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Research ArticleArticle

Evidence for a Second Receptor for Prostacyclin on Human Airway Epithelial Cells That Mediates Inhibition of CXCL9 and CXCL10 Release

Sylvia M. Wilson, Neil A. Sheddan, Robert Newton and Mark A. Giembycz
Molecular Pharmacology March 2011, 79 (3) 586-595; DOI: https://doi.org/10.1124/mol.110.069674

Abstract

Herein we provide evidence for the coexpression of two distinct prostacyclin (PGI2) receptors (IP) on BEAS-2B human airway epithelial cells. IP receptor heterogeneity initially was suggested by the finding that the rank orders of potency of PGI2 and three structurally similar analogs [taprostene, iloprost, 15-deoxy-16-(m-tolyl)-17,18,19,20-tetranorisocarbacyclin (15-deoxy-TIC)] for the inhibition of chemokine (CXCL9 and CXCL10) release and for transcriptional activation/augmentation of cAMP response element and glucocorticoid response element luciferase reporters were distinct. Indeed, PGI2, taprostene, and iloprost activated both reporters whereas 15-deoxy-TIC was inert. Conversely, 15-deoxy-TIC, PGI2, and taprostene (but not iloprost) suppressed chemokine release. Further experiments established that iloprost did not antagonize the inhibitory effect taprostene or 15-deoxy-TIC on chemokine output. Likewise, 15-deoxy-TIC failed to antagonize taprostene- and iloprost-induced reporter transactivation. Thus, iloprost- and 15-deoxy-TIC-induced responses were apparently mediated via pharmacologically distinct receptors. In human embryonic kidney 293 cells overexpressing the human recombinant IP receptor receptor, 15-deoxy-TIC was considerably less potent (>10,000-fold) than iloprost and taprostene in promoting cAMP accumulation, yet in BEAS-2B cells, these analogs were equipotent. IP receptor heterogeneity was also supported by the finding that the affinity of the IP receptor antagonist R-3-(4-fluorophenyl)-2-[5-(4-fluorophenyl)-benzofuran-2-yl-methoxycarbonyl-amino] propionic acid (RO3244794) for the receptor mediating inhibition of chemokine release was approximately 10-fold lower than for the receptor mediating both transcriptional outputs. Finally, small interfering RNAs directed against the IP receptor gene, PTGIR, failed to block the suppression of chemokine output induced by taprostene and 15-deoxy-TIC, whereas taprostene- and iloprost-induced transcriptional responses were markedly attenuated. Collectively, these results indicate that PGI2, taprostene and 15-deoxy-TIC suppress chemokine release from BEAS-2B cells by interacting with a novel IP receptor that we denote here as the “IP2” subtype.

Introduction

Prostacyclin (PGI2) receptor (IP) heterogeneity is controversial. To date, cloning studies have not provided any substantive data indicative of IP receptor subtypes. Likewise, the results of pharmacological investigations are inconclusive because of the paucity of selective ligands that can be used explicitly to classify responses mediated by IP receptors (Wise and Jones, 2000). Interpreting data across species may also be problematic. Indeed, the atypically low sequence homology between IP receptors [the human variant is only 73% identical at the amino acid level to the murine ortholog (Katsuyama et al., 1994)] could fundamentally alter the pharmacological behavior of synthetic IP receptor agonists and antagonists and, potentially, create the false impression of receptor multiplicity.

Nevertheless, several studies conducted in the same species have provided evidence for multiple IP receptors. Using the IP receptor agonists cicaprost and 2-[3-[4-[4,5-di(phenyl)-1,3-oxazol-2-yl]-1,3-oxazol-5-yl]phenoxy]acetic acid (BMY 45778) Wise et al. (1995) discriminated extraneuronal from neuronal IP receptor in the rat using neutrophils and enteric nerves in the colon as representative tissues, respectively. In another study, Hébert et al. (1995) proposed the existence of three IP receptors to account for the complex behavior of carbacyclin and iloprost at inhibiting vasopressin-induced water conductivity in rabbit cortical collecting ducts. Unfortunately, the results of these studies are difficult to interpret unambiguously. For example, the variable sensitivity of the rat neutrophil and colon to BMY 45578 (Wise et al., 1995) could be a function of agonist efficacy (Wise and Jones, 2000). Thus, cicaprost could behave as a full agonist in both tissues, whereas BMY 45778 may only have sufficient efficacy in the colon to act as a partial agonist. With respect to the IP receptor subtypes proposed by Hébert et al., (1995), there is evidence that the murine IP receptor can couple to several G-proteins and, hence, multiple second messenger-generating enzymes (Namba et al., 1994). If the human ortholog is similarly promiscuous, the need to invoke multiple IP receptors is negated.

IP receptor subtypes in the rodent CNS have also been proposed. Using the radiolabeled IP receptor agonists [3H]isocarbacyclin and [3H]iloprost, Takechi et al. (1996) identified two, apparently distinct, binding sites in rat brain. Specifically, [3H]isocarbacyclin bound to a site in the thalamus and nucleus tractus solitarius (NTS) with similar Kd values. In contrast, [3H]iloprost discriminated between these brain regions, having 23-fold higher affinity for sites in the NTS relative to the thalamus. Based on these data, it was concluded that the high-affinity and relatively low-affinity [3H]iloprost binding sites identified in the NTS and thalamus, respectively, represented different IP receptor subtypes (Takechi et al., 1996). Additional evidence for this taxonomy is that the 15R epimer of 16-m-tolyl-17,18,19,20-tetranor isocarbacyclin (15R-TIC) (Suzuki et al., 1996) and the achiral 15-deoxy derivative (15-deoxy-TIC; Fig. 1) were significantly more potent at binding to the site in the thalamus compared with the NTS (Takechi et al., 1996; Suzuki et al., 1999; Watanabe et al., 1999). In considering the implications of these data, at least four issues arise that are potential causes for concern. First, the assignment of the two binding sites in the CNS as distinct IP subtypes is critically dependent upon the exclusive interaction of [3H]isocarbacyclin with the IP receptor. However, [3H]isocarbacyclin has affinity for the EP2 and EP3 receptor subtypes (Dong et al., 1986; Kiriyama et al., 1997) and so this assumption may be flawed. Second, iloprost has appreciable affinity at EP1 and EP4 subtypes (Wilson et al., 2004; Wilson and Giles, 2005; Jones et al., 2009). Thus, the radioligand binding data may not reflect an exclusive interaction of iloprost with IP receptors. Third, the selectivity of 15R-TIC and 15-deoxy-TIC for the known prostanoid receptors is undefined. Fourth, the activity of naturally occurring prostanoids at their cognate receptors requires a hydroxyl-bearing chiral center at position C15 that, typically, is in the S configuration (Fig. 1; Monneret et al., 2003). The potent activity of 15R-TIC and 15-deoxy-TIC, therefore, is difficult to explain unless 1) the 15S-configuration is not critical for IP receptor agonism [as has been shown for DP2 receptor activation (Monneret et al., 2003)] or 2) [3H]isocarbacyclin and [3H]iloprost label a site in the CNS that is distinct from known prostanoid receptors. Thus, IP receptor heterogeneity is neither established nor disproved.

Fig. 1.
Fig. 1.

Chemical structures of PGI2, iloprost, taprostene, and 15-deoxy-TIC. *, chiral center.

In the present study, using a panel of IP receptor agonists (PGI2, iloprost, taprostene, 15-deoxy-TIC; Fig. 1) and the surmountable, competitive, and selective IP receptor antagonist R-3-(4-fluorophenyl)-2-[5-(4-fluorophenyl)-benzofuran-2-yl-methoxycarbonyl-amino] propionic acid (RO3244794) (Bley et al., 2006), we now provide clear functional evidence for the coexpression of two IP receptor subtypes on the human airway epithelial cell line, BEAS-2B, that mediate distinct functional responses.

Materials and Methods

Reagents.

15-Deoxy-TIC (15-deoxy-16-(m-tolyl)-17,18,19,20-tetranorisocarbacyclin) was synthesized using a novel ω-side chain addition strategy as detailed previously (Sheddan and Mulzer, 2005; Sheddan et al., 2007). 3-Benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-ydroxyethylamino) hydantoin (BWA A868C), PGE2, and iloprost were purchased from Cayman Chemicals (Ann Arbor, MI). (Z)-7-[(1R,2S,3R,5R)-5-Chloro-3-hydroxy-2-[(E,4S)-4-hydroxy-4-(1-prop-2-enylcyclobutyl)but-1-enyl]cyclopentyl]hept-5-anoic acid (ONO-AE1-259) and RO3244794 were provided by ONO Pharmaceuticals (Osaka, Japan) and Roche (Palo Alto, CA) respectively. 5-Bromo-2-methoxy-N-[3-(2-naphthalen-2-yl-methylphenyl)acryloyl]-benzene sulfonamide (L-798,106) and 5-butyl-2,4-dihydro-[[2′-[N-(5-methyl-2-thiophene-carbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-[(2-trifluoromethyl) phenyl]-1,2,4-triazol-3-one (L-161,982) were donated by Merck Frosst (Montreal, QC, Canada). All other reagents were from Sigma/Aldrich (Oakville, ON, Canada).

CRE and GRE Luciferase Reporters.

Stable transfection was used to generate CRE and GRE BEAS-2B reporter cell lines (Chivers et al., 2004; Meja et al., 2004). Luciferase activity was measured by luminometry and expressed as fold induction relative to unstimulated cells.

Cell Culture.

BEAS-2B cells and HEK-293 cells overexpressing the hrIP receptor (IPR-HEK), DP1 receptor, EP2 receptor, and EP4 receptor subtypes were cultured as described previously (Ayer et al., 2008. Wilson et al., 2009).

Measurement of Chemokines.

CXCL9 (monokine induced by γ-interferon) and CXCL10 (10-kDa IFNγ-induced protein) were measured by sandwich ELISA (Human DuoSet; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Transfection of Cells with siRNAs.

RNAiMax (Invitrogen, Burlington, ON, Canada), the siRNA of interest (10 nM; Table 1), and relevant controls were added to subconfluent (∼70%), growth-arrested BEAS-2B cells as described previously (Wilson et al., 2009). Effective “knockdown” of the IP receptor protein was assessed by Western blotting using a murine monoclonal IP receptor antibody from Abgent (San Diego, CA). IPR-HEK cells were used as a positive control.

View this table:
TABLE 1

siRNA oligonucleotide sequences

Measurement of cAMP.

Cells were pretreated (30 min) with the PDE3 and PDE4 inhibitors siguazodan (10 μM) and rolipram (10 μM), respectively. IP receptor agonists and/or antagonists were then added for an additional 45 min, a time when steady-state levels were achieved. cAMP was quantified by enzyme immunoassay (Cayman Chemicals, Ann Arbor, MI) according to the manufacturer's instructions.

Infection of Cells with Adenovirus Vectors.

Subconfluent BEAS-2B cells were infected (>90% efficiency) with an adenovirus vector (Ad5.CMV.PKIα) and a null control (Ad5.CMV.Null) encoding the α-isoform of cAMP-dependent protein kinase (PKA) inhibitor as described previously (Meja et al., 2004).

Curve Fitting and the Estimation of Antagonist Affinity.

Monophasic agonist concentration-effect (E/[A]) curves were fitted by least-squares, nonlinear iterative regression to the following form of the Hill equation (Prism 4, GraphPad Software Inc., San Diego, CA): Embedded Image where E is the effect, Emin and Emax are the lower and upper asymptote (i.e., the basal response and maximum agonist-induced response, respectively), p[A] is the log molar concentration of agonist, p[A]50 is a location parameter equal to the log molar concentration of agonist producing (Emax − Emin)/2, and n is the gradient of the E/[A] curve at the p[A]50 level.

The affinity of RO3422794 was estimated by least-squares nonlinear regression using a modification (Waud et al., 1978) of the Hill and Gaddum/Schild equations (Lazareno and Birdsall, 1993). Each IP receptor agonist was employed at a fixed submaximal concentration (∼p[A]95) in the absence and presence of increasing concentrations of RO3244794. For this modified Schild analysis, knowledge of the precise location of the agonist E/[A] curve is also required, and this was determined in parallel on the same batch of cells. Each pair of E/[A] curves (i.e., the control E/[A] curve and the E/[A] curve constructed in the presence of increasing concentrations of RO3244794) was fitted simultaneously to eq. 2. Thus, Embedded Image where [A] and [B] are the molar concentration of agonist and RO3244794, respectively; S is the Schild slope factor, which indicates the nature of antagonism; and pA2 is the affinity of the antagonist when S = 1, which is equivalent to the pKb. To determine whether S deviated significantly from unity, the pair of E/[A] curves that made up an individual experiment was fitted globally to eq. 2 under two conditions: one in which S was constrained to a constant equal to 1 and another in which S was a shared value for all data sets. The F test was applied to determine which equation gave the best fit, and that condition was used in the final analysis.

Statistics.

Data points, and values in the text and figure legends, represent the mean ± S.E.M. of N independent determinations. Where appropriate, data were analyzed statistically using Student's paired t test or by one-way ANOVA/Bonnferoni's multiple comparison test. The null hypothesis was rejected when p < 0.05.

Results

Selectivity of 15-Deoxy-TIC, Iloprost, and Taprostene for the DP1, EP2, and EP4 Receptor Subtypes.

To establish the selectivity of the IP receptor agonists shown in Fig. 1 for Gs-coupled prostanoid receptors, cAMP accumulation was measured in HEK-293 cells overexpressing the DP1, EP2, and EP4 subtypes. As shown in Supplemental Fig. S1, A to C, 15-deoxy-TIC was inactive at each of these receptor at concentrations 100 to 1000-times higher than maximally effective concentrations of both the natural ligands and representative synthetic agonists. Iloprost, similarly, did not elevate cAMP in hrEP2-HEK cells but did display some activity at the DP1 and EP4 subtypes (Supplemental Fig. S1, D–E). Indeed, iloprost was a potent (p[A]50 ∼ 11.4), albeit partial, DP1 receptor agonist (Supplemental Fig. S1D). We have reported previously that taprostene does not increase cAMP in HEK-293 cells overexpressing the EP2 receptor but is a weak agonist of very low potency in HEK-293 cells overexpressing the DP1 and EP4 receptor subtypes (see Fig. 2 in Wilson et al., 2009). Collectively, these data show that iloprost and 15-deoxy-TIC are selective IP receptor agonists but nevertheless may have the ability to activate DP1- and EP4 receptors at high concentrations. Accordingly, unless stated otherwise, all experiments were performed under conditions of concurrent DP1 (BWA 868C, 1 μM) and EP4 (L-161,982, 500 nM) receptor blockade. The EP3 receptor antagonist L-798,106 (100 nM) was also included in all experiments to eliminate the possible contribution of those splice variants of the EP3 receptor that can couple to adenylyl cyclase (Kotani et al., 1995).

Effect of IP Receptor Agonists on Chemokine Release and on Reporter Activity.

The effects of the IP receptor agonists shown in Fig. 1 were examined on two classes of functional response in BEAS-2B cells: 1) inhibition of IFNγ (100 ng/ml; p[A]85) induced CXCL9 and CXCL10 release, and 2) activation of a CRE reporter and augmentation of a dexamethasone (1 μM)-driven GRE reporter (Chivers et al., 2004; Meja et al., 2004; Ayer et al., 2008; Kaur et al., 2008).

Taprostene, PGI2, iloprost, and 15-deoxy-TIC inhibited the release of CXCL9 and CXCL10 in a concentration-dependent manner (Fig. 2, A–H; Table 2). In the presence of BWA 868C, L-798,106, and L-161,982, neither the p[A]50 nor the Emax values of taprostene, PGI2, and 15-deoxy-TIC were significantly affected (Table 2). In contrast, the inhibitory effects of iloprost were abolished (Fig. 2, C and G) indicating that these responses were not mediated by the IP receptor subtype. Indeed, further studies with BWA 868C and L-161,982 established that these effects of iloprost were mediated by both DP1 and EP4 receptors (see Supplemental Fig. S2 for CXCL10 data and Wilson et al., 2004, 2005). Taprostene, PGI2, and iloprost also promoted CRE-dependent transcription and augmented dexamethasone-stimulated GRE-reporter activity. Consistent with the chemokine release data described above, the concentration-effect (E/[A]) relationships that described these responses were not modified by BWA 868C, L-798,106, and L-161,982 (Fig. 2, I–K, M–O; Table 2). In contrast to its inhibitory effect on chemokine release, 15-deoxy-TIC was inactive on both reporter constructs (Fig. 2, L and P). Thus, these ligands were categorized into those that 1) inhibited chemokine output and promoted CRE- and GRE-dependent transcription (PGI2, taprostene); 2) activated the reporters only (e.g., iloprost); and 3) suppressed chemokine output only (e.g., 15-deoxy-TIC). Accordingly, the rank orders of agonist potency for the inhibition of chemokine release (15-deoxy-TIC > taprostene > PGI2) and for the activation of both reporters (iloprost > taprostene > PGI2) were distinct.

Fig. 2.
Fig. 2.

Functional effects of IP receptor agonists. A to H, BEAS-2B cells were pretreated (30 min) with vehicle or a combination of BWA 868C (1 μM), L-798,106 (100 nM), and L-161,982 (500 nM). Taprostene (A and E), PGI2 (B and F), iloprost (C and G), and 15-deoxy-TIC (D and H) were then added for an additional 30 min followed by IFNγ (100 ng/ml; p[A]85). At 24 h, the supernatant was harvested, and the amount of CXCL9 and CXCL10 was quantified by ELISA. I to L, CRE BEAS-2B reporter cells were pretreated (30 min) with vehicle or a combination of BWA 868C, L-798,106, and L-161,982 and then exposed to taprostene (I), PGI2 (J), iloprost (K), and 15-deoxy-TIC (L). GRE-reporter cells (M–P) were treated identically except that the IP receptor agonists were added concurrently with dexamethasone (Dex; 1 μM). At 6 h, supernatants were harvested, and luciferase activity was determined. E/[A] curves were then constructed from which p[A]50 and Emax values were determined. Each data point represents the mean ± S.E.M. of N independent determinations. See Table 2 for quantification of these data.

View this table:
TABLE 2

Relative potencies and functional activity of IP receptor agonists in BEAS-2B cells

Iloprost and 15-Deoxy-TIC Do Not Recognize the Same Receptor on BEAS-2B Cells.

Pretreatment of BEAS-2B cells with a concentration (1 μM) of iloprost that maximally activated both the CRE and GRE reporters did not antagonize the inhibitory effects 15-deoxy-TIC or taprostene on CXCL9 or CXCL10 release (Fig. 3, A and B, and Supplemental Fig. 3, A and B). Likewise, on CRE- and GRE-dependent transcription, a concentration of 15-deoxy-TIC (1 μM) that maximally suppressed chemokine release failed to antagonize CRE or GRE reporter activation induced by taprostene and iloprost (Fig. 3, C and D, and Supplemental Fig. 3, C and D).

Fig. 3.
Fig. 3.

Iloprost and 15-deoxy-TIC do not interact with the same receptor on BEAS-2B cells. A and B, E/[A] curves were constructed to 15-deoxy-TIC for the inhibition of IFNγ-induced CXCL9 and CXCL10 release, respectively, in cells pretreated (30 min) with an antagonist cocktail consisting of BWA 868C (1 μM), L-161,982 (500 nM), and L-798,106 (100 nM) and the antagonist cocktail supplemented with iloprost (1 μM). C and D, CRE and GRE reporter cells were pretreated (30 min) with antagonist cocktail, and iloprost E/[A] curves for luciferase expression were then constructed in the absence and presence of 15-deoxy-TIC (1 μM). Each bar and data point represents the mean ± S.E.M. of N determinations. See legend to Fig. 2 for further details.

RO3244794 Antagonizes Iloprost- and 15-Deoxy-TIC-Induced Responses with Different Affinities.

In general, the affinity of an antagonist for a given receptor is invariant within species. Accordingly, a modification (Waud et al., 1978) of the Hill and Gaddum/Schild equations (Lazareno and Birdsall, 1993) was used to determine whether there were differences in the affinity of the competitive, surmountable IP receptor antagonist RO3244794 (Bley et al., 2006; Jones et al., 2009) for the receptor(s) mediating iloprost-, taprostene-, and 15-deoxy-TIC-induced responses. RO3244794 (10 pM–100 nM) had no effect on IFNγ-induced chemokine release from BEAS-2B cells at any concentration examined (data not shown). In contrast, RO3244794 added to cells 30 min before a fixed, submaximal concentration of taprostene (1 μM) antagonized the inhibitory effect of taprostene on IFNγ-induced CXCL9 and CXCL10 release in a concentration-dependent manner (Fig. 4, A and C). Enumeration of the Schild slope factor, S, by simultaneously fitting to eq. 2 each pair of RO3244794 and taprostene E/[A] curves, which were constructed simultaneously (see Materials and Methods), indicated that this parameter did not deviate significantly from unity for either chemokine. Thus, RO3244794 behaved as a surmountable competitive antagonist (Neubig et al., 2003) from which pKb values of 10.3 (CXCL9) and 10.2 (CXCL10) were derived (Fig. 4, A and C). Similar affinities were obtained when 15-deoxy-TIC was used as agonist (pKb values CXCL9 = 9.90; CXCL10 = 9.79; see Fig. 4, B and D).

Fig. 4.
Fig. 4.

Determination of the affinity of RO3244794 for antagonizing taprostene-, iloprost-, and 15-deoxy-TIC-induced responses. BEAS-2B cells were pretreated (30 min) with RO3244794 (10 pM–1 μM). Taprostene (A and C) or 15-deoxy-TIC (B and D; both 1 μM) was then added followed by IFNγ (100 ng/ml). In parallel, a different culture of cells was pretreated (30 min) with taprostene (30 pM–10 μM) and 15-deoxy-TIC (10 pM–1 μM) in the absence of RO3244794 before being exposed to IFNγ. After 24 h, the amount of CXCL9 (A and B) and CXCL10 (C and D) released into the culture supernatant was quantified by a sandwich ELISA. In E and F, CRE BEAS-2B reporter cells were pretreated (30 min) with RO3244794 and then exposed to taprostene (1 nM–10 μM) or iloprost (100 pM–1 μM. GRE-reporter cells (G and H) were treated identically, except that the IP receptor agonists were added concurrently with dexamethasone (1 μM). Each pair of E/[A] curves was then analyzed simultaneously using a modification (Waud et al., 1978) of the Hill and Gaddum/Schild equations (Lazareno and Birdsall, 1993), as described previously (Ayer et al., 2008), from which pKb values were derived. Each data point represents the mean ± S.E.M. of N paired experiments, respectively.

On CRE- and GRE-dependent transcription, RO3244794 also behaved competitively (Fig. 4, E–H). However, although the affinity of RO3244794 was the same irrespective of whether taprostene (pKb values: CRE, 8.87; GRE, 9.10) or iloprost (pKb values: CRE, 9.06; GRE, 9.53) was used as agonist, it was nevertheless ∼10-fold lower than the pKb determined from the chemokine release experiments. Thus, these data suggest that 15-deoxy-TIC and iloprost bind to different receptors.

15-Deoxy-TIC Is a Low-Potency Agonist Relative to Iloprost in Promoting cAMP Accumulation in IPR-HEK-293 Cells, but Is Equipotent with Iloprost in BEAS-2B Cells.

In HEK-293 cells overexpressing the human IP receptor, taprostene, iloprost, and 15-deoxy-TIC promoted cAMP accumulation in a concentration-dependent manner (Fig. 5A). 15-Deoxy-TIC was a full agonist (α = 1.05 relative to iloprost) in this system, with a p[A]50 of 8.55 ± 0.01. However, relative to iloprost (p[A]50 = 13.27 ± 0.02) and taprostene (p[A]50 = 12.67 ± 0.2), 15-deoxy-TIC was greater than 52,000- and 13,000-fold less potent, respectively. In contrast, iloprost, taprostene, and 15-deoxy-TIC increased the cAMP concentration in BEAS-2B cells in a concentration-dependent manner with similar p[A]50 values (8.63 ± 0.05, 8.00 ± 0.01, and 8.70 ± 0.02, respectively; Fig. 5B). Thus, in this system, 15-deoxy-TIC was equipotent with iloprost and 5-fold more potent than taprostene.

Fig. 5.
Fig. 5.

Effect of taprostene, iloprost, and 15-deoxy-TIC on cAMP accumulation in IPR-HEK-293 and BEAS-2B cells. A and B, IPR-HEK-293 and BEAS-2B cells were both pretreated (30 min) with the PDE inhibitors, rolipram (10 μM; R), and siguazodan (10 μM; S), and then exposed to taprostene, iloprost, and 15-deoxy-TIC at the concentrations shown. C and D, fixed concentrations of iloprost (Ilo; 100 nM), 15-deoxy-TIC (TIC, 100 nM), and taprostene (Tap, 1 μM) were examined alone and in combination on cAMP levels in BEAS-2B cells. These experiments were performed in the presence of PDEI, BWA 868C (1 μM), L-798,106 (100 nM), and L-161,982 (500 nM) and, where indicated, the IP receptor antagonist RO3244794 (RO; 1 μM). Cells were lysed after 45 min and cAMP was measured by enzyme immunoassay. Each bar and data point represent the mean ± S.E.M. of N determinations. *, P < 0.05, significant difference; one-way ANOVA/Bonferroni's multiple comparison test.

Effect of Iloprost and 15-Deoxy-TIC on cAMP Levels.

At their maximally effective concentrations, iloprost and 15-deoxy-TIC were almost equally effective at increasing cAMP in BEAS-2B cells (Fig. 5, B and C). Despite inducing comparable response, only iloprost activated the CRE and GRE reporter cells (Fig. 2, K and O), whereas only 15-deoxy-TIC suppressed chemokine release (Fig. 2, D and H). In contrast, taprostene, increased cAMP maximally by an amount that was approximately 1.8- and 2.5-fold greater than that produced by iloprost and 15-deoxy-TIC, respectively (Fig. 5, B and C) and was active in both functional assays (Fig. 2, A, E, I, and M). When examined in combination, iloprost (100 nM) and 15-deoxy-TIC (100 nM) increased the cAMP concentration in an additive manner and by an amount that was equivalent in magnitude to the maximum taprostene (1 μM)-induced response (Fig. 5C). However, neither iloprost nor 15-deoxy-TIC further increased the cAMP content in BEAS-2B cells produced by a maximally effective concentration of taprostene (Fig. 5C). Thus, in BEAS-2B cells, iloprost and 15-deoxy-TIC apparently generated distinct pools of cAMP that regulated different functional responses. In the presence of RO3244794 (1 μM), taprostene-, iloprost- and 15-deoxy-TIC-induced cAMP accumulations were abolished (Fig. 5D).

Iloprost and 15-Deoxy-TIC Act via cAMP-Dependent Protein Kinase Cascades.

It is well established that the biological actions of cAMP are mediated by several effectors. Thus, additional studies were performed to identify the downstream target(s) of the cAMP generated by iloprost and 15-deoxy-TIC. Infection of BEAS-2B cells with the adenovirus vector Ad5.CMV.PKIα (multiplicity of infection = 40), which directs overexpression of a selective inhibitor of PKA (Meja et al., 2004), prevented the ability of 15-deoxy-TIC to suppress IFNγ-induced CXCL9 and CXCL10 release under conditions where an empty vector, Ad5.CMV.Null, was inactive (Fig. 6, A and B). Likewise, the transactivation of the CRE and GRE reporters by iloprost was blocked in Ad5.CMV.PKIα- but not Ad5.CMV.Null-infected cells (Fig. 6, C and D). Thus, in BEAS-2B cells, these responses induced by both iloprost and 15-deoxy-TIC required the activation of cAMP/PKA signaling cascades.

Fig. 6.
Fig. 6.

Effect of PKIα on iloprost- and 15-deoxy-TIC-induced responses. BEAS-2B cells were infected with Ad5.CMV.PKIα or Ad5.CMV.Null (both multiplicity of infection = 40) or were left untreated (control) for 48 h. 15-Deoxy-TIC-induced inhibition of chemokine release (A and B) and iloprost-induced reporter activation (C and D) were then measured at 24 and 6 h, respectively. Data points in each panel represent the mean ± S.E.M. of N independent determinations.

“Silencing” the IP Receptor Gene with siRNAs Selectively Inhibits Iloprost- but not 15-Deoxy-TIC-induced Responses.

Lipid-mediated transfection of BEAS-2B cells with siRNAs (PTGIR-1, PTGIR-2; see Table 1 for sequences) directed against PTGIR and all reported putative splice variants thereof (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?exdb=AceView&db=36a&term=ptgir) significantly reduced the expression of the variously glycosylated IP receptor (Smyth et al., 1996) as assessed by Western blotting (Fig. 7A, lane 3), whereas the transfection lipid, RNAiMax, and a universal negative control (UC) oligonucleotide (AllStars; QIAGEN, Valencia, CA) were inactive (Fig. 7A). Both oligonucleotides inhibited (by >76%) taprostene (1 μM)- and iloprost (100 nM)-induced CRE and GRE reporter activation under conditions in which the same responses evoked by PGE2 (1 μM) were preserved (Fig. 7, B and C; see Supplemental Fig. S4, A and B, for control siRNA data). The PTGIR-1 siRNA duplex was the most effective (Supplemental Fig. S4, A and B), with the enhancement of GRE-reporter activity being particularly sensitive (∼99% inhibition) to this intervention (Fig. 7C). In contrast, the E/[A] curves that described the inhibitory actions of taprostene and 15-deoxy-TIC on chemokine release were unaffected in cells transfected with PTGIR-1 (Fig. 7, D–G) and PTGIR-2 (Supplemental Fig. S4, C and D).

Fig. 7.
Fig. 7.

Effect of silencing PTGIR on taprostene, iloprost, and 15-deoxy-TIC-induced responses. BEAS-2B cells were transfected with siRNAs (10 nM) directed against PTGIR (PTGIR-1) and a UC. A, effect of PTGIR-1 and relevant controls on the expression of the variously glycosylated IP receptor as measured by Western blotting. The effects of iloprost (Ilo; 100 nM) and taprostene (Tap; 1 μM) on reporter activation are shown in B and C. 1, Control cells; 2, cells + UC; 3, cells + PTGIR-1; 4, cells + RNAiMax; 5, IPR-HEKs (positive control). D to G, 15-deoxy-TIC and taprostene E/[A] curves are shown in untreated cells and in cells transfected with PTGIR-1 and the UC. Each bar and data point represents the mean ± S.E.M. of N independent determinations. *, P < 0.05, significant inhibition of transcriptional responses in PTGIR-1-transfected cells; one-way ANOVA/Bonferroni's multiple comparison test. NS, no stimulus; Dex, dexamethasone (1 μM).

Taprostene- and iloprost-induced cAMP accumulations also were inhibited (45.4 ± 0.5 and 63.7 ± 1.4%, respectively) in cells transfected with PTGIR-1 siRNAs (Fig. 8, A and B), whereas the increments in cAMP produced by 15-deoxy-TIC and PGE2 (a negative control) were unaffected (Fig. 8, C and D). The transfection lipid, RNAiMax, and siRNAs that target the genes encoding lamin A/C, green fluorescent protein, and the UC oligonucleotide had no effect on any of the functional outputs studied (Figs. 7, B–E, and 8, and Supplemental Fig S4, A–D).

Fig. 8.
Fig. 8.

Effect of silencing PTGIR on taprostene, iloprost, 15-deoxy-TIC-, and PGE2-induced cAMP accumulation. BEAS-2B cells were transfected with siRNAs directed against PTGIR (PTGIR-1), green fluorescent protein, and lamin A/C (Lamin), as well as UC). The transfection lipid RNAiMax was also tested in parallel. Cells were pretreated with the PDE inhibitors rolipram (10 μM; R) and siguazodan (10 μM; S), BWA 868C (1 μM), L-798,106 (100 nM), and L-161,982 (500 nM), and cAMP accumulation was induced by iloprost (Ilo, 100 nM; A), taprostene (Tap, 1 μM; B), and 15-deoxy-TIC (TIC, 100 nM; C). The effect of PGE2 (1 μM; D) was also examined but in the absence of BWA 868C (1 μM), L-798,106 (100 nM), and L-161,982 (500 nM). Each bar represents the mean ± S.E.M. of N independent determinations. *, P < 0.05, significant inhibition of cAMP accumulation in PTGIR-1-transfected cells. One-way ANOVA/Bonferroni's multiple comparison test.

To exclude the possibility that the generation of the stable transfectants in BEAS-2B cells perturbed the ability of the IP receptor to recognize agonists, the effects of the PGI2 analogs on chemokine release and on the CRE reporter were determined concurrently in the same cell cultures. As shown in Supplemental Fig. S5, A–C, CRE reporter cells responded to taprostene, iloprost, and 15-deoxy-TIC in the absence and presence of PTGIR-1 in an identical manner to nontransfected cells (see Figure 2).

Discussion

In the present study, we provide evidence for the coexpression of two IP receptors in the human airway epithelial cell line BEAS-2B. It is important to restate that these experiments were performed in the presence of L-798,106, L-161,982, and BWA 868C to eliminate any contribution of EP3, EP4, and DP1 receptors, which can couple positively to adenylyl cyclase. Our preliminary data also firmly established that none of the IP receptor agonists studied activated the hrEP2 receptor expressed in HEK-293 cells.

IP receptor multiplicity initially was suggested by differences in the rank orders of potency of PGI2 and three structurally related agonists for the inhibition of chemokine release [15-deoxy-TIC > taprostene > PGI2 (iloprost was inactive at 1 μM)] and the activation of CRE- and GRE-dependent transcription [iloprost > taprostene > PGI2 (15-deoxy-TIC was inactive at 1 μM)]. However, the classification of receptors using agonists alone is problematic because variability in potency orders between functional responses, even in the same cell type, may reflect differences in efficacy rather than receptor subtypes. Indeed, an alternative interpretation is that 15-deoxy-TIC has high affinity for the IP receptor but low efficacy in driving CRE- and GRE-dependent transcription compared with the inhibition of chemokine output. If this explanation is correct, then 15-deoxy-TIC should behave as an IP receptor antagonist. As shown in Fig. 3, a high concentration of 15-deoxy-TIC that maximally suppressed chemokine release failed to produce a dextral displacement of the taprostene and iloprost E/[A] curves that described the activation of the two reporter constructs. Thus, the affinity of 15-deoxy-TIC for the IP receptor that is sensitive to both taprostene and iloprost is, at best, weak (> 1 μM). This conclusion was corroborated by the finding that 15-deoxy-TIC was ∼52,000- and ∼13,000-fold less potent than iloprost and taprostene, respectively, in promoting cAMP accumulation in HEK-293 cells overexpressing the hrIP receptor. Differences in efficacy could also account for the inability of iloprost to suppress chemokine release. However, iloprost did not display measurable affinity for the 15-deoxy-TIC-sensitive IP receptor subtype as evinced from its inability to antagonize the inhibition of chemokine generation (Fig. 3). Collectively, therefore, these pharmacological data suggest the presence of two taprostene (and PGI2) -sensitive receptors on BEAS-2B cells, which are denoted here as IP1 (iloprost-sensitive) and “IP2” (15-deoxy-TIC–sensitive) subtypes.

Further studies using a surmountable, competitive IP receptor antagonist, RO3244794 (Bley et al., 2006), which has a high affinity (pKb = 8.50–9.24) and selectivity for the IP receptor subtype (Bley et al., 2006; Jones et al., 2009), provided additional evidence for IP receptor heterogeneity. Thus, RO3244794 antagonized taprostene- and iloprost-induced CRE and GRE reporter activation with an affinity (pKb ∼ 9) consistent with an interaction with the known, cloned IP receptor (IP1) (Katsuyama et al., 1994). RO3244794 also antagonized the ability of taprostene and 15-deoxy-TIC to suppress CXCL9 and CXCL10 release. However, in these experiments, RO3244794 had significantly higher affinity (pKb ∼ 10), suggesting that chemokine output was regulated by a receptor (“IP2”) that was distinct from the IP1-subtype recognized by iloprost and taprostene in the reporter cells. There are limited data with 15-deoxy-TIC from which pharmacodynamic parameters can accurately be derived. Nevertheless, applying the “IP1/IP2” nomenclature introduced above, our results are supported by the finding that 15-deoxy-TIC is a weak inhibitor of platelet aggregation (IP1-mediated) relative to isocarbacyclin (Suzuki et al., 1996; Suzuki et al., 1999). Conversely, 15-deoxy-TIC rescues cultured gerbil hippocampal neurons from oxygen-induced apoptosis (“IP2”-mediated) under conditions in which iloprost is inactive (Satoh et al., 1999). This classification is also supported by Takechi et al. (1996), who suggested the existence of a second IP receptor subtype in the rat CNS based on radioligand binding data (see Introduction).

Iloprost, taprostene, and 15-deoxy-TIC elevated cAMP in BEAS-2B by a mechanism that was abolished by the IP receptor antagonist RO3244794. Moreover, adenovirus-delivery of PKIα, a highly selective inhibitor of PKA (Meja et al., 2004), to these cells similarly blocked the effects of 15-deoxy-TIC and iloprost on chemokine release and reporter activity, respectively. Thus, the “IP2” receptor as well as the well characterized IP1-subtype mediated these functional effects by activating classic cAMP/PKA cascades.

At maximally effective concentrations, cAMP accumulation induced by taprostene was 2.5- and 1.8-fold higher than that elicited by 15-deoxy-TIC and iloprost, respectively. These data led us to speculate that the increments in cAMP induced by iloprost and 15-deoxy-TIC were due to selective agonism of IP1 and “IP2” receptors, respectively, whereas taprostene activated both subtypes, resulting in an additive cAMP response. Indeed, when used in combination at their maximally effective concentrations, iloprost and 15-deoxy-TIC produced cAMP signals that were the sum of the increments produced by both drugs alone and equivalent in magnitude to the taprostene-induced response.

To consolidate the evidence that BEAS-2B cells express two pharmacologically distinct IP receptors, the gene encoding the IP1 subtype PTGIR (Katsuyama et al., 1994) was “silenced” using siRNAs. Under stringently controlled conditions, CRE- and GRE-dependent transcription induced by taprostene and iloprost were markedly inhibited (74–100%) in cells transfected with PTGIR-1 oligonucleotides. Conversely, the same responses evoked by PGE2 (mediated by EP2 and EP4 receptors) were unaffected, indicating that these PTGIR-targeting siRNAs produced a selective functional knockdown of the desired molecular target. In contrast, the inhibition by taprostene and 15-deoxy-TIC of chemokine output was unaffected in cells in which PTGIR was “silenced.” Entirely consistent results were obtained when cAMP was selected as the functional readout. Thus, the PTGIR-1 oligonucleotides did not block the cAMP signal induced by 15-deoxy-TIC, whereas the increases in cAMP levels induced by iloprost and taprostene were inhibited by 64 and 44%, respectively. These findings cannot easily be reconciled with a single IP receptor system. Initially, in considering how best to interpret these data, it was thought that a large IP receptor reserve might exist on BEAS-2B cells for 15-deoxy-TIC-induced inhibition of chemokine release such that the degree of gene silencing produced by PTGIR-1 siRNAs was insufficient to impair function. However, this possibility was discounted, because silencing PTGIR will reduce IP functional receptor number such that PTGIR-1 should behave in the same way as an inactivating alkylating agent (Kenakin, 1987). Thus, if the known IP receptor mediates the effect of taprostene and 15-deoxy-TIC on chemokine output, then the E/[A] curves that describe these effects should have been displaced to the right in PTGIR-1-transfected cells and, if receptor number is limiting, should be associated with a reduction in the maximal asymptote. However, as shown in Fig. 7, D to G, the PTGIR-1 oligonucleotides had no effect on the taprostene or 15-deoxy-TIC E/[A] curves as assessed by p[A]50 values and maximum inhibition produced. Thus, these findings are consistent, again, with the expression of a hitherto-undefined IP receptor (“IP2”) on BEAS-2B cells.

We submit that the PTGIR silencing studies also indicate that 15-deoxy-TIC cannot be acting through a splice variant of the known IP-subtype. Indeed, the PTGIR-1 oligonucleotides target sequence in the 5′-untranslated region of PTGIR and would “silence” all reported putative splice variants. The likelihood that 15-deoxy-TIC suppressed chemokine output by interacting with a heterodimer composed of the IP1 subtype and an unknown G protein-coupled receptor was also excluded because inactivation of PTGIR would reduce the number of dimers and therefore the responses they mediate.

In conclusion, the present study provides pharmacological evidence for IP receptor heterogeneity in BEAS-2B cells. Two functionally distinct receptors have been characterized that couple to the activation of canonical cAMP/PKA cascades and independently regulate reporter (CRE and GRE) activation and inhibition of chemokine (CXCL9 and CXCL10) release. These receptors can be distinguished with iloprost (IP1 receptor agonist), 15-deoxy-TIC (“IP2” receptor agonist), the IP receptor antagonist,RO3244794 (affinity at “IP2” > IP1), as well by silencing PTGIR. We submit that these data cannot readily be explained by mRNA splicing or G protein-coupled receptor heterodimerization. Thus, we propose that the RO3244794-sensitive, 15-deoxy-TIC-induced responses in BEAS-2B cells are mediated by the activation of a second receptor for PGI2 that is distinct from the known IP-subtype encoded by PTGIR.

Authorship Contributions

Participated in research design: Newton and Giembycz.

Conducted experiments: Hill.

Contributed new reagents or analytic tools: Sheddan.

Performed data analysis: Hill and Giembycz.

Wrote or contributed to the writing of the manuscript: Newton and Giembycz.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported by the Canadian Institutes of Health Research (CIHR) [Grants MOP 68828 (to R.N.), MOP 93742 (to M.G.)]. R.N. is a CIHR New Investigator and an Alberta Innovates—Health Solutions Senior Scholar. M.G. is the recipient of a Tier 1 Canada Research Chair in Pulmonary Pharmacology.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.110.069674.

  • ABBREVIATIONS:

    PGI2
    prostacyclin
    IP
    prostacyclin receptor
    BMY 45778
    2-[3-[4-[4,5-di(phenyl)-1,3-oxazol-2-yl]-1,3-oxazol-5-yl]phenoxy] acetic acid
    CNS
    central nervous system
    CXCL9
    monokine induced by IFNγ
    CXCL10
    10-kDa IFNγ-induced protein
    NTS
    nucleus tractus solitarius
    15R-TIC
    15R epimer of 16-m-tolyl-17,18,19,20-tetranor isocarbacyclin
    15-Deoxy-TIC
    15-deoxy-16-(m-tolyl)-17,18,19,20-tetranorisocarbacyclin
    RO3244794
    R-3-(4-fluoro-phenyl)-2-(5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino)-propionic acid
    BWA A868C
    3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-ydroxyethylamino) hydantoin
    PGE2
    prostaglandin E2
    ONO-AE1-259
    (Z)-7-[(1R,2S,3R,5R)-5-chloro-3-hydroxy-2-[(E,4S)-4-hydroxy-4-(1-prop-2-enylcyclobutyl)but-1-enyl]cyclopentyl]hept-5-anoic acid
    L-798,106
    5-bromo-2-methoxy-N-[3-(2-naphthalen-2-yl-methylphenyl)acryloyl]-benzene sulfonamide
    L-161,982
    5-butyl-2,4-dihydro-[[2′-[N-(5-methyl-2-thiophene-carbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-[(2-trifluoromethyl) phenyl]-1,2,4-triazol-3-one
    CRE
    cAMP response element
    GRE
    glucocorticoid response element
    HEKm
    human embryonic kidney
    IPR-HEK
    HEK-293 cells overexpressing the hrIP receptor
    IFN
    interferon
    ELISA
    enzyme-linked immunosorbent assay
    siRNA
    small interfering RNA
    PDE
    phosphodiesterase
    CMV
    cytomegalovirus
    PKA
    cAMP-dependent protein kinase
    ANOVA
    analysis of variance
    UC
    universal negative control.

  • Received October 27, 2010.
  • Accepted December 20, 2010.

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Evidence for a Second Receptor for Prostacyclin on Human Airway Epithelial Cells That Mediates Inhibition of CXCL9 and CXCL10 Release

Sylvia M. Wilson, Neil A. Sheddan, Robert Newton and Mark A. Giembycz
Molecular Pharmacology March 1, 2011, 79 (3) 586-595; DOI: https://doi.org/10.1124/mol.110.069674
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