Abstract
Prostacyclin receptor (IP-receptor) agonists display anti-inflammatory and antiviral activity in cell-based assays and in preclinical models of asthma and chronic obstructive pulmonary disease. In this study, we have extended these observations by demonstrating that IP-receptor activation also can enhance the ability of glucocorticoids to induce genes with anti-inflammatory activity. BEAS-2B bronchial epithelial cells stably transfected with a glucocorticoid response element (GRE) luciferase reporter were activated in a concentration-dependent manner by the glucocorticoid dexamethasone. An IP-receptor agonist, taprostene, increased cAMP in these cells and augmented luciferase expression at all concentrations of dexamethasone examined. Analysis of the concentration-response relationship that described this effect showed that taprostene increased the magnitude of transcription without affecting the potency of dexamethasone and was, thus, steroid-sparing in this simple system. RO3244794, an IP-receptor antagonist, and oligonucleotides that selectively silenced the IP-receptor gene, PTGIR, abolished these effects of taprostene. Infection of BEAS-2B GRE reporter cells with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA) also prevented taprostene from enhancing GRE-dependent transcription. In BEAS-2B cells and primary cultures of human airway epithelial cells, taprostene and dexamethasone interacted either additively or cooperatively in the expression of three glucocorticoid-inducible genes (GILZ, MKP-1, and p57kip2) that have anti-inflammatory potential. Collectively, these data show that IP-receptor agonists can augment the ability of glucocorticoids to induce anti-inflammatory genes in human airway epithelial cells by activating a cAMP/PKA-dependent mechanism. This observation may have clinical relevance in the treatment of airway inflammatory diseases that are either refractory or respond suboptimally to glucocorticoids.
Prostacyclin (PGI2)4 is a labile eicosanoid derived from arachidonic acid following the sequential action of cyclooxygenase and PGI2 synthase (1). The biological actions of PGI2 are mediated primarily through the PGI2 receptor (IP-receptor), which typically couples to Gs for the activation of adenylyl cyclase (2). Although the role of PGI2 in the regulation of vascular homeostasis has, for many years, been a primary research focus, it is now appreciated that PGI2 and its cognate receptor may play important (patho)physiological roles in a variety of other processes, including airway inflammatory diseases (2).
The IP-receptor subtype is expressed on many immune and proinflammatory cells, including the monocyte (3), T lymphocyte (4, 5), dendritic cell (6), epithelial cell (7), and airway (8) and pulmonary vascular smooth muscle cells (9). Moreover, accumulated evidence suggests that IP-receptor agonism in the lung may exert anti-inflammatory and/or antiviral activity. For example, the IP-receptor agonists cicaprost and iloprost attenuate cytokine production from anti-CD3-stimulated, murine CD4+ T lymphocytes cultured under conditions that cause Th1 or Th2 cell polarization (5). Cytokine secretion from murine bone marrow-derived dendritic cells in response to LPS is also inhibited by iloprost (6, 10). A central role for the IP-receptor in regulating allergic inflammatory responses in vivo similarly has been documented. Thus, IP-receptor gene deficiency in allergen-challenged, sensitized mice is associated with a phenotype characterized by exaggerated pulmonary inflammation and airway hyper-responsiveness when compared with wild-type animals (11, 12). These data are consistent with the finding that PGI2 attenuates allergen-induced inflammation in mice by limiting pulmonary CD4+ T cell recruitment (4, 13), which may be due to an inhibitory effect on dendritic cell function (14). Activation of the IP-receptor may also be beneficial in chronic obstructive pulmonary disease (COPD). Indeed, taprostene, an IP-receptor agonist, suppressed the generation of the CD8+ T cell chemoattractants CXCL9 and CXCL10 from human airway epithelial cells in vitro (7). Moreover, another IP-receptor agonist, beraprost, protected rats against the development of experimental cigarette smoke-induced emphysema, possibly by means of a concerted inhibitory action on alveolar epithelial cell apoptosis, oxidative burden, matrix metalloproteinase expression, and proinflammatory cytokine generation (15). Finally, there is evidence from animal studies that IP-receptor agonists protect against the deleterious effects of respiratory syncytial virus infection (16). Potentially, this is an important observation because viral infections are believed to precipitate exacerbation in many individuals with asthma and COPD (17). Collectively, therefore, these findings suggest that selective IP-receptor agonists could be exploited to therapeutic advantage in the treatment of airway diseases where chronic inflammation with or without associated parenchymal destruction is a defining characteristic (11, 14).
Unlike asthma, COPD is an example of a chronic inflammatory disease that is relatively insensitive to inhaled glucocorticoids (ICSs) (18). Clearly, therefore, a drug that can boost or reveal latent sensitivity to ICSs while having intrinsic anti-inflammatory and/or antiviral activity could be a significant pharmacotherapeutic advance. Repression of inflammatory gene expression by ICSs is believed to occur by at least two general mechanisms. The classical repressive mode of glucocorticoid action is termed trans-repression, in which the activity of key proinflammatory transcription factors, such as NF-κB and AP-1, is inhibited via direct interactions with the ligand-bound glucocorticoid receptor (GR) (19). However, in simple model systems, glucocorticoids often are relatively weak (partial) inhibitors of inflammatory gene transcription, implying that processes in addition to trans-repression must be operative to account for the anti-inflammatory effects seen in bona fide models of inflammation (20, 21, 22, 23). In this respect, the induction (trans-activation) by glucocorticoids of anti-inflammatory genes that then repress proinflammatory processes is now believed to be a major mechanism of glucocorticoid action (20, 21, 22, 23). Moreover, in the context of the present study, cAMP-elevating agents can, in gene expression studies, interact positively with glucocorticoids (20, 24). Accordingly, we have tested the hypothesis, using human bronchial epithelial cells as a model system, that selective agonism of the IP-receptor will enhance the anti-inflammatory activity of a glucocorticoid to a level that cannot be achieved by the glucocorticoid alone. We submit that the demonstration of such a phenomenon could have clinical utility in the treatment of airway inflammatory diseases, including COPD, that are either refractory or respond suboptimally to glucocorticoids. Such a finding would also provide a precedent for the idea that novel glucocorticoid combination therapies could be developed in which the cAMP-elevating drug is tailored to the inflammatory disease of interest.
Materials and Methods
Culture of BEAS-2B cells
Cells were cultured for 2 days under a 5% CO2/air atmosphere at 37°C in 6- or 24-well plastic plates containing DMEM/F12 (Invitrogen) supplemented with 10% FBS (Invitrogen), l-glutamine (2.5 mM), and 0.15% (v/v) sodium bicarbonate. The cells were then growth-arrested for 24 h in serum-free medium (SFM). At this time, cultures were tightly confluent and were processed for biochemical and functional measurements as described below.
Culture of human primary airway epithelial cells (HpAECs)
Cells were obtained by proteinase digestion of nontransplanted normal human lung (International Institute for the Advancement of Medicine, Edison, NJ), as previously described (25). Cells were seeded in 12-well plates (Corning Life Sciences) containing bronchial epithelial cell growth medium (Lonza) supplemented with penicillin (50 μg/ml) and streptomycin (10 μg/ml), cultured under a 5% CO2/air atmosphere at 37°C until confluent (typically 14 days; medium was changed every 3 to 4 days), growth arrested for 24 h in supplement-free, bronchial epithelial cell basal medium (Lonza), and processed for biochemical and functional measurements as described below. Ethics approval for the use of human tissues has been granted by the Conjoint Health Research Ethics Board of the University of Calgary (Calgary, Alberta, Canada).
Culture of human embryonic kidney (HEK)-293 Epstein-Barr nuclear Ag cells
HEK-293 Epstein-Barr nuclear Ag cells expressing the human recombinant D prostanoid 1-receptor (DP1R-HEK), E prostanoid 2-receptor (EP2R-HEK), E prostanoid 4-receptor (EP4R-HEK), or IP-receptor (IPR-HEK) subtype were cultured for 2 days under a 5% CO2/air atmosphere at 37°C in 24-well plastic plates containing DMEM supplemented with 10% (v/v) FBS, geneticin (200 μg/ml), and hygromycin B (200 μg/ml). Confluent cells were then growth-arrested in DMEM for 24 h under a 5% CO2 atmosphere at 37°C in the absence of serum and antibiotics before processing for cAMP experiments as described below.
Generation of a glucocorticoid response element (GRE) reporter
Stable transfection was used to generate a GRE reporter cell line as described previously (26). The construct, pGL3.neo.TATA.2GRE, contains two copies of a consensus simple GRE site (sense strand, 5′-TGTACAGGATGTTCT-3′) positioned upstream of a minimal β-globin promoter driving a luciferase gene and a separate neomycin gene to confer resistance to geneticin (26). BEAS-2B cells at ∼70% confluence in T162 flasks were transfected with 8 μg of plasmid DNA and 20 μl of Lipofectamine 2000 (Invitrogen). After 24 h, geneticin (100 μg/ml) was added until foci of stable transfectants appeared that were harvested to create heterogeneous populations of cells in which the site of integration was randomized.
Transfection of BEAS-2B GRE reporter cells with siRNAs
RNAiMax (Invitrogen) and the small interfering RNA (siRNA) of interest (20 nM) (see Table I⇓ for siRNA oligonucleotide sequences) were diluted to 2× the desired final concentration with antibiotic-free, serum-free DMEM:F12 (Invitrogen) combined in a 1:1 ratio and left at room temperature for 30 min. Subconfluent (∼70%) BEAS-2B GRE reporter cells in 24-well plates were exposed to the siRNA/lipid mix for 6 h at 37°C under a 5% CO2/air atmosphere. The medium was then replaced with fresh DMEM/F12 supplemented with 1% FBS (v/v) and left for 42 h. Cells were then growth arrested in SFM for a further 48 h before beginning the experiment.
siRNA oligonucleotide sequences
Treatment of GRE BEAS-2B reporter cells and measurement of luciferase
Confluent, growth-arrested cells were treated with dexamethasone, taprostene, PGE2, and the IP-receptor antagonist RO3244794 (27) as indicated in the text and figure legends. Unless stated otherwise, cells were incubated at 37°C under a 5% CO2 atmosphere and harvested 5 h later in 1× reporter lysis buffer (Promega). Luciferase activity was then measured using a Monolight luminometer (BD Biosciences) according to the manufacturer’s instructions. Data are expressed as the fold induction of luciferase relative to that of unstimulated cells.
Infection of BEAS-2B Cells with Ad5.CMV.PKIα
Subconfluent (∼70%) BEAS-2B GRE reporter cells were infected (multiplicity of infection (MOI) = 40) with an E1−/E3− replication-deficient adenovirus vector (Ad5.CMV.PKIα) containing a 251-base pair DNA fragment encoding the complete amino acid sequence of the α-isoform of cAMP-dependent protein kinase (PKA) inhibitor α (PKIα) downstream of the constitutively active CMV immediate early promoter (28). After 48 h, cells were processed for the assessment of GRE-dependent transcription as described above. To control for biological effects of the virus per se, the vector Ad5.CMV.Null, expressing no transgene, was used in parallel. Using this experimental protocol, we have previously reported that >90% of BEAS-2B cells are infected with Ad5.CMV.PKIα, resulting in the expression of a completely functional transgene with no adverse effect on cell viability (28).
cAMP mass determination
BEAS-2B and HEK-293 cells in 24-well plates were growth arrested for 24 h and then pretreated (30 min) with the phosphodiesterase (PDE) inhibitors rolipram (10 μM) and siguazodan (10 μM). Prostanoid receptor agonists and antagonists were then added at the concentrations indicated in the text and figure legends in the continued presence of PDE inhibitors. After 45 min of incubation at 37°C under 5% CO2, cells were lysed with HCl (100 mM) and cAMP mass was measured by enzyme immunoassay (Cayman Chemical) according to the manufacturer’s instructions.
Measurement of CXCL10
Growth-arrested BEAS-2B cells were pretreated (30 min) with GW9662 (1 μM), a peroxisome proliferator-activated receptor (PPAR) γ antagonist (29), or its vehicle followed by a PARγ agonist, rosiglitazone (1 μM), for a further 30 min. IFN-γ (100 ng/ml; p[A]90) was then added and the cells incubated at 37°C under a 5% CO27 for details).
RNA isolation, reverse transcription, and real-time quantitative PCR
Total RNA was extracted from BEAS-2B cells and HpAECs that had been treated with dexamethasone (1 μM) and taprostene (1 μM) or forskolin (10 μM) alone or in combination using RNeasy mini kits (Qiagen) and was reverse transcribed to cDNA as described previously (26). Real-time quantitative PCR analysis of cDNA using the primer sequences shown in Table II⇓ (designed using Primer Express software; Applied Biosystems) encoding glucocorticoid-induced leucine zipper (GILZ; also known as TGF-β-stimulated clone 22, domain family member 3 or TSC22D3), MAPK phosphatase (MKP)-1 (also known as dual-specificity phosphatase 1 or DUSP-1), and kinase inhibitor protein 2 of 57 kDa (p57kip2; also known as cyclin-dependent kinase inhibitor 1C or CDKN1C) was performed using an ABI 7900HT instrument (Applied Biosystems) on 2.5 μl of cDNA in 20-μl reactions using SYBR GreenER chemistry (Invitrogen) according to the manufacturer’s guidelines. Relative cDNA concentrations were determined from a cDNA standard curve that was analyzed simultaneously with the test samples. Amplification conditions were as follows: 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Dissociation (melt) curves (95°C for 15 s and 60°C for 20 s with ramping to 95°C over 20 min and then 95°C for 15 s) were constructed to confirm primer specificity.
Primer pairs for real-time qPCRa
Curve fitting
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) shown in Equation 1,
where E is the effect, Emin and Emax are the lower and upper asymptotes (i.e., the basal response and maximum agonist-induced response, respectively), p[A] the log molar concentration of agonist, p[A]50 a location parameter equal to the log molar concentration of agonist producing Emax/2, and n the gradient of the E/[A] curve at the p[A]50 level.
The antagonism of taprostene-induced responses by RO3244794 was evaluated by least-squares, nonlinear regression using a modification of the Hill and Gaddum/Schild equations derived by Waud et al. (30). Each family of E/[A] curves (i.e., the control E/[A] curve and E/[A] curves constructed in the presence of increasing concentrations of RO3244794) were fitted simultaneously to Equation 2. Thus,
where [A] and [B] are the molar concentrations of taprostene 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 entire family of E/[A] curves that made up an individual experiment was fitted globally to Equation 2 under two conditions: one where S was constrained to a constant equal to 1 and the other where it was a shared value for all data sets. The F test was applied to determine which equation gave the best fit, which was then used for the analysis.
Assessment of cell viability
Cell viability was evaluated colorimetrically by measuring the reduction of the tetrazolium salt, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, to formazan by mitochondrial dehydrogenases.
Drugs, Abs, and analytical reagents
ONO-AE1–259 ((Z)-7-[(1R,2S,3R,5R)-5-chloro-3-hydroxy-2-[(E,4S)- 4-hydroxy-4-(1-prop-2-enylcyclo butyl)but-1-enyl]cyclopentyl]hept-5-enoic acid) and ONO-AE1–329 (2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxo-cyclopentyl]sulfanylpropylsulfanyl]acetic acid) were from ONO Pharmaceuticals. RO3244794 ((R-3-(4-fluoro phenyl)-2-[5-(4-fluorophenyl)-benzofuran-2-ylmethoxycarbonylamino]propionic acid) was provided by Roche Pharmaceuticals. Iloprost, PGD2, PGE2, BW245C (7-[3-(3-cyclohexyl-3-hydroxy-propyl)-2,5-dioxo-imidazolidin-4-yl]heptanoic acid), rosiglitazone, and GW9662 (2-chloro-5-nitrobenzanilide) were obtained from Cayman Chemical and rolipram and siguazodan were purchased from Calbiochem (EMD Biosciences). Goat anti-human PKIα (code 1944) and goat anti-human β-actin (code 1615) were from Autogen Bioclear. Taprostene, forskolin, and all other reagents were from Sigma- Aldrich. Drugs were dissolved in DMSO and diluted to the desired working concentration in the appropriate culture medium.
Definitions and statistics
In the text, the term “additivity” refers to two drugs that, when combined, produce an effect that is the sum of their individual components. In contrast, the term “positive cooperativity” is used when the biological response of two drugs given in combination is greater than the sum of their individual effects.
Data points and values in the text and figure legends represent the mean ± SEM of N independent determinations. Data were analyzed by Student’s t test or ANOVA (one-way or two-way as indicated) followed, when appropriate, by Bonferroni’s multiple comparison test. The null hypothesis was rejected when p < 0.05.
Results
None of the compounds or their vehicles used in the experiments described herein significantly affected cell viability.
Selection of taprostene as an IP-receptor agonist
The explicit classification of responses mediated by IP-receptors is hindered by a paucity of suitable pharmacological tools. In the present study, we established that a synthetic PGI2 analog, taprostene (31), is a highly selectively agonist suitable for examining the (patho)physiological role of the IP-receptor subtype (Fig. 1⇓). Thus, in IPR-HEK cells, taprostene increased the cAMP content in a concentration-dependent manner with a p[A]50 of 12.7 ± 0.2 (Fig. 2⇓a). Relative to iloprost, taprostene was a full agonist in these cells (intrinsic activity (α) = 1.01; Fig. 2⇓a). In contrast, taprostene failed to increase cAMP mass in HEK-293 cells expressing the recombinant DP1-, EP2-, and EP4-receptor subtypes (all adenylyl cyclase coupled) at a concentration (1 nM) that maximally increased the cAMP level in IPR-HEK cells (Fig. 2⇓, b–d). Indeed, even at a concentration of 10 μM, taprostene was weak, eliciting a cAMP response (ρ) that was 13% of that produced by PGD2 in DP1R-HEK cells and 4.9 and 10% of that produced by PGE2 in EP2R-HEK and EP4R-HEK cells, respectively (Fig. 2⇓, b–d). In contrast, at a concentration of 100 nM, BW245C (DP1-selective), ONO-AE1–259 (EP2-selective), and ONO-AE1–329 (EP4-selective) were full agonists in DP1R-HEK, EP2R-HEK, and EP4R-HEK cells, respectively (Fig. 2⇓, b–d).
Chemical structure of PGI2, taprostene, and RO3244794. Asterisk (∗) denotes chiral center. Optically active isomers are indicated.
Effect of taprostene on cAMP accumulation in HEK-293 cells stably expressing adenylyl cyclase-coupled prostanoid receptor subtypes. Cells expressing the human recombinant IP-(IPR-HEK) (a), DP1-(DP1R-HEK) (b), EP2-(EP2R-HEK) (c), and EP4-receptor (EP4R-HEK) (d) were pretreated (30 min) with the PDE3 and PDE4 inhibitors and siguazodan (S; 10 μM) and rolipram (R; 10 μM), respectively, to prevent cAMP hydrolysis. E/[A] curves to taprostene were then constructed (IP: from 100 aM to 10 nM; DP1, EP2, and EP4: from 100 pM to 10 μM) using cAMP and measured after 45 min as a biochemical output. The open and filled/hatched bars in each panel represent the cAMP response in cells treated with the PDE inhibitors in the absence or presence of the natural prostaglandin and/or synthetic agonist, respectively, as indicated (each at 100 nM). The intrinsic activity (α) of taprostene in IPR-HEK relative to iloprost is indicated in a. In b–d, the cAMP response (ρ) of taprostene (10 μM) is shown relative to that of PGD2 (DP1) and PGE2 (EP2 and EP4), which are used as reference full agonists. The dashed line in each panel indicates the baseline cAMP level. Data points and bars represent the mean ± SEM of N independent determinations.
Effect of taprostene on GRE-dependent transcription
Treatment of BEAS-2B reporter cells with dexamethasone (0.1 nM to 1 μM) for 5 h induced GRE-dependent transcription in a concentration-dependent manner with a p[A]50 and Emax of 7.96 ± 0.11 and 19.3 ± 3.8-fold, respectively (Fig. 3⇓a). Taprostene (1 μM) alone was without effect on the GRE reporter but, when added to cells concurrently with dexamethasone, significantly augmented GRE-dependent transcription above that produced by the glucocorticoid alone (Fig. 3⇓a). Thus, the interaction of taprostene and dexamethasone was one of positive cooperativity. Analysis of the E/[A] relationship that described this effect showed that taprostene enhanced the magnitude (Emax) of transcription (from 19.3 ± 3.8- to 37.3 ± 5.5-fold) without affecting the potency of dexamethasone (p[A]50 = 7.73 ± 0.2) (Fig. 3⇓a). Further studies established that in the presence of a maximally effective concentration of dexamethasone (1 μM), the ability of taprostene to augment GRE-dependent transcription was concentration-dependent, with a p[A]50 of 6.82 ± 0.12 (Fig. 3⇓b).
Effect of taprostene on GRE-dependent transcription. BEAS-2B GRE reporter cells were treated respectively with either dexamethasone (0.1 to 1 μM) in the absence and presence of taprostene (1 μM) (a) or with taprostene (1 nM to 10 μM) in the presence of dexamethasone (1 μM) (b). After 5 h cells were lysed and luciferase activity was determined. The filled bar and dashed line in b indicate the effect on luciferase expression of dexamethasone alone. Data points and bars represent the mean ± SEM of N independent determinations.
Kinetics of the enhancement by taprostene of GRE-dependent transcription
Treatment of the BEAS-2B reporter with dexamethasone (1 μM) induced the luciferase gene in a time-dependent manner (Fig. 4⇓). This effect reached a maximum at 5 h and was maintained for a further 11 h. Thereafter, the luciferase signal declined. In the presence of taprostene (1 μM), which did not activate the reporter, the induction by dexamethasone of GRE-dependent transcription was augmented at all time points (Fig. 4⇓). The greatest positive cooperativity was seen at the 2 and 5 h time points when taprostene (inactive by itself) augmented luciferase expression from 6- to 14-fold and from 10- to 18.5-fold, respectively.
Kinetics of GRE-dependent transcription in BEAS-2B GRE reporter cells. Cells were treated with dexamethasone (Dex; 1 μM), taprostene (Tap; 1 μM), a combination of both drugs (each 1 μM), or vehicle (Control). Cells were then incubated for 1–19 h and harvested for luciferase activity. Data points represent the mean ± SEM of three independent determinations. ∗, p < 0.05, significant enhancement of transcription relative to time-matched, unstimulated cells; +, p < 0.05, significant enhancement of transcription relative to time-matched, dexamethasone-treated cells. Data were analyzed by using two-way ANOVA followed by Bonferroni’s multiple comparison test.
Affinity of RO3244794 for antagonizing taprostene-induced responses in BEAS-2B GRE reporter cells
Pretreatment of BEAS-2B GRE reporter cells with the selective IP-receptor antagonist RO3244794 (10 and 30 nM; Ref. 27), had no effect on the expression of luciferase measured at 5 h or on the ability of dexamethasone to promote GRE-dependent transcription (data not shown). These data are thus consistent with the inability of BEAS-2B cells to synthesize PGI2 (32). However, in dexamethasone (1 μM)-treated cells RO3244794 produced a graded, parallel rightwards displacement (12- and 36-fold at 10 and 30 nM, respectively) of the taprostene E/[A] curve (Fig. 5⇓). Enumeration of the Schild slope factor S (which indicates the nature of the antagonism) by simultaneously fitting to Equation 2 each RO3244974 and agonist E/[A] curve indicated that this parameter did not deviate significantly from unity. Thus, RO3244794 behaved in a manner that was consistent with surmountable competitive antagonism (33). Accordingly, S was constrained to a value of 1 from which a pKb value of 9.21 ± 0.25 was derived.
Schild analysis of the antagonism by RO3244794 of taprostene-induced responses in BEAS-2B cells. BEAS-2B GRE cells were pretreated (30 min) with RO3244794 at the concentrations indicated in each panel. Taprostene E/[A] curves were then constructed in the continued presence of RO3244794 for the enhancement of dexamethasone (1 μM)-induced GRE-dependent transcription. Modified Schild analysis was then performed (30 ) that yielded a pKb of 9.21. The filled bar and dashed line indicate the effect on luciferase expression of dexamethasone alone. Data points and bars represent the mean ± SEM of five independent determinations.
Effect of “silencing” PTGIR on the enhancement by taprostene of GRE-dependent transcription
Lipid-mediated transfection of BEAS-2B GRE reporter cells with siRNAs (each 20 nM) directed against the gene of interest PTGIR, the gene in the jellyfish Aequorea victoria that encodes GFP or a universal negative control oligonucleotide (AllStars; Qiagen) that is reported to not recognize any human mRNA, affected neither basal nor dexamethasone (1 μM)-induced luciferase expression (Fig. 6⇓). The transfection lipid RNAiMax was also inactive.
Effect of silencing PTGIR by using siRNA on the enhancement by taprostene of GRE-dependent transcription. Subconfluent (70%) BEAS-2B GRE reporter cells were transfected (6 h) using RNAiMax with siRNAs (each 20 nM) directed against PTGIR, GFP, and a universal negative control oligonucleotide. Cells were washed, incubated for 18 h in DMEM/F12 supplemented with 1% (v/v) FBS and then growth arrested for 48 h in SFM. The effect of dexamethasone (Dex: 1 μM) alone and in the presence of taprostene (Tap; 1 μM) and PGE2 (100 nM) on GRE-dependent luciferase expression at 5 h was then determined. The dashed line indicates the effect on luciferase expression of dexamethasone alone. Bars represent the mean ± SEM of seven independent determinations. See Materials and Methods for further details. NS, No stimulation representing basal luciferase expression; ∗, p < 0.05, significant inhibition of the enhancement by taprostene of GRE-dependent transcription relative to cells transfected with a universal control oligonucleotide. Data were analyzed by using one-way ANOVA followed by Bonferroni’s multiple comparison test.
Treatment of BEAS-2B reporter cells with taprostene (1 μM) or PGE2 (100 nM) did not promote GRE-dependent transcription but significantly augmented (from 13.0 ± 0.4- to 24.9 ± 2.9- and 25.4 ± 1.9-fold, respectively) the transcriptional response produced by a maximally effective concentration of dexamethasone (1 μM). In cells transfected with siRNAs (PTGIR-1 and PTGIR-2) that target PTGIR, the ability of taprostene to enhance GRE-dependent transcription was abolished whereas RNAiMax alone and in the presence of the “control” siRNAs was without effect (Fig. 6⇑). In contrast, the augmentation of dexamethasone-induced reporter activity by PGE2 was not affected by PTGIR-1, PTGIR-2, the “control” siRNAs, or RNAiMax (Fig. 6⇑), thereby confirming the selectivity of the knockdown.
Role of the cAMP/PKA pathway in the enhancement by taprostene of GRE-dependent transcription
The IP-receptor couples typically to Gs for the stimulation of adenylyl cyclase, although in some systems it can also promote phospholipase C-dependent inositol phosphate production (34, 35). To establish the mechanism by which activation of the IP-receptor enhanced GRE-dependent transcription, BEAS-3B cells were exposed to taprostene and cAMP mass was determined. As shown in Fig. 7⇓a, taprostene increased cAMP in a concentration-dependent manner with a p[A]50 of 8.01 ± 0.01. In subsequent experiments, BEAS-2B GRE reporter cells were infected with an adenovirus vector, Ad5.CMV.PKIα, which directs overexpression of PKIα, a highly selective inhibitor of PKA (28). In uninfected cells, PKIα was not detected by Western blotting in any experiment. However, 48 h after infection of BEAS-2B cells with Ad5.CMV.PKIα (MOI = 40), a single peptide was labeled by the anti-PKIα Ab that migrated as a 12-kDa band on SDS-polyacrylamide gels (Fig. 7⇓c). As shown in Fig. 7⇓b, taprostene enhanced dexamethasone (1 μM)-induced GRE-dependent transcription in a concentration-dependent manner (p[A]50 = 6.91 ± 0.14; Emax = 24.0 ± 0.9-fold), which was prevented in cells expressing the PKIα transgene (Fig. 7⇓b). In contrast, cells infected with a null virus, Ad5.CMV.Null, responded to taprostene in a manner that was not significantly different from uninfected cells (p[A]50 = 6.86 ± 0.08; Emax = 26.1 ± 1.6-fold; Fig. 8⇓b). Forskolin, a direct activator of adenylyl cyclase, also augmented GRE-dependent transcription by 2.78-fold (from 25.0 ± 5.8 to 69.5 ± 11.3; n = 5), and this effect also was abolished in cells expressing the PKIα transgene (data not shown).
Effect of taprostene on the cAMP/PKA cascade in BEAS-2B cells. a, BEAS-2B cells were pretreated (30 min) with the PDE3 and PDE4 inhibitors and siguazodan (S; 10 μM) and rolipram (R; 10 μM), respectively, to prevent cAMP hydrolysis and then exposed to taprostene (0.1 nM to 10 μM) for 45 min. Cells were lysed and cAMP mass was measured by enzyme immunoassay according to the manufacturer’s instructions (Cayman Chemical). b, Subconfluent cells were infected with Ad5.CMV.PKIα, Ad5.CMV.Null (both at MOI = 40), or left untreated for 48 h. Cells were then treated with dexamethasone (Dex; 1 μM) alone (filled bar) or taprostene (1 nM to 10 μM) in the presence of dexamethasone (1 μM). After 5 h cells were lysed and luciferase activity was determined. c, Western blot, representative of three experiments, of the expression of the PKIα transgene in BEAS-2B cells 48 h after infection with Ad5.CMV.PKIα. The filled bar and dashed line in b indicate the effect on luciferase expression of dexamethasone alone. Data points and bars represent the mean ± SEM of N independent determinations. Lane 1, Naive cells; lane 2, Ad5.CMV.Null-infected cells; lane 3, Ad5.CMV.PKIα-infected cells.
Effect of the PPARγ antagonist GW9662 on the enhancement by taprostene of GRE-dependent transcription and on the suppression by rosiglitazone of IFN-γ-induced CXCL10 release. a, BEAS-2B GRE reporter cells were pretreated with GW9662 (1 μM; 30 min) or vehicle. Dexamethasone (1 μM) was then added alone (bars) or in combination with taprostene (1 nM to 10 μM). After 5 h cells were lysed and luciferase activity was determined. b, BEAS-2B cells were pretreated with GW9662 (1 μM; 30 min) or vehicle and exposed to rosiglitazone (100 nM) for a further 30 min. IFN-γ (100 ng/ml) was then added and the amount of CXCL10 released into the supernatant at 24 h was measured by ELISA. The bars and dashed line in panel a indicate the effect on the luciferase expression of dexamethasone (± GW9662) alone. Data points and bars represent the mean ± SEM of N independent determinations. ∗, p < 0.05, significant inhibition of CXCL10 release; ∗∗, p < 0.05, significant reversal of CXCL10 release by GW9662. Data were analyzed by using one-way ANOVA followed by the Bonferroni multiple comparison test.
Role of PPARγ in the enhancement by taprostene of GRE-dependent transcription
Agonism of the human IP-receptor subtype is reported to activate the nuclear hormone receptor PPARγ (36). Accordingly, using GW9662 as a selective antagonist (29), studies were performed to establish whether PPARγ mediated the augmentation by taprostene of dexamethasone-induced GRE-dependent transcription. GW9662 (1 μM) had no effect on basal or dexamethasone-induced luciferase expression (Fig. 8⇑a). Similarly, GW9662 did not affect the E/[A] relationship that described the enhancement by taprostene of GRE-dependent transcription (Fig. 8⇑a). Indeed, neither the p[A]50 (6.68 ± 0.07) nor Emax (38.2 ± 5.5-fold) were altered by GW9662 (p[A]50 = 6.81 ± 0.19; Emax = 39.3 ± 6.3-fold). In contrast, GW9662 (1 μM) abolished the inhibitory effect of rosiglitazone, a PPARγ agonist, on IFN-γ-induced CXCL10 output from BEAS-2B cells under similar experimental conditions (Fig. 8⇑b).
Effect of taprostene and forskolin on the expression of glucocorticoid-inducible anti-inflammatory genes in BEAS-2B cells
The data presented in the preceding sections indicate that taprostene enhanced dexamethasone-induced transcription from a conventional simple GRE reporter. To determine whether this finding is applicable to real genes, we took advantage of data derived from a prior microarray analysis in which dexamethasone-inducible genes were identified in pulmonary type II A549 cells (see Ref. 20 for details). Several of these genes have anti-inflammatory potential, including GILZ, MKP-1, and p57kip2 (22, 37, 38, 39, 40), and were selected to examine the interaction between dexamethasone and taprostene (or forskolin).
The data derived from the GRE reporter showed substantial positive cooperativity between dexamethasone (1 μM) and taprostene (1 μM) at 1 h, 2 h and 5 h. Accordingly, the expression of GILZ, MKP-1 and p57kip2 were examined over the same time-frame. Relative to unstimulated cells, dexamethasone (1 μM) strongly induced GILZ in a time-dependent manner; the mRNA level peaked at 2 h (∼75-fold induction) and remained elevated up to 6 h (Fig. 9⇓a). In contrast, taprostene (1 μM) was inactive over the same time frame but augmented the transcriptional activity of dexamethasone at 1 and 2 h in a manner reminiscent of that produced in GRE reporter cells (Fig. 9⇓a). Similarly, forskolin, a direct activator of adenylyl cyclase, failed to induce GILZ at any time point but, like taprostene, enhanced the effect of dexamethasone (Fig. 9⇓b). Thus, taprostene and forskolin interacted with dexamethasone in the regulation of GILZ in a positive cooperative fashion.
Effect of taprostene and forskolin on the expression of glucocorticoid-inducible genes in BEAS-2B cells. Cells were treated with dexamethasone (Dex; 1 μM), taprostene (Tap; 1 μM), both agents simultaneously (Dex + Tap), or vehicle (control), and RNA was harvested at 1, 2, and 6 h. (a, c, and e) A similar experiment was also performed in which taprostene was replaced with forskolin (Forsk; 10 μM) (b, d, and f). After cDNA synthesis, real-time RT-PCR was performed for GILZ (a and b), p57kip2 (c and d), and MKP-1 (e and f) and normalized to GAPDH. Data are expressed as fold stimulation at each time point relative to GAPDH and are plotted as means ± SEM of six independent observations. The horizontal dashed line in each panel indicates basal gene expression at the 0-h time point, which has been normalized to a value of 1. The nature of the interaction between dexamethasone and the cAMP-elevating agent at one or more time points is indicated in each panel. ∗, p < 0.05, significant enhancement of transcription relative to time-matched, unstimulated cells; +, p < 0.05, significant enhancement of transcription relative to time-matched, dexamethasone-treated cells. Data were analyzed by using two-way ANOVA followed by Bonferroni’s multiple comparison test.
p57kip2 also was rapidly induced by dexamethasone (1 μM) in a time-dependent manner, although the inducibility and kinetics of the response were different from those for GILZ. Thus, mRNA levels increased almost linearly over the course of the experiment (∼12-fold at 6 h) and were significantly lower than the GILZ transcripts matched for time (Fig. 9⇑c). Taprostene (1 μM) alone had a negligible effect on the expression of p57kip2 (∼1.4-fold induction) but augmented the transcriptional activity of dexamethasone at all time points. Qualitatively comparable data were obtained when forskolin was used as a cAMP-elevating stimulus (Fig. 9⇑d), but the magnitude of this positive cooperative interaction was far more pronounced and greatest at the 6-h time point (∼43- vs ∼17-fold for taprostene; compare GILZ, Fig. 9⇑b).
The third gene examined, MKP-1, was induced by dexamethasone (1 μM) in a transient manner; mRNA levels peaked at the 2-h time point and then slowly decayed (Fig. 9⇑e). Taprostene (1 μM) and forskolin (10 μM) also induced MKP-1 (maximally by 2.1- and 4.1-fold, respectively, at 1 h), but this effect also was short-lived and was essentially lost by 2 h (Fig. 9⇑, e and f). When used in combination, dexamethasone (1 μM) and taprostene (1 μM) or dexamethasone (1 μM) and forskolin (10 μM) at 1 h interacted in a purely additive fashion (Fig. 9⇑, e and f). At later time points (2 and 6 h) the expression of MKP-1 induced by both drug combinations was not different from the effect produced by dexamethasone alone (Fig. 9⇑, e and f).
Effect of taprostene and forskolin on the expression of glucocorticoid-inducible anti-inflammatory genes in HpAECs
Additional studies were performed to determine whether the drug interactions found in BEAS-2B cells also occurred in HpAECs. To this end, GILZ and MKP-1 were selected as genes that were regulated by glucocorticoid and a cAMP-elevating agent in a positive cooperative and additive manner, respectively. As shown in Fig. 10⇓, a and b, dexamethasone (1 μM) induced GILZ in a time-dependent manner. Consistent with the data obtained with BEAS-2B cells (Fig. 9⇑, a and b), both taprostene (1 μM) and forskolin (10 μM) were inactive but, in the presence of dexamethasone (1 μM), augmented (markedly at 6 h) the expression of GILZ. However, several notable differences were apparent. First, GILZ was at least an order of magnitude less inducible in HpAECs at all time points (compare Fig. 10⇓a with Fig. 9⇑a). Second, although taprostene and dexamethasone interacted cooperatively in HpAECs, this was seen only at the 6-h time point whereas cooperativity was seen at all time points when forskolin and dexamethasone were combined (compare Fig. 10⇓, a and b with Fig. 9⇑, a and b). Third, the kinetics of GILZ induction in response to dexamethasone in combination with taprostene or forskolin appeared to be slower in HpAECs when compared with BEAS-2B cells (compare Fig. 10⇓, a and b with Fig. 9⇑, a and b).
Effect of taprostene (Tap) and forskolin (Forsk) on the expression of glucocorticoid-inducible genes in HpAECs. Cells were treated and cDNA prepared as described in the legend to Fig. 9. Real-time RT-PCR was performed for GILZ (a and b) and MKP-1 (c and d) and normalized to GAPDH. Data are expressed as fold stimulation at each time point relative to GAPDH and are plotted as means ± SEM of three independent observations. The horizontal dashed line in each panel indicates basal gene expression at the 0-h time point, which has been normalized to a value of 1. The nature of the interaction between dexamethasone (Dex) and a cAMP-elevating agent at one or more time points is indicated in each panel. ∗, p < 0.05, significant enhancement of transcription relative to time-matched, unstimulated cells; +, p < 0.05, significant enhancement of transcription relative to time-matched, dexamethasone-treated cells. Data were analyzed by using two-way ANOVA followed by Bonferroni’s multiple comparison test.
In HpAECs the MKP-1 gene was also induced by dexamethasone (1 μM) and, additionally, by taprostene (1 μM) and forskolin (10 μM; Fig. 10⇑, c and d). For all three stimuli, MKP-1 mRNA appeared very transiently (more so than in BEAS-2B cells); peak levels were achieved at 1 h and had declined to baseline at 2 h. In combination, taprostene and dexamethasone, or forskolin and dexamethasone interacted additively on MKP-1 expression with similarly transient kinetics (Fig. 10⇑, c and d). In contrast to GILZ, the inducibility of MKP-1 in HpAECs was similar to that found in BEAS-2B cells (compare Fig. 10⇑, c and d with Fig. 9⇑, e and f).
Discussion
The results of the present study show that the selective agonism of the IP-receptor on the human bronchial epithelial cell line BEAS-2B with taprostene enhanced dexamethasone-induced transcription of a simple GRE reporter by activating the cAMP/PKA cascade. Similarly, taprostene augmented the expression of certain glucocorticoid-inducible genes in BEAS-2B cells and HpAECs, including GILZ and MKP-1, which have potential anti-inflammatory activity. These findings support and extend the existing body of knowledge, by demonstrating that IP-receptor agonists may have utility in treating chronic airway inflammation by boosting the clinical efficacy of glucocorticoids as part of an IP-receptor agonist/ICS combination therapy.
Selection of taprostene as an IP-receptor agonist
A major problem in classifying responses mediated by IP-receptors has been a paucity of suitable pharmacological tools. Indeed, PGI2 and many PGI2 analogs and mimetics, including iloprost, carbacyclin, AFP-07, and TEI-9063, are not sufficiently selective for their biological actions to be diagnostic of IP-receptor agonism (41, 42). Even cicaprost, which is often the agonist of choice in studies examining IP-receptor pharmacology, must be used with caution at it does not definitively discriminate IP-, EP4-, and, to a lesser extent, EP3-receptor-mediated responses (41, 42). To overcome these problems, we used, in the present study, a synthetic PGI2 analog, taprostene (31). Two sets of data led us to select this ligand. First, taprostene showed high selectivity for the human recombinant IP-receptor expressed in HEK-293 cells relative to the other prostanoid receptors that couple positively to adenylyl cyclase. Although absolute selectivity ratios cannot be calculated from these data, taprostene at its ∼p[A]95 (i.e., 100 pM) was inactive at the DP1-, EP2-, and EP4-subtypes (Fig. 2⇑). Second, the ability of taprostene to enhance GRE-dependent transcription in BEAS-2B reporter cells was antagonized in a competitive manner by RO3244794, an IP-receptor-blocking drug, with an affinity (pKb = 9.21) consistent with an interaction at IP-receptors (27, 43). Collectively, these results strongly suggest that in BEAS-2B cells, taprostene augments GRE-dependent transcription by an IP-receptor-mediated mechanism. This conclusion was confirmed in gene silencing studies where, under stringently controlled conditions, two siRNAs that target PTGIR abolished the ability of taprostene to enhance GRE-dependent transcription, whereas the same response evoked by PGE2 was preserved.
Interaction of taprostene and dexamethasone
Using a simple GRE-reporter construct stably transfected into BEAS-2B cells, taprostene augmented the ability of dexamethasone to promote the induction of the luciferase gene in a time- and concentration-dependent manner. Inspection of the E/[A] curves showed that taprostene increased the ability of dexamethasone to “drive” the luciferase gene (i.e., Emax was increased) without significantly affecting its potency (i.e., p[A]50 was unchanged). Of potential clinical significance is that this interaction is one of positive cooperativity, because taprostene alone did not promote GRE-dependent transcription (see below). Moreover, taprostene was “steroid-sparing” in this model system. For example, dexamethasone, at its p[A]95, produced a 17.4-fold induction of the luciferase gene. However, in the presence of taprostene (1 μM), which was inactive by itself, the same degree of gene induction was achieved at a concentration of dexamethasone that was significantly lower (7.3-fold if the p[A]95 of the glucocorticoid response alone is taken as reference) (see Fig. 11⇓).
Interaction between taprostene and dexamethasone on GRE-dependent transcription. The graph in Fig. 3a has been redrawn to illustrate that taprostene augments dexamethasone-induced GRE reporter activity in a steroid-sparing manner. BEAS-2B cells stably expressing a GRE-reporter were treated with taprostene (1 μM) in the absence otr presence of dexamethasone (100 pM to 1 μM). After 6 h, cells were harvested for luciferase assay. The data show that the effect of taprostene and dexamethasone in combination promotes GRE-dependent transcription above the maximum effect achieved by the glucocorticoid alone (1.93-fold at the Emax). Taprostene was also glucocorticoid sparing in this model. Thus, dexamethasone at a concentration that evoked 95% of the maximum response produced a 19.3-fold induction of the luciferase gene. However, in the presence of taprostene (1 μM), which was inactive by itself, the same degree of gene induction was achieved at a concentration of glucocorticoid that was significantly (7.3-fold) lower.
Prior microarray analysis of genes induced by dexamethasone in A549 cells identified several candidates for subsequent analysis that are predicted to exert anti-inflammatory activity. The three genes studied here were found to be regulated in an additive or positive cooperative manner by glucocorticoid and taprostene in combination. Thus, GILZ and p57kip2 were induced in a positive cooperative fashion in BEAS-2B, and a similar effect was seen with GILZ in HpAECs. Although there were differences in the magnitude and kinetics of gene induction between BEAS-2B cells and HpAECs, both cell types responded at certain time points in a manner that resembled activation of the GRE reporter. Indeed, a functional conventional simple GRE is present in the p57kip2 and GILZ promoters (44, 45), which implies that taprostene and dexamethasone in combination can induce in a positive cooperative manner real anti-inflammatory genes. GILZ has clear anti-inflammatory potential through its ability to attenuate the activation of NF-κB and/or AP-1, which are critical transcription factors that control the expression of many proinflammatory genes (37, 38). Similarly, p57kip2 encodes a cell cycle kinase inhibitor that regulates the anti-proliferative effects of glucocorticoids (39). Given that airway remodeling in COPD could be explained, in part, by the increase in airway wall volume occupied by bronchial epithelial cells as well as smooth muscle (46), it is possible that an IP-receptor agonist/ICS combination therapy could be disease modifying through its ability to promote the expression of p57kip2. The p57kip2 gene product also blocks JNK (47) suggesting that it may suppress proinflammatory events known to be regulated by this MAPK.
The third gene examined was MKP-1. This gene encodes an enzyme that dephosphorylates (inactivates) the three core mammalian MAPKs (p38 MAPK, ERK, and JNK) that are central to the induction of many proinflammatory genes (e.g., growth-related oncogene-α) (Ref. 40). As shown in Figs. 9⇑ and 10⇑, the regulation of MKP-1 was distinct from that of p57kip2 and GILZ. Thus, MKP-1 was induced by both taprostene and dexamethasone, and in combination the interaction of these stimuli was transient and purely additive. These data indicate that that unlike p57kip2 and GILZ, the glucocorticoid-dependent regulation of MKP-1 is not primarily mediated via simple GREs, which is consistent with the finding that the promoter of this and many other glucocorticoid-inducible genes do not feature a conventional GRE consensus sequence (i.e., sense strand, 5′-TGTACAGGATGTTCT-3′) (48).
It is noteworthy that the transcription of glucocorticoid-inducible genes that mediate adverse effects may also be augmented by an IP-receptor agonist, thereby limiting the clinical efficacy of such a combination therapy. For example, the expression of metabolic genes such as phosphoenol pyruvate carboxykinase and glucose-6-phosphastase, which regulate gluconeogenesis, are induced by cAMP-elevating stimuli and glucocorticoids and, in combination, act at least additively (49, 50, 51). Clearly, therefore, the pharmacokinetics of a glucocorticoid will dictate the propensity to which such adverse effect genes may be induced. In respiratory diseases, a glucocorticoid should be delivered directly to the lung and have low oral absorption, high plasma protein binding to limit systemic exposure, and high first-pass hepatic metabolism. Under these conditions, the ability of an IP-receptor agonist to enhance the transcriptional activity of that glucocorticoid should be retained predominantly in the lung, allowing the superior clinical efficacy of the combination therapy to be achieved.
Mechanism of action
It has been reported that PGI2 analogs, including carbacyclin and treprostinil, are PPARγ agonists (36). However, no evidence for such a mechanism was obtained in the present study based on the inability of GW9662, a PPARγ antagonist (29), to inhibit taprostene-induced transcriptional responses. Indeed, our data suggest that the mechanistic basis for the enhancement by taprostene of GRE-reporter activity involves the activation of the classical cAMP/PKA pathway. Thus, this effect was mimicked by forskolin and abolished in cells infected with Ad5.CMV.PKIα. Similarly, the ability of forskolin to interact either additively or cooperatively with dexamethasone on the expression of GILZ, p57kip2, and MKP-1 in BEAS-2B cells and HpAECs confirms that cAMP can, in some way, augment the transcription of real glucocorticoid-inducible genes.
We have reported previously that salmeterol, a β2-adrenoceptor agonist, and forskolin also augment GRE-dependent transcription in BEAS-2B cells, but only a certain, undefined population of glucocorticoid-inducible genes are affected (20). Indeed, genes that encode tristetraprolin, aminopeptidase N, and plasminogen activator inhibitor-1 are not regulated in an additive or positive cooperative manner by either of these cAMP-elevating agents (20). Current data indicate that β2-adrenoceptor agonists and forskolin display an identical, qualitative profile of activity on a panel of glucocorticoid-inducible genes (20). Whether taprostene shares this defined activity or displays a distinct or overlapping profile is unknown. Although the present study was undertaken to establish the concept that an IP-receptor agonist can augment GRE-dependent trans-activation, this is nevertheless an important objective for future research. Indeed, it is conceivable that different cAMP-elevating drugs may enhance the transcription of distinct populations of glucocorticoid-inducible genes. Clearly, this raises the possibility of selectively regulating the expression of certain genes, which would have clear therapeutic potential.
The identity of the targets downstream of PKA that more directly up-regulate GRE-dependent transcription is vague. It has been proposed that cAMP-elevating agents enhance the translocation of the GR from the cytosol to the nucleus and that this boosts transcription (52, 53, 54). Indeed, the cAMP-elevating drug formoterol augments the ability of budesonide to promote the binding of the GR to GREs on target genes above the level produced by the glucocorticoid alone (55). Such an effect would be consistent with the enhanced GR:DNA binding seen in cells overexpressing PKA (56). Another possibility is that cAMP-elevating agents increase the expression of functional GR (57) and/or stabilize the interaction of glucocorticoid-bound GR with DNA through activation of PKA (58). Although these are plausible ideas, both mechanisms necessarily imply that the transcription of all glucocorticoid-inducible genes would be enhanced by a cAMP-elevating drug, which is not the case (20). To account for this, we propose that taprostene and other cAMP-elevating agents may act predominantly within the nucleus by regulating the activity and/or recruitment of specific cofactors that affect only the transcription of a specific subset of glucocorticoid-inducible genes (59, 60, 61). Although this proposal remains to be investigated, it would confer promoter specificity and explain why the expression of some glucocorticoid-inducible genes is augmented in a positive cooperative fashion by cAMP-elevating agents whereas others are not.
Targeting the IP-receptor in COPD
COPD is a multifaceted disorder. Typically, neutrophilic inflammation of the small airways and lungs is a defining pathological feature that is associated with chronic airflow limitation (60). Many individuals with COPD also have systemic inflammation (62) that is positively related to disease severity (63). Pulmonary vascular remodeling leading to impaired gas exchange and/or pulmonary hypertension is also frequently seen. In many cases, such abnormalities occur in conjunction with right-side heart failure (64, 65) and platelet hyper-reactivity, rendering afflicted individuals at increased risk of developing thromboses (66, 67, 68). Unlike asthma, stable COPD is an example of a chronic inflammatory disease that is relatively insensitive to ICSs (18). Conceivably, an “add-on” therapy that is known to effectively target the cardiovascular pathologies and concurrently augment the anti-inflammatory effect of an ICS could provide an effective and potentially disease-modifying treatment. We submit that a selective IP-receptor agonist could fulfill this role for several tangible reasons. In particular, the expression of PGI2 synthase and the level of 6-keto-PGF1α, the primary metabolite of PGI2, are reduced in whole lung lysates prepared from subjects with emphysema (69). Conversely, the urinary excretion of 11-dehydro-thromboxane (TX) B2, the major enzymatic metabolite of TXA2 in subjects with COPD, is significantly enhanced relative to that in healthy individuals matched for age and gender (70). Thus, there appears to be a PGI2 deficiency in emphysematous COPD and a reciprocal increase in TXA2 generation with predicted undesirable effects on platelet reactivity and pulmonary vascular smooth muscle function. Clearly, a selective IP-receptor agonist could restore this PGI2/TXA2 imbalance and, thereby, reduce the threshold for platelet aggregation (71), lower pulmonary vascular resistance (72) (through smooth muscle relaxation and, long term, by inhibiting mitogenesis), and even promote bronchodilatation (73). The results of this study indicate that an IP-receptor agonist could also suppress airway and pulmonary vascular inflammation by enhancing the clinical efficacy of an ICS. It is noteworthy that IP-receptor agonists are effective in the treatment of pulmonary arterial hypertension (74). However, there have been few controlled trials in pulmonary hypertension associated with COPD (65), and the suitability of these drugs for treating cardiovascular defects in this pathological setting would need to be tested empirically in well-designed clinical trials.
Recently, there has been a considerable effort in medicinal chemistry to develop new PGI2 mimetics for pulmonary arterial hypertension that are both stable and selective for the IP subtype. For example, chemists at Nippon Shinyaku have discovered a number of nonprostanoid pro-drugs that are metabolized in vivo into potent and selective IP-receptor agonists (75). One of these, NS-304, is an N-acylsulphonamide that is slowly hydrolyzed by the liver to produce the corresponding carboxylic acid MRE-269, which has a t1/2 in blood of 7.9 h (75). In the event that IP-receptor agonists are shown to be efficacious in treating the cardiovascular abnormalities in COPD, a drug exemplified by NS-304 could be suitable for twice a day dosing with an existing ICS.
Conclusions
In the present study we report that a selective IP-receptor agonist, taprostene, enhances the ability of a glucocorticoid to transcribe genes, in airway epithelial cells, with potential anti-inflammatory activity. This novel mechanism of action has clear clinical relevance in diseases, such as COPD, that are generally refractory to glucocorticoids as a monotherapy. Moreover, these data complement a growing body of literature in which PGI2 analogs are reported to have both anti-inflammatory and antiviral activity in preclinical models of airway inflammation (see Introduction). We submit that because activation of the IP-receptor also elicits beneficial effects on platelet reactivity, pulmonary vascular smooth muscle tone. and, indirectly, cardiac function, ICS/IP-receptor agonist combination therapy could provide an effective treatment option in COPD that may be particularly effective in subjects with hematological and cardiovascular dysfunction.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 This study was funded in part by the Canadian Institutes of Health Research (CIHR) and the GlaxoSmithKline/Collaborative Innovation Research Fund. R.N. is a CIHR New Investigator and an Alberta Heritage Foundation for Medical Research (AHFMR) Scholar. M.A.G. is an AHFMR Senior Scholar. D.P. holds a Canadian Research Chair in Inflammatory Lung Diseases.
↵2 Current Address: Department of Pathology and Molecular Medicine, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada.
↵3 Address correspondence and reprint requests to Dr. Mark A. Giembycz, Department of Physiology and Pharmacology, Airways Inflammation Research Group, Institute of Infection, Immunity, and Inflammation, 3280 Hospital Drive Northwest, Calgary, Alberta, Canada T2N 4N1. E-mail address: giembycz{at}ucalgary.ca
↵4 Abbreviations used in this paper: PGI2, prostacyclin; COPD, chronic obstructive pulmonary disease; GILZ, glucocorticoid-inducible leucine zipper; GR, glucocorticoid receptor; GRE, glucocorticoid response element; E/[A], agonist concentration effect; HEK, human embryonic kidney; HpAEC, human primary airway epithelial cell; ICS, inhaled glucocorticoid; IP-receptor, PGI2 receptor; MKP, MAPK phosphatase; MOI, multiplicity of infection; p57kip2, kinase inhibitor protein 2 of 57 kDa; PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase A; PKI, PKA inhibitor; PPAR, peroxisome proliferator-activated receptor; SFM, serum-free medium; siRNA, small interfering RNA; TX, thromboxane.
- Received August 19, 2009.
- Accepted September 21, 2009.
- Copyright © 2009 by The American Association of Immunologists, Inc.
References
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