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Published ahead of print on August 9, 2007, doi:10.1165/rcmb.2006-0295OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 143-152, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2006-0295OC

8-iso-PGE2 Stimulates Anion Efflux from Airway Epithelial Cells via the EP4 Prostanoid Receptor

Andrew P. Joy1 and Elizabeth A. Cowley1

1 Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada

Correspondence and requests for reprints should be addressed to Dr. Elizabeth A. Cowley, Department of Physiology and Biophysics, Dalhousie University, Halifax, NS, B3H 4H7 Canada. E-mail: elizabeth.cowley{at}dal.ca


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Isoprostanes are biologically active molecules, produced when reactive oxygen species mediate the peroxidation of membrane polyunsaturated fatty acids. Previous work has demonstrated that the isoprostane 8-iso-prostaglandin E2 (PGE2) stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial anion secretion across the human airway epithelial cell line, Calu-3. Since isoprostanes predominantly achieve their effects via binding to prostanoid receptors, we hypothesized that this 8-iso-PGE2 stimulation of CFTR activity was the result of the isoprostane binding to a prostanoid receptor. Using RT-PCR, immunoblotting, and immunofluorescence, we here demonstrate that Calu-3 cells express the EP1-4 and FP receptors, and localize these proteins in polarized cell monolayers. Using iodide efflux as a marker for CFTR-mediated Cl– efflux, we investigate whether prostanoid receptor agonists elicit a functional response from Calu-3 cells. Application of the agonists PGE2, misoprostol (EP2, EP3, and EP4) and PGE1-OH (EP3 and EP4) stimulate iodide efflux; however, iloprost, butaprost, sulprostone, and fluoprostenol (agonists of the EP1, EP2, EP3, and FP receptors, respectively) have no effect. The iodide efflux seen with 8-iso-PGE2 is abolished by the EP4 receptor antagonist AH23848, the CFTR inhibitor 172, and inhibition of PKA and the PI3K pathway. In conclusion, we demonstrate that although Calu-3 cells possess numerous prostanoid receptors, only the EP4 subtype appears capable of eliciting a functional iodide efflux response, which is mediated via the EP4 receptor. We propose that 8-iso-PGE2, acting via EP4 receptor, could play an important role in the CFTR-mediated response to oxidant stress, and which would be compromised in the CF airways.

Key Words: oxidant stress • Calu-3 cells • iodide efflux • isoprostane



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
This article contains important new information that will be of considerable interest to researchers in the cystic fibrosis community, and to scientists concerned with airway physiology and inflammatory lung disease.

 
Oxidative stress plays a central role in the pathogenesis and progression of a number of pulmonary diseases, including asthma (1, 2), chronic obstructive pulmonary disease (COPD; 3) and cystic fibrosis (CF; 4–6). Such oxidative stress arises from exposure to reactive oxygen species (ROS), highly unstable compounds capable of directly oxidizing proteins, DNA, or lipids and consequently initial tissue damage. Isoprostanes are prostaglandin-like compounds, generated when ROS react with the unsaturated bonds of polyunsaturated fatty acids, for example membrane bound arachidonic acid (7–9) or docosahexaenoic acid (10). Isoprostanes have been described as markers of oxidative stress and lipid peroxidation in an enormous number of pathologies; indeed, markedly elevated levels of these compounds have been reported in exhaled breath condensates from patients with asthma (11), COPD (12), and CF (13, 14). Thus isoprostanes appear to be consistent markers of oxidant stress in a variety of inflammatory lung conditions. In addition, we and others (15–19) have reported that isoprostanes elicit a variety of physiologic responses, suggesting that they may act as important mediators of oxidant stress. Within the airways, the Helli and coworkers (20) and Janssen and colleagues (21) have described their effects as regulators of airway and pulmonary vascular smooth muscle tone, hypothesizing their potential important physiological role as candidate endothelium-derived hyperpolarizing and contracting factors (22). Work from our own laboratory has focused on the possible role of the isoprostane 8-iso-prostaglandin E2 (8-iso-PGE2) in mediating a host defense response to combat oxidant stress in airway epithelial cells (15). We previously reported that application of 8-iso-PGE2 to polarized monolayers of the human airway epithelial cell line, the Calu-3 cell, results in an increase in net transepithelial anion secretion, mediated via the co-ordinated increased activity of apically located cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel and basolateral K+ channels (23). Since mutations in CFTR result in CF (23), we proposed that the anion secretory response seen in with 8-iso-PGE2 would be compromised in the CF airways, reducing the effectiveness of host defense mechanisms and potentially exposing the CF lung to extended periods of oxidant stress (15). The aim of the present study was to investigate specifically the molecular mechanisms underlying one aspect of this 8-iso-PGE2–mediated transepithelial response; namely, how 8-iso-PGE2 application results in increased CFTR activity in Calu-3 cells.

There is considerable evidence that isoprostanes achieve their effects via activation of prostanoid receptors (reviewed in Ref. 9). These are G protein–coupled receptors, classified as DP, EP, FP, IP, or TP, for which the agonists with the highest affinity are PGD2, PGE2, PGF2{alpha}, PGI2, and thromboxane A2, respectively (24, 25). Prostanoid receptors are expressed in many tissues throughout the body (24), and mediate the enormous number of cellular functions attributed to these agents. Our previous work clearly demonstrated that Calu-3 cells possess the TP{alpha} isoform of the thromboxane A2 receptor and that application of a TP receptor–specific agonist stimulated a functional response in these cells: namely, an increase in transepithelial anion secretion (15). However, this earlier study also demonstrated that the TP receptor was responsible for mediating only a small amount of the CFTR-mediated anion secretion seen in response to 8-iso-PGE2 application; clearly, another mechanism was responsible for mediating the majority of the isoprostane effect we observed. Recent reports have described the expression of members of the EP receptor subtype in Calu-3 cells (26, 27). Therefore, we hypothesized that the majority of the increased CFTR activity seen in response to 8-iso-PGE2 application in Calu-3 cells was produced via activity of this isoprostane at the FP or an EP receptor subtype. In this article we investigate prostanoid receptor expression and localization in polarized Calu-3 cell monolayers, as well as the role these receptors play in mediating 8-iso-PGE2–stimulated CFTR activity. Parts of this study have been published in abstract form (28, 29).


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Culture
Calu-3 cells (American Type Culture Collection, Rockville, MD) were cultured in 1:1 Dulbecco's modified Eagle's medium:Ham's F-12 nutrient mixture supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin (all Invitrogen, Burlington, ON, Canada). Cells were incubated at 37°C in humidified 5% CO2/95% air. For RNA and protein extraction, cells were cultured on 100-mm-diameter Falcon culture dishes (Becton Dickinson, Franklin Lanes, NJ). For immunohistochemistry, cells were plated on Snapwell inserts (Corning Costar, Cambridge, MA) and maintained at an air–liquid interface with medium present only on the basolateral side, as previously described (30).

RNA Extraction
Total RNA was extracted from Calu-3s cells using TRIzol reagent (Gibco BRL, Burlington, ON, Canada). RNA was then DNase-treated with RQ1 RNase-free DNase (Promega, Madison, WI), and 2 µg DNase-treated RNA was then reverse transcribed using M-MLV reverse transcriptase (Invitrogen) in the presence of 5 mM dNTP (Invitrogen) and 1 µM oligo dT (Amersham Pharmacia, Baie d'Urfe, PQ, Canada) to produce cDNA.

Polymerase Chain Reaction
After reverse transcription, PCR was performed to amplify DNA fragments. All custom primers were obtained from Invitrogen and reactions were performed using primer pairs at 10 µM with 2.5 units Taq polymerase (MBI Fermentas, Burlington, ON, Canada), 25 mM MgCl2, and 5 mM dNTP in a total reaction volume of 25 µl. Reactions were run for 40 cycles using the amplification conditions outlined in Table 1. Primer sequences and conditions were taken from either Timoshenko and coworkers (31) or Schlötzer-Schrehardt and colleagues (32). PCR products were visualized by loading an 8-µl sample on a 1.5% agarose gel containing 250 µg l–1 ethidium bromide, alongside a 100–base pair DNA ladder (Gibco BRL). To confirm the identity of the amplified PCR fragments, the product was isolated from the gel using the QIAquick gel extraction kit (Qiagen, Mississauga, ON, Canada) and sequenced at a commercial sequencing facility (DalGEN Microbial Genomics Centre, Dalhousie University, Halifax, NS, Canada). For each PCR reaction, a negative control in which water was substituted for the cDNA template was also run. Each reaction was run on at least three different cDNA samples extracted from different passages of cells.


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  		TABLE 1. PRIMER SEQUENCES AND FRAGMENT SIZE FOR THE PROSTANOID RECEPTORS DETECTED BY RT-PCR

 
Western Blotting
Cells were grown to confluence before removal with a cell scraper, spun down, and the pellet resuspended in a lysis buffer containing 10% SDS and 15 mg/ml DTT. Complete protease inhibitor (Roche Applied Science, Indianapolis, IN) was added and the mixture sonicated. Total protein (50 µg) was run on a 7.5% polyacrylamide gel and transferred to Immobilon-P membrane (Millipore, Bedford, MA). Immunoblotting for prostanoid receptors (EP1, EP2, EP3, EP4, FP) was performed using affinity-purified rabbit polyclonal anti-human antibodies per manufacturer's instructions at 1:200 dilution (Cayman Chemical, Ann Arbor, MI) followed by incubation with horseradish peroxide–conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) at 1:5,000 dilution. Detection was carried out using the ECL Plus kit, following manufacturer's instructions (Amersham Pharmacia). In addition, immunoblots were also run in the presence of appropriate blocking peptides designed to block protein-antigen complex formation (Cayman Chemical). Each Western blot was performed at least four times, using different passages of Calu-3 cells.

Immunohistochemistry
Calu-3 cells grown on Snapwell filters were submerged in OCT embedding compound and frozen in liquid nitrogen. Sections (10 µm thickness) were fixed in 4% paraformaldehyde (Sigma Aldrich, Oakville, ON, Canada) and permeabilized with 0.3% Triton-X 100 (Sigma Aldrich). Nonspecific binding was prevented with a 1-hour incubation with 10% goat serum (Invitrogen). Sections were incubated with rabbit polyclonal anti-human prostanoid receptor primary antibodies (EP1, EP2, EP3, FP) all at 1:200 dilution in 2% goat serum overnight in a humid chamber with the exception of the anti-EP4 receptor antibody (Sigma Aldrich), which was used at 1:400 dilution. Secondary antibodies were prepared at 1:1,000 dilution by diluting Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) in PBS containing 2% goat serum. Sections were incubated for 1 hour before mounting with Citifluor (Marivac, Montreal, PQ, Canada). Proteins were visualized using a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss Canada, North York, ON, Canada).

For nuclear counterstaining, after incubation with the EP4 receptor antibody, sections were additionally stained with propidium iodide. Here, sections were equilibrated in 2x SSC buffer (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) before incubation with 100 µg/ml RNase A (MBI Fermentas; also in 2x SSC) at 37°C for 20 minutes. Sections were then rinsed three times with 2x SSC buffer and incubated for 3 minutes in propidium iodide (1.0mg/ ml solution in water; Invitrogen). For the EP4 receptor and CFTR co-staining, sections were incubated with anti-EP4 receptor antibody and the anti-CFTR antibodies concurrently, followed by two separate incubations with appropriate secondary antibodies, namely goat anti-rabbit Alexa Fluor 488 (for the EP4 receptor) and goat anti-mouse Alexa Fluor 633 (for CFTR).

A series of immunocytochemistry experiments were also performed to determine that Calu-3 cell monolayers grown on Snapwell inserts were fully polarized under our culture conditions. Specifically, we used CFTR as an apical membrane marker, the Na-K-ATPase as a basolateral membrane marker, and zonula occludens-1 (ZO-1) as a tight junction marker. In addition, propidium iodide was used as a nuclear stain, as described above. Incubations were performed with either an anti-CFTR monoclonal antibody (Chemicon International, Tenecula, CA) at 1:200 dilution, a mouse anti-human zonula occludens-1 monoclonal antibody (ZO-1; Clontech, Mountain View, CA) at 1:200 dilution, or a mouse anti-human Na-K-ATPase monoclonal antibody (Upstate Biotechnology, Lake Placid, NY) at 1:200 dilution. The secondary antibody was Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) at 1:1,000 dilution, with the exception being Alexa Fluor 488 goat anti-mouse IgG (1:1,000 dilution) used for the CFTR staining. Sections were visualized using a Zeiss LSM 510 laser scanning confocal microscope.

Iodide Efflux
Calu-3 cells were cultured to confluence on 35-mm-diameter plates. Cells were loaded with iodide by incubation with 2 ml of the iodide loading buffer (composition in mM: 136 NaI, 3 KNO3, 2 Ca(NO3)2, 11 glucose, and 20 Hepes, pH 7.4) for 1 hour at room temperature. The loading buffer was then removed by rapidly washing the cells three times with iodide efflux buffer (composition in mM: 136 NaNO3, 3 KNO3, 2 Ca(NO3)2, 11 glucose, and 20 Hepes, pH 7.4). Samples were then collected by repeatedly replacing the efflux buffer with fresh solution every 1 minute, and the iodide concentration determined using an iodide-sensitive electrode (Orion Research, Inc., Boston, MA). Pharmacologic agents under investigation were added to the efflux buffer at 3 minutes, and 1.5-ml samples were then collected every minute for a further 12 minutes in the presence of the agent under investigation. For each experiment, a negative control was run which had no agonist added and which represented basal iodide efflux, while the cAMP-elevating agent forskolin was added as a positive control. Stimulated effluxes were measured in triplicate or quadruplicate per experiment, for a total of at least three experiments. Data are represented both as examples of individual iodide efflux experiments (mean ± SEM of the triplicate or quadruplicate samples run during that individual experiment) and also as the cumulative percentage of total iodide efflux released by the cells over the course of the experiment after the pharmacologic agent was applied at 3 minutes (mean ± SEM of at least three different experiments). Here, the percentage iodide efflux is calculated from the cumulative iodide efflux up to time point X divided by the total amount of iodide effluxed from the cells over the course of the experiment. The absolute iodide efflux rate was calculated at the time of peak efflux (2–4 min after application of the agonist) by calculating the slope of percent iodide efflux/time.

For experiments using the CFTR inhibitor 172 (CFTRinh172), wortmannin, and LY294002, these agents were present throughout the 1-hour iodide-loading period, since they required pre-incubation.

Statistical significance was tested by initially performing a one-way ANOVA across likewise groups, followed by post hoc Student's t test to investigate for differences between the control and treated group, with significance determined as P < 0.05.

cAMP Assay
Calu3 cells were grown to confluence and washed twice with PBS before 5 minutes of incubation at 37°C with serum-free media containing the treatment of interest. The agonists 8-iso-PGE2 (1 µM), misoprostol (10 µM), PGE2 (10 µM), and PGE1-OH (1 µM) were all applied to the cells for 5 minutes, while IRS-1 was applied for 20 minutes before aspiration of the medium. Forskolin was used as a positive control. The reaction was then terminated by aspirating the medium and adding 0.5 ml of 0.1 M HCl with 0.5% Triton X 100. The cell lysate was then collected and spun for 2 minutes at greater than 600 G. cAMP levels were then measured by immunoassay using the Direct Cyclic AMP Enzyme Immunoassay kit (Assay Designs, Ann Arbor, MI). Each treatment was run in triplicate from three different samples of cells using two separate kits. Results are expressed as a percentage in relation to the level of intracellular cAMP in control, nontreated, Calu-3 cells.

Chemicals
Prostaglandin E2 (PGE2), 8-iso-PGE2, PGE1-OH, butaprost, sulprostone, iloprost, misoprostol, fluprostenol, and 17-phenyl trinor PGE2 were all from Cayman Chemical. AH23848, forskolin, diphenylamine-2-carboxylate (DPC), CFTRinh172, wortmannin, and the PKA inhibitor fragment 6-22 amide were from Sigma-Aldrich. LY294002 was from Calbiochem (EMD Bioscience, La Jolla, CA). Insulin receptor substrate-1 (IRS-1) was from Biomol (Plymouth Meeting, PA) and was made to a 1 mM stock solution in PBS. Propidium iodide was from Invitrogen. Stock solutions of at least 1,000-fold were made up in either ethanol or dimethyl sulfoxide (for AH23848, forskolin, LY294002, and CFTRinh172) so that the final concentration of solvent added to cells never exceeded 0.1%.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Expression of Prostanoid Receptors in Calu-3 Cells by RT-PCR
We were able to detect fragments of the EP1, EP2, EP3, EP4, and FP receptors by performing RT-PCR on total RNA extracted from Calu-3 cells (Figure 1). All bands were of the predicted size (Table 1), and the DNA was excised, subcloned, and sequenced to confirm identity by comparison with published sequences (National Center for Biotechnology Information, at http://www.ncbi.nlm.nih.gov). Fragments were not detected when water was substituted for the template.


Figure 1
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  		Figure 1. Calu-3 cells express transcripts for a number of prostanoid receptor subtypes. cDNA transcripts were detected for the EP1 (324 bp), EP2 (655 bp), EP3 (398 bp), EP4 (366 bp), and FP receptors (510 bp). M, 100 bp marker; N, negative control (water).

 
Expression of Prostanoid Receptors in Calu-3 Cells by Immunoblotting
We next determined that the EP1, EP2, EP3, EP4, and FP receptor proteins were expressed in total lysate from Calu-3 cells (Figure 2). In all cases, bands were detected at the correct size (EP1 = 42 kD; EP2 = 52 kD; EP3 = 53 kD; EP4 = 65 kD; FP = 64 kD) and bands were blocked when the blot was incubated in the presence of a control peptide supplied by the manufacturer.


Figure 2
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  		Figure 2. Calu-3 cells express protein for a number of prostanoid receptors. Immunoblotting of protein from total cell lysate from Calu-3 cells demonstrated the presence of the EP1 (42 kD), EP2 (52 kD), EP3 (53 kD), EP4 (65 kD), and FP receptors (64 kD). In all cases, bands were detected at the correct size and were blocked when the blot was incubated in the presence of an excess of control peptide (indicated by +P).

 
Localization of Prostanoid Receptors in Calu-3 Cells by Immunofluorescence
Frozen sections of polarized Calu-3 cell monolayers were incubated with the same anti-prostanoid receptor antibodies used above for immunoblotting (Figure 3). In the case of the EP1 receptor (Figure 3A), strong positive staining was detected localized exclusively to the apical membrane, or immediately subapical. A very similar distribution pattern was seen for the EP2 receptor (Figure 3B), with a strict localization to the apical membrane observed. In the case of the EP3 receptor (Figure 3C), positive staining was observed throughout the cell, and in a number of cells, staining did appear more intense closer to the plasma membrane. However, there was no discernable difference between the apical and basolateral membrane staining. The distribution of staining for the FP receptor (Figure 3D) appeared similar to that of the EP3 receptor, with staining again associated with the cell membrane. Strong staining was also seen for the EP4 receptor, which appeared at or toward the cell membrane (Figure 3E). In all cases, experimental controls were run in which the primary antibody was omitted and also when sections were incubated with antibody in the presence of a blocking peptide. No fluorescence was observed in any of these controls (representative example shown in Figure 3F). Co-staining experiments were also performed to examine the expression of the EP4 receptor in relation to the nucleus, using propidium iodide as a nuclear stain (Figure 3G) and in relation to the apical membrane, using the CFTR Cl– channel as an apical marker (Figure 3H). The EP4 receptor appears to not be co-localized with CFTR, with the most intense staining being somewhat beneath the cell membrane.


Figure 3
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  		Figure 3. Cellular localization of prostanoid receptors in Calu-3 cells. Calu-3 cells were cultured on permeable cell culture supports at an air-liquid interface to permit polarization. Positive immunostaining was detected for the EP1 (A), EP2 (B), EP3 (C), FP (D), and EP4 (E) receptors, while no staining was detected when the primary antibody was incubated in the presence of an excess of control peptide (F is a representative example). To further examine EP4 receptor expression, co-staining was performed with the EP4 receptor antibody together with the nuclear stain propidium iodide (G), and the CFTR Cl– channel, which is present only at the apical membrane (H). AP, apical; BL, basolateral. Scale bar = 10 µm.

 
In addition, a series of experiments was also performed to confirm that Calu-3 cell monolayers were polarized using the present culture conditions (Figure 4). Using markers specific for the apical membrane (the CFTR Cl– channel; Figure 4A), the basolateral membrane (the Na+-K+-ATPase, Figure 4B), and the tight junction protein zonula occludens-1 (ZO-1; Figure 4C), these polarization markers all showed characteristic distribution of staining, confirming Calu-3 monolayer polarization under our culture conditions. Negative control experiments were also run in which sections were not incubated with the primary antibody; a representative example (in this case from a section incubated with Alexa Fluor 488 goat anti-rabbit IgG at 1:1,000) is shown in Figure 4D. Sections were also stained with propidium iodide, to demonstrate nuclear staining (Figure 4E).


Figure 4
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  		Figure 4. Expression of polarization markers in Calu-3 cells. After culture at an air–liquid interface, Calu-3 cells form polarized monolayers as determined by staining for the apical membrane marker the CFTR Cl– channel (A), the basolateral membrane marker, the Na-K-ATPase (B), and the tight junction marker ZO-1 (zonula occludens; C). Negative controls were run in which the primary antibody was omitted (representative example in D). Nuclei were also stained with propidium iodide (E). AP, apical; BL, basolateral. Scale bar = 10 µm.

 
Iodide Efflux
To next examine whether the prostanoid receptors we have identified in Calu-3 cells are capable of eliciting a functional response, we investigated the ability of a number of known prostanoid receptor agonists to elicit an iodide efflux response. This response is a standard technique used as a direct measurement of Cl– channel activity, particularly CFTR (33, 34). Therefore, we are able to isolate and investigate in more detail one of the factors, namely enhanced CFTR activity, we previously identified as playing a role in stimulating transepithelial anion secretion in response to 8-iso-PGE2 (15). Since the technique directly measures stimulated anion conductance by imposing a large chemical halide gradient across nonpolarized cells, its major advantage is that it is largely unaffected by factors influencing the electrochemical driving forces across intact epithelia, for example inhibition of K+ channels (34) or the basolateral Na-K-ATPase. Indeed, application of the hIK channel inhibitor clotrimazole, which has a profound effect on the transepithelial secretory response to 8-iso-PGE2 (15), or the Na-K-ATPase inhibitor ouabain, has no effect on the iodide efflux response (results not shown).

Every time an iodide efflux experiment was performed, the cAMP-elevating agent forskolin (at 10 µM) was applied to stimulate CFTR-mediated efflux and confirm the integrity of our cells (n = 17). A representative forskolin response is shown in Figure 5A. Furthermore, to confirm that this stimulated efflux is occurring via CFTR, a series of experiments were performed using the CFTR blocker DPC (0.5 mM; n = 3; Figure 5M), and the more selective CFTR channel inhibitor CFTRinh172 (10 µM), both of which significantly inhibited the forskolin-stimulated response (n = 3; Figures 5B, 5C, and 5M, P < 0.05). Finally, since CFTR activity is PKA dependent (35), we investigated the effects of the PKA inhibitor (6–22 amide, 10 µM) on the forskolin-stimulated iodide efflux. In the presence of the PKA inhibitor, the rate of forskolin-stimulated efflux was significantly reduced (efflux rate = 5.39 ± 0.58, n = 3 with PKA inhibitor versus 7.80 ± 0.24 forskolin alone; n = 17; P < 0.05, results not shown).


Figure 5
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  		Figure 5. Prostaglandin receptor agonists stimulate iodide efflux in Calu-3 cells via the EP4 receptor. For all iodide efflux experiments, a positive stimulation was induced with the cAMP elevating agent forskolin. A shows a representative trace of the iodide efflux seen when forskolin (10 µM) is applied at 3 minutes after loading of cells with NaI for 1 hour (n = 17), while B shows that this forskolin response is essentially abolished in the presence of the CFTR inhibitor 172 (10 µM; 60 minutes preincubation, n = 3). C compares the cumulative iodide efflux from Calu-3 cells in the presence of forskolin alone and in the presence of CFTR inhibitor 172, the higher curve reflecting increased iodide efflux due to CFTR activity. Iodide efflux (%) is calculated from the cumulative iodide efflux up to time point X/total amount of iodide effluxed from the cells over the course of the experiment. Application of PGE2 (10 µM; n = 3) stimulates an iodide efflux response (E and F) when compared with the basal, nonstimulated iodide efflux (D and F). Misoprostol (10 µM; n = 5), which can act at the EP2, EP3, or EP4 receptors, similarly stimulated iodide efflux (G and I), which was inhibited by the EP4 antagonist AH23848 (10 µM; n = 3; H and I). Application of a number of prostanoid receptor agonists, for example the EP2 receptor agonist butaprost shown in J, failed to elicit any iodide efflux response and produced iodide efflux traces identical to basal efflux. Finally, application of the EP4 receptor agonist PGE1-OH (1 µM) also stimulated efflux (K and L). Arrow indicates application of drug. The absolute iodide efflux rates were calculated at the time of peak efflux (2–4 min after application of the agonist) by calculating the slope of percent iodide efflux/time and are shown collectively in M. An ANOVA was performed, and the asterisk represents a significant difference in the initial rate of iodide efflux between two treatments as determined by a subsequent unpaired Student's t test (P < 0.05).

 
An example of a basal, nonstimulated efflux is shown in Figure 5D and the absolute iodide efflux rate (RI) shown in Figure 5M (n = 14). Application of PGE2 (10 µM; n = 3) produced a stimulation of iodide efflux (Figures 5E and 5M) compared with the basal efflux alone (n = 14; Figure 5F). This confirms that at least one functional EP receptor subtype was present. Misoprostol (10 µM), a nonselective EP receptor agonist that can act at EP2, EP3, or EP4 receptors, also stimulated an iodide efflux response (Figures 5G, 5I, and 5M; n = 5). This misoprostol response was significantly abolished in the presence of the EP4 receptor antagonist AH23848 (Figures 5H, 5I, and 5M; n = 3, P < 0.05) implicating the EP4 receptor in the stimulatory response to misoprostol. Application of the more selective EP1, EP2, and EP3 receptor agonists iloprost, butaprost, and sulprostone, respectively (all at 10 µM), 17-phenyl trinor PGE2 (a synthetic PGE2 analogue which acts at the EP1 and EP3 receptors at 10 µM), and also the FP receptor agonist fluprostenol (10 µM) all failed to elicit an iodide efflux response from Calu-3 cells. A representative example is shown in Figure 5J, which shows that the application of butaprost (10 µM; n = 3) failed to stimulate an iodide efflux response. Finally, we investigated the effects of PGE1-OH (1 µM), often described as an EP4 receptor agonist (36, 37), though it does also bind to the EP3 receptor at lower affinity. This agent induced an iodide efflux response (Figures 5K, 5L, and 5M; n = 3), indicating that EP4 receptors are functionally active in Calu-3 cells. This PGE1-OH response was not inhibited by the presence of AH6809 (15 µM) an EP1, EP2, and EP3 receptor antagonist (Figure 5M; n = 3).

Having demonstrated the presence of functionally active EP4 receptors, we next wished to directly investigate whether the increased CFTR activity seen previously in response to 8-iso-PGE2 (15) could be the result of stimulation of the EP4 receptor subtype. Application of 8-iso-PGE2 at either 1 µM (Figures 6A, 6C, and 6J, n = 6) or 10 µM (Figure 6J, n = 3) elicited an iodide efflux response, significantly reduced in the presence of CFTRinh172 (10 µM; Figures 6B, 6C, and 6J; n = 3, P < 0.05), confirming that efflux is occurring via CFTR. This response was also significantly inhibited in the presence of the PKA inhibitor (Figure 6J) as would be predicted for any CFTR-mediated event. When 8-iso-PGE2 (1 µM) was applied in the presence of the selective EP4 receptor antagonist AH23848 (at 10 µM), the efflux response to the isoprostane was eliminated (Figures 6D, 6E, and 6J; n = 3, P < 0.05) confirming that the 8-iso-PGE2 response is mediated via the EP4 receptor in Calu-3 cells. In contrast, the response to 8-iso-PGE2 was not abolished in the presence of AH6809, an EP1, EP2, and EP3 receptor antagonist (15 µM), suggesting these receptor subtypes are not mediating the response (Figures 6F,6G, and 6J). Finally, since activation of the EP4 receptor has been associated with activation of the phophatidylinositol-3-kinase (PI3K) pathway (25), we preincubated cells for 60 minutes in the presence of the PI3K inhibitors wortmannin (at 1 µM) or LY290042 (10 µM) before application of 8-iso-PGE2 (1 µM). The presence of LY290042 significantly reduced the iodide efflux response to 8-iso-PGE2 (Figures 6H, 6I, and 6J; n = 3), as did wortmannin (Figure 6J; n = 3; P < 0.05). As an additional control to confirm that these agents were not targeting CFTR directly, we also examined the response to forskolin in the presence of both LY290042 and wortmannin. Neither agent affected the iodide efflux response forskolin (Figure 6J; n = 3 for both LY2009 and wortmannin and n = 17 for forskolin alone), strongly suggesting that the PI3K signaling pathway is somehow involved in mediating the response to 8-iso-PGE2.


Figure 6
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  		Figure 6. The 8-iso-PGE2 response in Calu-3 cells is inhibited by EP4 receptor inhibition. Application of 8-iso-PGE2 at 1 µM (A) stimulates iodide efflux from Calu-3 cells. This stimulation (1 µM) is inhibited in the presence of both the CFTR inhibitor 172 (B and C) and the EP4 receptor antagonist AH23848 (D and E). However, the presence of the EP1, EP2, and EP3 receptor antagonist AH6809 did not abolished the response (F), particularly apparent in the cumulative efflux trace (G), which shows that the 8-iso-PGE2 response in the presence of AH6809 was identical to that of 8-iso-PGE2 alone. Finally, when 8-iso-PGE2 (1 µM) was applied after pre-incubation with the PI3K inhibitor LY290042 for 60 minutes, the stimulated iodide efflux response was abolished (H and I). Arrow indicates application of drug. J shows the initial rate of efflux for the various treatments. An ANOVA was performed, and the asterisk represents a significant difference in the initial rate of iodide efflux between two treatments as determined by a subsequent unpaired Student's t test (P < 0.05).

 
Application of DMSO alone produced no iodide efflux from Calu-3 cells, while the response to 8-iso-PGE2 was unaffected by pre-incubation with DMSO alone (results not shown). Finally, when the EP4 receptor antagonist AH23848 (10 µM) was applied to Calu-3 cells at the same time as the positive control agent forskolin, it had no effect on the stimulated iodide efflux response, confirming that the effect of AH23848 is not occurring via a nonspecific inhibitory effect on CFTR (RI = 7.27 ± 0.55; n = 3; results not shown).

cAMP Assays
Since CFTR is a PKA-dependent channel (33), we wished to investigate whether application of 8-iso-PGE2 to Calu-3 cells resulted in an increase in intracellular cAMP levels, which would stimulate CFTR activity. In addition, since our iodide efflux results strongly implicate that inhibition of the PI3K pathway abolishes response to 8-iso-PGE2, we additionally wished to investigate whether a PI3K activator could affect cytosolic cAMP levels. For this experiment we utilized IRS-1. When IRS-1 undergoes tyrosine phosphorylation it binds to the SH2-containing p85 regulatory subunit of PI3K, thus activating the enzyme (38, 39). While the positive control forskolin produced a large increase in cAMP (3,357.8 ± 887.6%), none of the agonists of interest produced an increase in intracellular cAMP (8-iso-PGE2 = 127.4 ± 15.7%; misoprostol = 106.7 ± 15.1%; PGE2 = 102.2 ± 9.2%; PGE1-OH- 239.4 ± 46.6%; IRS-1 = 120.6 ± 28.9%; all n = 6).


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Previous work from our own laboratory has demonstrated that application of the isoprostane 8-iso-PGE2 results in an increased transepithelial anion secretion across polarized Calu-3 cells (15). This response consisted of increased activity of both apical CFTR Cl– channels and basolateral K+ channels, which together resulted in the enhanced anion secretion (15). The aim of the presence study was to investigate the molecular mechanisms connecting 8-iso-PGE2 application to enhanced CFTR activity, in isolation from other confounding factors that would influence transepithelial secretion. To do this, we have used iodide efflux as a marker of CFTR activity, since it enables us to directly examine enhanced anion conductance in response to 8-iso-PGE2. Furthermore, since isoprostanes are believed to exert their biological effects via binding to the series of G protein–coupled receptors that make up the prostanoid receptor family (9) and our earlier work had demonstrated that the predominant effect of 8-iso-PGE2 was not mediated via the TP receptor (15), we were particularly interested in whether a member of the EP receptor family, or the FP receptor, was involved in mediating the increased CFTR activity seen in response to this isoprostane.

Our finding that Calu-3 cells express all four EP receptor subtypes is in contrast to the recent work of two other groups. Using PCR, the study of Palmer and coworkers (27) described the presence of the EP2 and EP4 receptors in Calu-3 cells, but these authors were unable to detect EP1 or EP3. Different results again were described by Clayton and colleagues (26), who found that Calu-3 cells express the EP4, but not the EP2 subtype, while EP1 and EP3 expression was not investigated. We were therefore initially surprised that were we able to detect mRNA transcripts for all four EP receptor subtypes in Calu-3 cells (Figure 1). However, all positive PCR fragments were excised, sequenced, and their identity confirmed by comparison with the National Center for Biotechnology Information database. Unlike the above studies, we used primers taken from published work, which described positive expression for all four EP receptor subtypes in human breast cancer epithelial cells (31). Therefore we are confident that our positive PCR findings do indeed reflect that, at least in our hands, Calu-3 cells express mRNA for the EP1, EP2, EP3, and EP4, as well as FP, prostanoid receptors.

The additional protein work presented here further confirms that Calu-3 cell possess a full complement of EP receptor subtypes, as well as the FP receptor (Figure 2). While RT-PCR demonstrated mRNA expression, the earlier studies of Clayton and coworkers (26) and Palmer and colleagues (27) do not demonstrate protein expression. In all cases in our study, the band detected was of the correct, predicted size; the band was eliminated when the Western blot was performed in the presence of a peptide designed to block protein–antigen complex formation; and protein BLAST searches performed on the peptide antigen revealed no cross-specificity with other peptides (National Center for Biotechnology Information database). Therefore, these experiments represent strong evidence that all the prostanoid receptors investigated are present at the protein level in Calu-3 cells.

Our immunolocalization experiments on Calu-3 monolayers further strengthen the above protein findings. The EP1 and EP2 receptors both exhibited a very similar distribution pattern, with immunostaining strictly limited to the apical, or immediately subapical, compartment. Activation of the EP1 receptor results in increases in phosphatidyl inositol turnover and an increase in intracellular Ca2+ (24, 40), while activation of the EP2 receptor results in increases in cAMP via activation of adenylate cyclase (24). Given this apical distribution pattern, and the possibility that these receptors are co-localized with the CFTR Cl– channel (Figure 3), we were optimistic that activation of one of these receptors would produce a functional response from Calu-3 cells. In particular, we hypothesized that activation of the EP2 subtype would lead to increased cAMP and subsequent activation of CFTR, known to be a PKA-dependent channel (35). However, this was not the case and application of the specific EP1 and EP2 agonists iloprost and butaprost to Calu-3 cells failed to produce a stimulation of iodide efflux, reflecting increased CFTR activity.

Immunolocalization experiments also demonstrated a faint and diffuse pattern of staining throughout the entire cell for the EP3 and FP receptor types, though again, stimulation with the selective agonists sulprostane and fluprostenol did not elicit an iodide efflux response. Therefore, while our data does demonstrate the presence of EP1, EP2, EP3, and FP receptor proteins in Calu-3 cells, even suggesting a distinctly polarized distribution for the apical EP1 and EP2, the functional significance of these receptors is not apparent from our studies.

We additionally confirm the findings of earlier studies (26, 27) that EP4 receptors are present in Calu-3 cells, but expand this observation by demonstrating their subcellular localization. Furthermore, of all the prostanoid receptors we detected, activation of the EP4 receptor is unique in that it elicits a functional iodide efflux response.

The Calu-3 cell line is a widely used model of the human submucosal gland serous cell, since it retains several aspects of the native tissue (41). Basbaum and coworkers (42) described the ability of prostaglandins, most significantly PGE1 and PGE2, to mediate glycoprotein release from human serous cells, indicating the presence of at least one prostaglandin receptor subtype on serous cells. The physiologic significance of prostaglandin receptors on serous cells is unclear, but likely relates to the control of secretion when inflammatory mediators are generated. Therefore, the presence of EP4 receptors on Calu-3 cells may provide important insights into how serous cells respond to the presence of inflammatory products in vivo. Exposure to the pro-inflammatory cytokine IL-1β down-regulates EP4 receptor expression in Calu-3 cells, resulting in a net decrease in Cl– efflux in response to PGE2, while concurrently increasing β2-adrenergic receptors, permitting an increased Cl– efflux to isoprenaline (26). Therefore, in vivo, the presence of inflammatory mediators may influence EP4 receptor expression and consequent activity, permitting differential CFTR-mediated Cl– secretory responses to occur.

Importantly, we provide evidence here that the isoprostane 8-iso-PGE2 is mediating its effects via the EP4 receptor in Calu-3 cells, since the EP4 receptor antagonist AH23848 effectively abolished the iodide efflux response to 8-iso-PGE2. Although AH23848 has some activity blocking the TP receptor (43), our earlier work demonstrated that the majority of the increased transepithelial anion secretion in response to 8-iso-PGE2 was not occurring via the TP receptor (15); therefore, we believe the inhibitory effects of AH23848 shown in Figures 6D and 6E represent an EP4 receptor–mediated phenomenon. The ability of 8-iso-PGE2 to increase CFTR activity in Calu-3 cells led us earlier to propose a potential role for this molecule in airways host defense, which would be impacted in CF, since this enhanced CFTR activity would be lost (15). Here, we further delineate its mechanism of action, by implicating the EP4 receptor as the molecular target coupled to increased CFTR activity. This suggests the possibility that 8-iso-PGE2 generated by oxidant stress in vivo could be a physiologically relevant agonist of this receptor.

Our immunolocalization experiments place the EP4 receptor at, or near, the cell membrane (Figure 3E); therefore, EP4 receptors could be responding to 8-iso-PGE2 generated locally in response to oxidant stressors. In terms of its signal transduction mechanism, activation of the EP4 receptor has been linked to a moderate increase in intracellular cAMP levels (44), which could directly activate CFTR and result in the increased activity we see. However, our direct measurements of intracellular cAMP in Calu-3 cells after exposure to 8-iso-PGE2 failed to detect any stimulation of cAMP production. Similarly, none of the agonists that we believe are working via the EP4 receptor produced a detectable increase in intracellular cAMP. Therefore, the precise mechanism by which EP4 receptor activation by 8-iso-PGE2 results in increased CFTR activity is not immediately apparent. One possibility is there is a small increase in cAMP in response to EP4 receptor activation in Calu-3 cells, but that it is below the limits of sensitivity of the assay system we used. Alternatively, the generation of cAMP may be extremely localized to an EP4 receptor–CFTR complex, making it undetectable in as a global increase throughout the cell, but being sufficient to activate CFTR.

EP4 receptor activation has additionally been associated with activation of the phophatidylinositol-3-kinase (PI3K) pathway (44, 45), and subsequent phosphorylation of the extracellular signal–regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK; 44). We found that the iodide efflux response to 8-iso-PGE2 was reduced by the PI3K inhibitor wortmannin and the more specific PI3K inhibitor LY290042, suggesting that we are looking at a PI3K-coupled event at some level. This suggestion is not unprecedented: a investigation of the ability of the β3-adrenoceptor to activate CFTR revealed that channel activity was blocked by inhibition of either PI3K or ERK1/2 MAPK (46). These authors concluded that CFTR activity can also be regulated via a Gi/o/PI3K/ERK1/2 MAPK pathway, entirely independent of the cAMP-dependent phosphorylation of CFTR more generally associated with its physiologic regulation (46). One possibility presented by our results is that the EP4 receptor could potentially be acting in a similar manner, raising the possibility that this is a more widespread regulatory mechanism that has previously been appreciated.

We previously reported that the apical Cl– efflux seen in response to 8-iso-PGE2 application was inhibited in the presence of a PKA inhibitor (15), which would be expected to inhibit CFTR activity. Similarly in the present study, inhibition of PKA also inhibited 8-iso-PGE2–stimulated iodide efflux (Figure 6J). However, we here also describe the presence of basolaterally located receptors, which would be more responsive to systemic oxidant stress, but are more distant to CFTR and are unlikely to be directly coupled. However, activation of basolaterally located G protein–coupled receptors, for example the vasoactive intestinal polypeptide receptor VPAC1, is known to induce CFTR-dependent Cl– secretion in polarized Calu-3 cells (47). In intact epithelia in vivo, basolaterally located receptors could lead to activation of basolateral K+ channels, since increased K+ efflux hyperpolarizes the cell, thereby enhancing the driving force for anion exit via CFTR. Indeed, our earlier report (15) showed that both apical and basolateral application of 8-iso-PGE2 stimulated anion secretion across intact Calu-3 monolayers, which is consistent with the presence of the EP4 receptor in both membranes. Stimulation of basolateral K+ channels by 8-iso-PGE2 would have an overall pro-secretory effect, since increasing K+ efflux would hyperpolarize cells and increase the driving force for anions exit through apical located CFTR (15, 30). Therefore, activation of EP4 receptors at either the apical or basolateral membrane could be predicted to be strongly pro-secretory across polarized, intact Calu-3 monolayers. While previous work has indicated that changes in electrical driving forces have some effect on the rate of iodide efflux from airway cells (48), efflux is predominantly dependent on the chemical gradient for iodide and is, therefore, less dependent on K+ channel activity than anion efflux from intact epithelia. Furthermore, the results of the iodide efflux experiments were obtained from nonpolarized cells. However, in both the present study and our previous work using polarized epithelial cells (15), application of 8-iso-PGE2 results in an increase in CFTR-mediated anion conductance. Since the EP4 receptor appears to have no specific apical or basolateral distribution in polarized monolayers (Figure 3), we are confident that results from the present study have physiologic relevance.

EP4 receptor activation has also been associated with subsequent induction of the zinc finger transcription factor ERG-1 (early growth factor-1; 44), an early gene product implicated in regulating the expression of numerous downstream genes, including several proinflammatory mediators (49). Since the CF airways are the site of repeated neutrophil-dominated inflammatory responses and the consequent release of ROS, longer-term activation of the EP4 receptor in the CF airways could potentially result in pathologically significant changes in the transcription of numerous pro-inflammatory mediators, which would be disadvantageous to their host defense.

The physiologic effect of increased CFTR activity as a consequence of EP4 receptor stimulation would be to increase glandular fluid secretion and enhance fluid movement. In addition to producing the majority of glandular secretions, serous cells have been implicated strongly in the pathogenesis of CF lung disease (41) since they possess a large amount of the CFTR Cl– channel, mutations which result in CF. Thus we believe it is possible that this response represents a host defense mechanism on the part of the airway epithelial serous cells to remove the oxidant stressors producing the 8-iso-PGE2 and which would be absent in the CF airways.

In conclusion, we demonstrate that Calu-3 cells demonstrate EP4 receptors capable of mediating a Cl– efflux response; we further demonstrate that the isoprostane, 8-iso-PGE2, stimulates an increased anion conductance mediated via CFTR. This suggests a novel role for the EP4 receptor in the airways. We propose that when the airway epithelium is exposed to ROS, generated 8-iso-PGE2 could activate the EP4 receptor and result in increased CFTR activity. In intact epithelia, this activity would enhance anion and fluid movement. Since activation of the EP4 receptor would be unable to stimulate Cl– conductance in CF airways, these results have significant implications for the pathogenesis of CF lung disease.


   Acknowledgments
 
The authors thank Brenna Vantol and Kellie Davis for technical assistance, and they are indebted to Dr. Valerie Chappe for extensive help with establishing the iodide efflux protocol.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research, the Nova Scotia Health Research Foundation, the Canadian Cystic Fibrosis Foundation, and the Nova Scotia Lung Association. A.P.J. was supported by an NSERC studentship.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0295OC on August 9, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 10, 2006

Accepted in final form August 1, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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