-BrcAMP enhanced the release of G-CSF from IL-1{beta} (10 pg/ml)-stimulated HASM cells in a concentration-dependent manner with a –log EC50 of 4.56 ± 0.25 (Fig. 8). At the maximally effective concentration of drug tested (1 mM), 8-BrcAMP potentiated the IL-1{beta}-stimulated response by 261% (from 3.25 ± 0.93 to 8.47 ± 1.22 ng/ml, Fig. 8).



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  		Fig. 8. Effect of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) on IL-1{beta}-induced G-CSF release. Adherent cells were pretreated for 30 min with 8-BrcAMP (10 µM–1 mM) before being exposed to IL-1{beta} (10 pg/ml) for 24 h. The G-CSF released in to the culture medium was quantified at 24 h by a sandwich ELISA. Each data point represents the mean ± SE of 3 independent determinations using tissue from different donors. Indomethacin (10 µM) was present throughout the experiment. See MATERIALS AND METHODS for further details.

 
Role of PKA in the action of PGE2 on G-CSF release. To determine the role of PKA in mediating the effect of PGE2 on G-CSF release from HASM cells, we employed a virus vector, Ad5.CMV.PKI{alpha}, containing DNA encoding the complete amino acid sequence of PKI{alpha}, an endogenous, potent and highly selective inhibitor of PKA (29, 56). In uninfected cells PKI{alpha} was not detected by Western blotting in any experiment. However, 48 h after infection with Ad5.CMV.PKI{alpha} (MOI = 100), a single peptide was labeled by the anti-PKI{alpha} antibody that migrated as a 12-kDa band on SDS polyacrylamide gels (Fig. 9A). Confocal microscopy confirmed that at this time the number of PKI{alpha}+ cells expressed as a percentage of cells counterstaining for the nuclear marker DAPI was >90% (Fig. 9B).



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  		Fig. 9. Expression of cAMP-dependent protein kinase inhibitor (PKI{alpha}) in HASM cells infected with Ad5.CMV.PKI{alpha}. Adherent cells were cultured until 50% confluent and then infected with Ad5.CMV.PKI{alpha} (multiplicity of infection = 100) or left untreated (naïve) for 48 h at 37°C. Cells were growth arrested in serum-free medium and processed for PKI{alpha} expression by Western blotting (A) and confocal microscopy (B). A: lanes 1 and 2, naïve; lanes 3 and 4, Ad5.CMV.Null; lanes 5 and 6, Ad5.CMV.PKI{alpha}. B: 4',6'-diamidino-2-phenylindole stained cell nuclei (blue, a), PKI{alpha} (green, b), and the composite (c), respectively. Data are representative of 3 determinations. See MATERIALS AND METHODS for further details.

 
HASM cells exposed to IL-1{beta} elaborated G-CSF in an amount that was not significantly altered following infection with Ad5.CMV.PKI{alpha} or Ad5.CMV.Null (MOI = 100, 48 h) (Fig. 10, A and B). PGE2 (10 µM) and 8-BrcAMP (1 mM) enhanced IL-1{beta}-stimulated G-CSF release by a mechanism that was prevented in cells expressing the PKI{alpha} transgene but not those infected with the empty virus (Fig. 10, A and B). Moreover, the phosphorylation by 8-BrcAMP and PGE2 of CREB, a well-established substrate for PKA, was also abolished in HASM cells infected with Ad5.CMV.PKI{alpha} (Fig. 10C).



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  		Fig. 10. Effect of PKI{alpha} on 8-BrcAMP- and PGE2-induced G-CSF release and cAMP response element binding protein/activating transcription factor-1 (CREB/ATF-1) phosphorylation in HASM cells. Adherent cells were infected with Ad5.CMV.Null or Ad5.CMV.PKI{alpha} (MOI = 100, 48 h). 8-BrcAMP (1 mM) or PGE2 (10 µM) was then added for 30 min. At this point cells were either exposed to IL-1{beta} (10 pg/ml) for 24 h to promote G-CSF release (A), which was measured by ELISA, or alternatively processed by Western blotting for CREB/ATF-1 phosphorylation and {beta}-actin to confirm equal loading of sample (B and C). Each bar represents the mean ± SE of 4 independent determinations using tissue from different donors. Indomethacin (10 µM) was present throughout the experiment. See MATERIALS AND METHODS for further details. *P < 0.05, significant augmentation in G-CSF release over the effect of IL-1{beta} alone. Lanes 1 and 2, naïve; lanes 3 and 4, Ad5.CMV.Null; lanes 5 and 6, Ad5.CMV.PKI{alpha}.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
We (11) and others (2, 3) have reported previously that PGE2 and other agents that elevate cAMP enhance from HASM cells the elaboration of certain cytokines including G-CSF. The experiments described herein were designed to further this work by characterizing the prostanoid receptor(s) and the biochemical mechanism through which PGE2 mediates this effect. To this end pharmacological and biochemical approaches were adopted using naturally occurring and synthetic prostanoid agonists and antagonists, allied with RT-PCR and immunocytochemistry. PGE2 and cicaprost were the most potent prostanoids at enhancing G-CSF release, which may reflect the activation of an EP and/or IP receptor subtype. Support for this conclusion was that both PGE2 and cicaprost were approximately one order of magnitude more potent than U-46619 (TP agonist), PGD2, and PGF2{alpha} (Table 2). Although HASM cells have functional TP receptors (27) and express DP1 receptor mRNA (this study), these results are consistent with the inability of a TP [SQ 29,548 (62)] and a DP1 receptor antagonist [BW A868C (28)] to block G-CSF output evoked by PGE2, cicaprost, PGD2, PGF2{alpha}, and U-46619. Similarly, fluprostenol, a selective FP receptor agonist (1), did not mimic PGF2{alpha}, which agrees with the restricted distribution of FP-receptor-expressing tissues. Recently, Hirai et al. (33) reported that indomethacin is a potent agonist at the human DP2 receptor subtype (aka CRTH2; GPR44). It was considered possible that since HASM cells express mRNA transcripts for this novel receptor (this study), albeit in a low abundance relative to many other human tissues (51), indomethacin, which was present in every culture to prevent endogenous prostanoid production, may have influenced the ability of PGE2 to augment G-CSF output. However, three separate experimental approaches failed to produce evidence for this idea. First, indomethacin did not promote G-CSF release in the absence of IL-1{beta}. Second, PGE2 augmented G-CSF release to the same extent in cells treated with diclofenac, a COX inhibitor devoid of agonist activity at the DP2 receptor (33), and indomethacin. Third, indomethacin neither inhibited nor enhanced IL-1{beta}-induced G-CSF release in diclofenac-treated cells. Thus, although HASM cells may express functional DP2 receptors, they do not regulate G-CSF release.

Of the IP and EP receptors thus far defined, RT-PCR of total RNA purified from HASM cells unequivocally yielded products that correspond to the predicted sizes and sequences of the human IP, EP2, and EP4 receptor subtypes. These data were corroborated at the protein level by immunocytochemistry and are essentially consistent with the EP receptor expression profile recently published by Burgess and colleagues (9), although in that study the EP4 receptor subtype was not found. The possible involvement of IP or one or more EP receptors in enhancing G-CSF release was investigated using a variety of agonists and antagonists that can discriminate between the EP receptor subtypes. Concentration-response curves constructed to seven EP receptor agonists yielded a rank order of potency of ONO-AE1-259 > 16,16-dimethyl PGE2 = PGE2 > misoprostol > 17-phenyl-{omega}-trinor PGE2 = butaprost > sulprostone. The activity of butaprost and high potency of ONO-AE1-259 is indicative, if not diagnostic, for EP2 receptors (8, 67). Moreover, butaprost and misoprostol were ~20- and 5-fold less potent than PGE2, which is consistent with their e.c.r on established EP2-containing tissues such as the rabbit ear artery, guinea pig ileum circular muscle, and cat trachea (13, 26, 47, 54). However, AH 6809, which can be used as a selective EP2 receptor antagonist in human tissues such as airway smooth muscle, was inactive at a concentration that should have produced a 10-fold rightward shift of the PGE2 concentration-response curve if EP2 receptors were involved. 8-BrcAMP mimicked the effect of PGE2, butaprost, and ONO-AE1-259 on G-CSF output, suggesting the involvement of IP or another EP receptor subtype that is positively coupled to adenylyl cyclase. Currently, selective antagonists at IP receptors have not been described, and so the role of this receptor in mediating the effect of PGE2 and related ligands on G-CSF output remains unclear. Nevertheless, the Ki of PGE2 for the human cloned IP receptor is in excess of 10 µM, which is ~2,000, 30,000, and 12,500 times lower than its affinity at the EP2, EP3, and EP4 receptor subtypes, respectively (8). On the basis of these affinity estimates it seems unlikely that PGE2 acts through IP receptors to enhance G-CSF release.

Of the four known EP receptors, the EP4 and certain splice variants of the EP3 receptor subtypes can also enhance cAMP synthesis (8, 53, 64). Despite the expression of EP3 receptor mRNA (this study) and protein (9) in HASM cells, sulprostone (a selective EP3 receptor agonist) was very weak at enhancing G-CSF secretion. Accordingly, we reasoned that the EP4 receptor subtype was a more likely candidate, and this was evaluated with L-161,982, a highly selective EP4 receptor antagonist (48). However, at a concentration that is reported to be ~500 times greater than its KB [calculated by Schild (60) analysis] for the human EP4 receptor subtype expressed in HEK-293 cells (48), L-161,982 failed to antagonize the enhanced secretion of G-CSF effected by PGE2. Although these data imply that agonism of neither EP2 nor EP4 receptors enhances G-CSF output, they are difficult to reconcile with the behavior of butaprost, ONO-AE1-259, and 8-BrcAMP seen in the present study. In view of these results, the role of EP2 and EP4 receptors was investigated further. Specifically, the effect of blocking the EP2 and EP4 receptors concurrently on the enhancement by PGE2 of G-CSF release in IL-1{beta}-stimulated HASM was studied. A precedent for performing this experiment is derived from several studies including those reported by Fukuroda and colleagues (24, 25). Thus endothelin (ET)-1-induced contractions of human bronchus and rabbit pulmonary artery are insensitive to selective ETA and ETB receptor antagonists, whereas a significant antagonism is seen when the antagonists were used in combination (24, 25). As shown in Fig. 5, the PGE2 concentration-response curve that described the augmentation of G-CSF release from HASM cells was not significantly affected by either the EP2 or EP4 receptor antagonist (AH 6809 and L-161,982, respectively). However, blockade of both receptor subtypes simultaneously significantly displaced the PGE2 concentration-response curve to the right by one order of magnitude. A separate series of experiments established that AH 6809 and L-161,982 blocked the effect of butaprost and PGE1-OH, respectively, confirming that both the EP2 and EP4 receptor populations on HASM cells can, in isolation, positively regulate G-CSF output. Collectively, these data demonstrate that selective antagonism of EP2 or EP4 receptors is insufficient to suppress G-CSF release evoked by PGE2 and that both subtypes need to be blocked to compromise signaling. In terms of receptor theory, these results may indicate that there exist on HASM cells a sufficient EP2 and EP4 receptor reserve at maximum response such that, in the presence of AH 6809 or L-161,982, the antagonist-free prostanoid receptor can still efficiently mediate the full effect of a high-efficacy agonist such as PGE2. However, even in the presence of "spare receptors" for PGE2, a parallel, rightward shift of their concentration-response curves would be expected unless the receptor reserve is so large at maximal response that the antagonism cannot be visualized. An alternative explanation is intramolecular cross talk between the EP2 and EP4 receptor populations. This phenomenon has been demonstrated for endothelin receptor subtypes in Girardi cardiac myocytes where ET-1, acting through the ETA receptor, reduces the affinity of BQ-788, a selective ETB receptor antagonist (57). Whether analogous signaling is elicited by PGE2 in tissues that express multiple EP receptor subtypes is unexplored. However, from an empirical standpoint, intramolecular cross talk could accommodate our data more readily given that the antagonism effected by AH 6809 and L-161,982 in combination was considerably less than would be predicted from their affinity for the human EP2 (pA2 = 6) and EP4 receptor subtypes (pA2 = 8.4) (14, 40, 48, 66).

Agonism of EP2 and EP4 receptors evokes many responses that are thought to rely exclusively on the activation of the cAMP/PKA cascade (64). However, persuasive evidence implicating this signaling pathway is difficult to obtain as many compounds marketed as PKA inhibitors such as H-89 are isoquinoline sulfonamides, which are extremely nonselective (17), presumably because they block a conserved ATP-binding site found among many protein kinases (20). The potential limitations of small-molecule protein kinase inhibitors prompted us to establish a role for PKA in the regulation of G-CSF output by PGE2 by using PKI{alpha}, an endogenous, potent (Ki = 50–100 pM), and highly selective, if not specific, inhibitor of PKA (16, 56, 59). Indeed, PKI{alpha} at a concentration ~106 times higher than its affinity for PKA fails to inhibit the highly homologous cGMP-dependent protein kinase (29) to which it is most closely related (63). To this end HASM cells were infected with an adenovirus vector encoding the complete amino acid sequence of PKI{alpha}. As shown in Fig. 10, CREB/activating transcription factor-1 phosphorylation and the enhancement of G-CSF release evoked by PGE2 and 8-BrcAMP were abolished in HASM cells expressing PKI{alpha}, indicating that the CSF3 gene is positively regulated, directly or indirectly, by PKA. Paradoxically, these results were inconsistent with data obtained with H-89, which failed to block the augmentation by cAMP-elevating drugs of G-CSF release from HASM cells (unpublished observations). H-89 also is reported to have no effect on the secretion of IL-6, GM-CSF, and eotaxin from vascular and HASM cells following agonism of a variety of Gs-coupled receptor (10, 38, 58). Thus, given the remarkable specificity of PKI{alpha} for PKA, these data clearly demonstrate that H-89 is an unsuitable pharmacological tool to assess the role of PKA in biological responses, and it is likely that many historical results obtained with this and other structurally similar compounds are erroneous. Indeed, we and others have recently shown that while H-89 blocks PKA it is completely unselective for this protein kinase (17, 49).

In 2002, evidence was first published that the EP4 receptor can also couple via Gs to the activation of the phosphatidylinositol 3-kinase/PKB{alpha} pathway independently of cAMP and PKA (21–23). However, the participation of this novel signaling pathway seems unlikely given that the enhancement of G-CSF output elicited by PGE2 was abolished by PKI{alpha} (16, 29, 56, 59). Moreover, if EP4 receptors regulate G-CSF secretion in HASM cells by coupling to a non-PKA-dependent mechanism, then PKI{alpha} will only block EP2 receptor signaling. Under that condition, G-CSF release should be preserved (at least in part), as agonism of the EP4 receptor subtype by PGE2 still produces the full functional response in the presence of EP2 receptor blockade. However, as shown in Fig. 10, PGE2-induced G-CSF release was, in fact, abolished in PKI{alpha}-expressing cells.

IL-1{beta} is known to activate multiple MAP kinase signaling cascades (31) and transcription factors (4, 43) in HASM cells, although the critical elements required for G-CSF release and the potentiation of this effect by the cAMP/PKA pathway have not yet been determined. Nevertheless, our data are reminiscent of the ability of cAMP-elevating agents to enhance TNF-{alpha}-induced IL-6 generation from HASM (2). In that investigation, elevation of the cAMP content was associated with an increase in IL-6 mRNA transcripts over that produced by TNF-{alpha} alone (2), indicating that PKA may enhance the expression of this and possibly other genes, such as CSF3, through increased transcription and/or stabilization of existing mRNA transcripts. Indeed, the inability of PGE2 and 8-BrcAMP to promote G-CSF release in the absence of IL-1{beta} is consistent with posttranscriptional mechanisms involving mRNA stabilization.

Cicaprost (61), which is known to elevate cAMP in many tissues, also enhanced G-CSF release from HASM cells, implicating adenylyl cyclase-coupled IP receptors in this response. Although selective antagonists that would confirm this assertion are unavailable, this interpretation is supported by several pieces of evidence. First, IP receptors are expressed by HASM cells (this study). Second, cicaprost is a potent and highly selective IP receptor agonist (8, 19). Third, cicaprost was equipotent with PGE2 in potentiating G-CSF release. Fourth, the affinity of cicaprost at the human EP2 receptor subtype is very weak (8).

In conclusion, PGE2 potentiated G-CSF release from IL-1{beta}-stimulated HASM cells by acting through prostanoid receptors of the EP2 and EP4 subtypes, and this effect was mediated by PKA. Moreover, studies with selective antagonists revealed that concurrent blockade of both receptor subtypes is necessary to antagonize this effect of PGE2.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a Medical Research Council/Aventis collaborative Studentship to D. L. Clarke. M. A. Giembycz and R. Newton are funded by the Canadian Institutes of Health Research.


   ACKNOWLEDGMENTS
 
The authors thank the Harefield Research Foundation and GlaxoSmithKline for financial support and Dr. Mark Birrell for helpful suggestions throughout these studies. The authors acknowledge Merck Frosst (Montreal, Canada) and Ono Pharmaceuticals (Osaka, Japan) for the generous provision of the EP4 receptor antagonist, L-161,982, and EP2 receptor agonist, ONO-AE1-259, respectively. M. A. Giembycz is an Alberta Heritage Senior Medical Scholar.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. Giembycz, Dept. of Pharmacology & Therapeutics, Respiratory Research Group, Univ. of Calgary, 3330 Hospital Dr., NW, Calgary, Alberta, T2N 4N1, Canada (E-mail giembycz{at}ucalgary.ca)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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