The ß-Blocker Carvedilol and Related Aryloxypropanolamines Promote ERK1/2 Phosphorylation in HEK293 Cells with K A Values Distinct From Their Equilibrium Dissociation Constants as ß 2-Adrenoceptor Antagonists: Evidence for Functional Affinity.

The determination of affinity by using functional assays is important in drug discovery because it provides a more relevant estimate of the strength of interaction of a ligand to its cognate receptor than radioligand binding. However, empirical evidence for so-called, "functional affinity" is limited. Herein, we determined whether the affinity of carvedilol, a ß-adrenoceptor antagonist used to treat heart failure that also promotes extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation, differed between these two pharmacological activities. Four structurally related ß-adrenoceptor antagonists (alprenolol, carazolol, pindolol, propranolol) that also activated ERK1/2 were included as comparators to enhance our understanding of how these drugs work in the clinical setting. In HEK293 cells stably expressing the human ß 2-adrenoceptor carvedilol and related aryloxypropanolamines were partial agonists of ERK1/2 phosphorylation with potencies ([A]50s) that were lower than their equilibrium dissociation constants (K Bs) as ß 2-adrenoceptor antagonists. As the [A]50 of a partial agonist is a good approximation of its K B, then these data indicated that the affinities of carvedilol and related ligands for these two activities were distinct. Moreover, there was a significant negative rank order correlation between the [A]50 of each ligand to activate ERK1/2 and their intrinsic activities (i.e., as intrinsic activity for ERK1/2 phosphorylation increased, so did affinity). Genome editing revealed that the transducer that coupled the ß 2-adrenoceptor to ERK1/2 phosphorylation in response to carvedilol and other ß 2-adrenoceptor antagonists was Gαs. Collectively, these data support the concept of "functional affinity" and indicate that the ability of the ß 2-adrenoceptor to recruit Gαs may influence the affinity of the activating ligand. SIGNIFICANCE STATEMENT: In HEK293 cells overexpressing the human ß2-adrenoceptor carvedilol and four related aryloxypropanolamines behaved as ß2-adrenoceptor antagonists and partial agonists of ERK1/2 phosphorylation with rank orders of affinity that were distinct. These data imply that carvedilol and other ß-blockers can stabilize the ß2-adrenoceptor in different affinity conformations that are revealed when functionally distinct responses are measured. This is the basis for the pharmacological concept of "functional affinity."

ß 2-Adrenoceptor Agonists Promote Extracellular Signal-Regulated Kinase 1/2 Dephosphorylation in Human Airway Epithelial Cells by Canonical, cAMP-Driven Signaling Independently of ß-Arrestin 2.

Chronic use of ß 2-adrenoceptor agonists as a monotherapy in asthma is associated with a loss of disease control and an increased risk of mortality. Herein, we tested the hypothesis that ß 2-adrenoceptor agonists, including formoterol, promote biased, ß-arrestin (Arr) 2-dependent activation of the mitogen-activated protein kinases, ERK1/2, in human airway epithelial cells and, thereby, effect changes in gene expression that could contribute to their adverse clinical outcomes. Three airway epithelial cell models were used: the BEAS-2B cell line, human primary bronchial epithelial cells (HBEC) grown in submersion culture, and HBEC that were highly differentiated at an air-liquid interface. Unexpectedly, treatment of all epithelial cell models with formoterol decreased basal ERK1/2 phosphorylation. This was mediated by cAMP-dependent protein kinase and involved the inactivation of C-rapidly-activated fibrosarcoma, which attenuated downstream ERK1/2 activity, and the induction of dual-specificity phosphatase 1. Formoterol also inhibited the basal expression of early growth response-1, an ERK1/2-regulated gene that controls cell growth and repair in the airways. Neither carvedilol, a ß 2-adrenoceptor agonist biased toward ßArr2, nor formoterol promoted ERK1/2 phosphorylation in BEAS-2B cells, although ß 2-adrenoceptor desensitization was compromised in ARRB2-deficient cells. Collectively, these results contest the hypothesis that formoterol activates ERK1/2 in airway epithelia by nucleating a ßArr2 signaling complex; instead, they indicate that ß 2-adrenoceptor agonists inhibit constitutive ERK1/2 activity in a cAMP-dependent manner. These findings are the antithesis of results obtained using acutely challenged native and engineered HEK293 cells, which have been used extensively to study mechanisms of ERK1/2 activation, and highlight the cell type dependence of ß 2-adrenoceptor-mediated signaling. SIGNIFICANCE STATEMENT: It has been proposed that the adverse effects of ß 2-adrenoceptor agonist monotherapy in asthma are mediated by genomic mechanisms that occur principally in airway epithelial cells and are the result of ß-arrestin 2-dependent activation of ERK1/2. This study shows that ß 2-adrenoceptor agonists, paradoxically, reduced ERK1/2 phosphorylation in airway epithelia by disrupting upstream rat sarcoma-C-rapidly accelerated fibrosarcoma complex formation and inducing dual-specificity phosphatase 1. Moreover, these effects were cAMP-dependent protein kinase-dependent, suggesting that ß 2-adrenoceptor agonists were not biased toward ß-arrestin 2 and acted via canonical, cAMP-dependent signaling.

Transcriptome-Level Interactions between Budesonide and Formoterol Provide Insight into the Mechanism of Action of Inhaled Corticosteroid/Long-Acting ß 2-Adrenoceptor Agonist Combination Therapy in Asthma.

In 2019, the Global Initiative for Asthma treatment guidelines were updated to recommend that inhaled corticosteroid (ICS)/long-acting ß 2-adrenoceptor agonist (LABA) combination therapy should be a first-in-line treatment option for asthma. Although clinically superior to ICS, mechanisms underlying the efficacy of this combination therapy remain unclear. We hypothesized the existence of transcriptomic interactions, an effect that was tested in BEAS-2B and primary human bronchial epithelial cells (pHBECs) using formoterol and budesonide as representative LABA and ICS, respectively. In BEAS-2B cells, formoterol produced 267 (212 induced; 55 repressed) gene expression changes (≥2/≤0.5-fold) that were dominated by rapidly (1 to 2 hours) upregulated transcripts. Conversely, budesonide induced 370 and repressed 413 mRNAs, which occurred predominantly at 6-18 hours and was preceded by transcripts enriched in transcriptional regulators. Significantly, genes regulated by both formoterol and budesonide were over-represented in the genome; moreover, budesonide plus formoterol induced and repressed 609 and 577 mRNAs, respectively, of which ∼one-third failed the cutoff criterion for either treatment alone. Although induction of many mRNAs by budesonide plus formoterol was supra-additive, the dominant (and potentially beneficial) effect of budesonide on formoterol-induced transcripts, including those encoding many proinflammatory proteins, was repression. Gene ontology analysis of the budesonide-modulated transcriptome returned enriched terms for transcription, apoptosis, proliferation, differentiation, development, and migration. This "functional" ICS signature was augmented in the presence of formoterol. Thus, LABAs modulate glucocorticoid action, and comparable transcriptome-wide interactions in pHBECs imply that such effects may be extrapolated to individuals with asthma taking combination therapy. Although repression of formoterol-induced proinflammatory mRNAs should be beneficial, the pathophysiological consequences of other interactions require investigation. SIGNIFICANCE STATEMENT: In human bronchial epithelial cells, formoterol, a long-acting ß 2-adrenoceptor agonist (LABA), enhanced the expression of inflammatory genes, and many of these changes were reduced by the glucocorticoid budesonide. Conversely, the ability of formoterol to enhance both gene induction and repression by budesonide provides mechanistic insight as to how adding a LABA to an inhaled corticosteroid may improve clinical outcomes in asthma.

Prostanoid Receptors of the EP4-Subtype Mediate Gene Expression Changes in Human Airway Epithelial Cells with Potential Anti-Inflammatory Activity.

There is a clear, unmet clinical need to identify new drugs to treat individuals with asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) in whom current medications are either inactive or suboptimal. In preclinical models, EP4-receptor agonists display efficacy, but their mechanism of action is unclear. In this study, using human bronchial epithelial cells as a therapeutically relevant drug target, we hypothesized that changes in gene expression may play an important role. Several prostanoid receptor mRNAs were detected in BEAS-2B cells, human primary bronchial epithelial cells (HBECs) grown in submersion culture and HBECs grown at an air-liquid interface with PTGER4 predominating. By using the activation of a cAMP response element reporter in BEAS-2B cells as a surrogate of gene expression, Schild analysis determined that PTGER4 mRNAs encoded functional EP4-receptors. Moreover, inhibitors of phosphodiesterase 4 (roflumilast N-oxide [RNO]) and cAMP-dependent protein kinase augmented and attenuated, respectively, reporter activation induced by 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxo-cyclopentyl]sulphanylpropylsulphanyl] acetic acid (ONO-AE1-329), a selective EP4-receptor agonist. ONO-AE1-329 also enhanced dexamethasone-induced activation of a glucocorticoid response element reporter in BEAS-2B cells, which was similarly potentiated by RNO. In each airway epithelial cell variant, numerous genes that may impart therapeutic benefit in asthma, COPD, and/or IPF were differentially expressed by ONO-AE1-329, and those changes were often augmented by RNO and/or dexamethasone. We submit that an EP4-receptor agonist, either alone or as a combination therapy, may be beneficial in individuals with chronic lung diseases in whom current treatment options are inadequate. SIGNIFICANCE STATEMENT: Using human bronchial epithelial cells as a therapeutically relevant drug target, we report that EP4-receptor activation promoted gene expression changes that could provide therapeutic benefit in individuals with asthma, COPD, and IPF in whom current treatment options are ineffective or suboptimal.

Impact of Phosphodiesterase 4 Inhibition on the Operational Efficacy, Response Maxima, and Kinetics of Indacaterol-Induced Gene Expression Changes in BEAS-2B Airway Epithelial Cells: A Global Transcriptomic Analysis.

The effects of phosphodiesterase (PDE) 4 inhibitors on gene expression changes in BEAS-2B human airway epithelial cells are reported and discussed in relation to the mechanism(s) of action of roflumilast in chronic obstructive pulmonary disease (COPD). Microarray-based gene expression profiling failed to identify mRNA transcripts that were differentially regulated by the PDE4 inhibitor 6-[3-(dimethylcarbamoyl)benzenesulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GSK 256066) after 1, 2, 6, or 18 hours of exposure. However, real-time polymerase chain reaction analysis revealed that GSK 256066 was a weak stimulus, and the negative microarray results reflected low statistical power due to small sample sizes. Furthermore, GSK 256066, roflumilast, and its biologically active metabolite roflumilast N-oxide generally potentiated gene expression changes produced by the long-acting ß 2-adrenoceptor agonists (LABAs) salmeterol, indacaterol, and formoterol. Many of these genes encode proteins with antiviral, anti-inflammatory, and antibacterial activities that could contribute to the clinical efficacy of roflumilast in COPD. RNA-sequencing experiments established that the sensitivity of genes to salmeterol varied by ∼7.5-fold. Consequently, the degree to which a PDE4 inhibitor potentiated the effect of a given concentration of LABA was gene-dependent. Operational model fitting of concentration-response curve data from cells subjected to fractional, ß 2-adrenoceptor inactivation determined that PDE4 inhibition increased the potency and doubled the efficacy of LABAs. Thus, adding roflumilast to standard triple therapy, as COPD guidelines recommend, may have clinical relevance, especially in target tissues where LABAs behave as partial agonists. Collectively, these results suggest that the genomic impact of roflumilast, including its ability to augment LABA-induced gene expression changes, may contribute to its therapeutic activity in COPD.

Long-Acting ß2-Adrenoceptor Agonists Enhance Glucocorticoid Receptor (GR)-Mediated Transcription by Gene-Specific Mechanisms Rather Than Generic Effects via GR.

In asthma, the clinical efficacy of inhaled corticosteroids (ICSs) is enhanced by long-acting ß2-adrenoceptor agonists (LABAs). ICSs, or more accurately, glucocorticoids, promote therapeutically relevant changes in gene expression, and, in primary human bronchial epithelial cells (pHBECs) and airway smooth muscle cells, this genomic effect can be enhanced by a LABA. Modeling this interaction in human bronchial airway epithelial BEAS-2B cells transfected with a 2× glucocorticoid response element (2×GRE)-driven luciferase reporter showed glucocorticoid-induced transcription to be enhanced 2- to 3-fold by LABA. This glucocorticoid receptor (GR; NR3C1)-dependent effect occurred rapidly, was insensitive to protein synthesis inhibition, and was maximal when glucocorticoid and LABA were added concurrently. The ability of LABA to enhance GR-mediated transcription was not associated with changes in GR expression, serine (Ser203, Ser211, Ser226) phosphorylation, ligand affinity, or nuclear translocation. Chromatin immunoprecipitation demonstrated that glucocorticoid-induced recruitment of GR to the integrated 2×GRE reporter and multiple gene loci, whose mRNAs were unaffected or enhanced by LABA, was also unchanged by LABA. Transcriptomic analysis revealed glucocorticoid-induced mRNAs were variably enhanced, unaffected, or repressed by LABA. Thus, events leading to GR binding at target genes are not the primary explanation for how LABAs modulate GR-mediated transcription. As many glucocorticoid-induced genes are independently induced by LABA, gene-specific control by GR- and LABA-activated transcription factors may explain these observations. Because LABAs promote similar effects in pHBECs, therapeutic relevance is likely. These data illustrate the need to understand gene function(s), and the mechanisms leading to gene-specific induction, if existing ICS/LABA combination therapies are to be improved.

Analysis of the Indacaterol-Regulated Transcriptome in Human Airway Epithelial Cells Implicates Gene Expression Changes in the Adverse and Therapeutic Effects of ß2-Adrenoceptor Agonists.

The contribution of gene expression changes to the adverse and therapeutic effects of ß2-adrenoceptor agonists in asthma was investigated using human airway epithelial cells as a therapeutically relevant target. Operational model-fitting established that the long-acting ß2-adrenoceptor agonists (LABA) indacaterol, salmeterol, formoterol, and picumeterol were full agonists on BEAS-2B cells transfected with a cAMP-response element reporter but differed in efficacy (indacaterol ≥ formoterol > salmeterol ≥ picumeterol). The transcriptomic signature of indacaterol in BEAS-2B cells identified 180, 368, 252, and 10 genes that were differentially expressed (>1.5- to <0.67-fold) after 1-, 2-, 6-, and 18-hour of exposure, respectively. Many upregulated genes (e.g., AREG, BDNF, CCL20, CXCL2, EDN1, IL6, IL15, IL20) encode proteins with proinflammatory activity and are annotated by several, enriched gene ontology (GO) terms, including cellular response to interleukin-1, cytokine activity, and positive regulation of neutrophil chemotaxis The general enriched GO term extracellular space was also associated with indacaterol-induced genes, and many of those, including CRISPLD2, DMBT1, GAS1, and SOCS3, have putative anti-inflammatory, antibacterial, and/or antiviral activity. Numerous indacaterol-regulated genes were also induced or repressed in BEAS-2B cells and human primary bronchial epithelial cells by the low efficacy LABA salmeterol, indicating that this genomic effect was neither unique to indacaterol nor restricted to the BEAS-2B airway epithelial cell line. Collectively, these data suggest that the consequences of inhaling a ß2-adrenoceptor agonist may be complex and involve widespread changes in gene expression. We propose that this genomic effect represents a generally unappreciated mechanism that may contribute to the adverse and therapeutic actions of ß2-adrenoceptor agonists in asthma.

GS-5759, a Bifunctional ß2-Adrenoceptor Agonist and Phosphodiesterase 4 Inhibitor for Chronic Obstructive Pulmonary Disease with a Unique Mode of Action: Effects on Gene Expression in Human Airway Epithelial Cells.

(R)-6-[(3-{[4-(5-{[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino}pent-1-yn-1-yl)phenyl] carbamoyl}phenyl)sulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide trifluoroacetic acid (GS-5759) is a bifunctional ligand composed of a quinolinone-containing pharmacophore [ß2-adrenoceptor agonist orthostere (ß2A)] found in several ß2-adrenoceptor agonists, including indacaterol, linked covalently to a phosphodiesterase 4 (PDE4) inhibitor related to 6-[3-(dimethylcarbamoyl)benzenesulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GSK 256066) by a pent-1-yn-1-ylbenzene spacer. GS-5759 had a similar affinity for PDE4B1 and the native ß2-adrenoceptor expressed on BEAS-2B human airway epithelial cells. However, compared with the monofunctional parent compound, ß2A, the KA of GS-5759 for the ß2-adrenoceptor was 35-fold lower. Schild analysis determined that the affinities of the ß-adrenoceptor antagonists, (2R,3R)-1-[(2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl) amino]-2-butanol (ICI 118551) and propranolol, were agonist-dependent, being significantly lower for GS-5759 than ß2A. Collectively, these data can be explained by "forced proximity," bivalent binding where the pharmacophore in GS-5759 responsible for PDE4 inhibition also interacts with a nonallosteric domain within the ß2-adrenoceptor that enhances the affinity of ß2A for the orthosteric site. Microarray analyses revealed that, after 2-hour exposure, GS-5759 increased the expression of >3500 genes in BEAS-2B cells that were highly rank-order correlated with gene expression changes produced by indacaterol and GSK 256066 in combination (Ind/GSK). Moreover, the line of regression began close to the origin with a slope of 0.88, indicating that the magnitude of most gene expression changes produced by Ind/GSK was quantitatively replicated by GS-5759. Thus, GS-5759 is a novel compound exhibiting dual ß2-adrenoceptor agonism and PDE4 inhibition with potential to interact on target tissues in a synergistic manner. Such polypharmacological behavior may be particularly effective in chronic obstructive pulmonary disease and other complex disorders where multiple processes interact to promote disease pathogenesis and progression.

Superiority of combined phosphodiesterase PDE3/PDE4 inhibition over PDE4 inhibition alone on glucocorticoid- and long-acting ß2-adrenoceptor agonist-induced gene expression in human airway epithelial cells.

Glucocorticoids, also known as corticosteroids, induce effector gene transcription as a part of their anti-inflammatory mechanisms of action. Such genomic effects can be significantly enhanced by long-acting ß2-adrenoceptor agonists (LABAs) and may contribute to the clinical superiority of inhaled corticosteroid (ICS)/LABA combinations in asthma and chronic obstructive pulmonary disease (COPD) over ICSs alone. Using models of cAMP- and glucocorticoid-induced transcription in human bronchial epithelial BEAS-2B cells, we show that combining inhibitors of phosphodiesterase (PDE) 3 and PDE4 provides greater benefits compared with inhibiting either PDE alone. In respect to cAMP-dependent transcription, inhibitors of PDE3 (siguazodan, cilostazol) and PDE4 (rolipram, GSK256066, roflumilast N-oxide) each sensitized to the LABA, formoterol. This effect was magnified by dual PDE3 and PDE4 inhibition. Siguazodan plus rolipram was also more effective at inducing cAMP-dependent transcription than either inhibitor alone. Conversely, the concentration-response curve describing the enhancement of dexamethasone-induced, glucocorticoid response element-dependent transcription by formoterol was displaced to the left by PDE4, but not PDE3, inhibition. Overall, similar effects were described for bona fide genes, including RGS2, CD200, and CRISPLD2. Importantly, the combination of siguazodan plus rolipram prolonged the duration of gene expression induced by formoterol, dexamethasone, or dexamethasone plus formoterol. This was most apparent for RGS2, a bronchoprotective gene that may also reduce the proinflammatory effects of constrictor mediators. Collectively, these data provide a rationale for the use of PDE3 and PDE4 inhibitors in the treatment of COPD and asthma where they may enhance, sensitize, and prolong the effects of LABA/ICS combination therapies.

Induction of regulator of G-protein signaling 2 expression by long-acting ß2-adrenoceptor agonists and glucocorticoids in human airway epithelial cells.

In asthma and chronic obstructive pulmonary disease (COPD) multiple mediators act on Gαq-linked G-protein-coupled receptors (GPCRs) to cause bronchoconstriction. However, acting on the airway epithelium, such mediators may also elicit inflammatory responses. In human bronchial epithelial BEAS-2B cells (bronchial epithelium + adenovirus 12-SV40 hybrid), regulator of G-protein signaling (RGS) 2 mRNA and protein were synergistically induced in response to combinations of long-acting ß2-adrenoceptor agonist (LABA) (salmeterol, formoterol) plus glucocorticoid (dexamethasone, fluticasone propionate, budesonide). Equivalent responses occurred in primary human bronchial epithelial cells. Concentrations of glucocorticoid plus LABA required to induce RGS2 expression in BEAS-2B cells were consistent with the levels achieved therapeutically in the lungs. As RGS2 is a GTPase-activating protein that switches off Gαq, intracellular free calcium ([Ca(2+)]i) flux was used as a surrogate of responses induced by histamine, methacholine, and the thromboxane receptor agonist U46619 [(Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid]. This was significantly attenuated by salmeterol plus dexamethasone pretreatment, or RGS2 overexpression, and the protective effect of salmeterol plus dexamethasone was abolished by RGS2 RNA silencing. Although methacholine and U46619 induced interleukin-8 (IL-8) release and this was inhibited by RGS2 overexpression, the repression of U46619-induced IL-8 release by salmeterol plus dexamethasone was unaffected by RGS2 knockdown. Given a role for Gαq-mediated pathways in inducing IL-8 release, we propose that RGS2 acts redundantly with other effector processes to repress IL-8 expression. Thus, RGS2 expression is a novel effector mechanism in the airway epithelium that is induced by glucocorticoid/LABA combinations. This could contribute to the efficacy of glucocorticoid/LABA combinations in asthma and COPD.

ß2-Adrenoceptor agonist-induced RGS2 expression is a genomic mechanism of bronchoprotection that is enhanced by glucocorticoids.

In asthma and chronic obstructive pulmonary disease, activation of G(q)-protein-coupled receptors causes bronchoconstriction. In each case, the management of moderate-to-severe disease uses inhaled corticosteroid (glucocorticoid)/long-acting ß(2)-adrenoceptor agonist (LABA) combination therapies, which are more efficacious than either monotherapy alone. In primary human airway smooth muscle cells, glucocorticoid/LABA combinations synergistically induce the expression of regulator of G-protein signaling 2 (RGS2), a GTPase-activating protein that attenuates G(q) signaling. Functionally, RGS2 reduced intracellular free calcium flux elicited by histamine, methacholine, leukotrienes, and other spasmogens. Furthermore, protection against spasmogen-increased intracellular free calcium, following treatment for 6 h with LABA plus corticosteroid, was dependent on RGS2. Finally, Rgs2-deficient mice revealed enhanced bronchoconstriction to spasmogens and an absence of LABA-induced bronchoprotection. These data identify RGS2 gene expression as a genomic mechanism of bronchoprotection that is induced by glucocorticoids plus LABAs in human airway smooth muscle and provide a rational explanation for the clinical efficacy of inhaled corticosteroid (glucocorticoid)/LABA combinations in obstructive airways diseases.

Phosphodiesterase 4 inhibitors augment the ability of formoterol to enhance glucocorticoid-dependent gene transcription in human airway epithelial cells: a novel mechanism for the clinical efficacy of roflumilast in severe chronic obstructive pulmonary disease.

Post-hoc analysis of two phase III clinical studies found that the phosphodiesterase 4 (PDE4) inhibitor, roflumilast, reduced exacerbation frequency in patients with severe chronic obstructive pulmonary disease (COPD) who were taking inhaled corticosteroids (ICS) concomitantly, whereas patients not taking ICS derived no such benefit. In contrast, in two different trials also performed in patients with severe COPD, roflumilast reduced exacerbation rates in the absence of ICS, indicating that PDE4 inhibition alone is sufficient for therapeutic activity to be realized. Given that roflumilast is recommended as an "add-on" medication to patients with severe disease who will inevitably be taking a long-acting ß2-adrenoceptor agonist (LABA)/ICS combination therapy, we tested the hypothesis that roflumilast augments the ability of glucocorticoids to induce genes with anti-inflammatory activity. Using a glucocorticoid response element (GRE) luciferase reporter transfected into human airway epithelial cells [both bronchial epithelium + adenovirus 12 - SV40 hybrid (BEAS-2B) cells and primary cultures], roflumilast enhanced fluticasone propionate-induced GRE-dependent transcription. Roflumilast also produced a sinistral displacement of the concentration-response curves that described the augmentation of GRE-dependent transcription by the LABA formoterol. In BEAS-2B cells and primary airway epithelia, roflumilast interacted with formoterol in a positive cooperative manner to enhance the expression of several glucocorticoid-inducible genes that have anti-inflammatory potential. We suggest that the ability of roflumilast and formoterol to interact in this way supports the concept that these drugs together may impart clinical benefit beyond that achievable by an ICS alone, a PDE4 inhibitor alone, or an ICS/LABA combination therapy. Roflumilast may, therefore, be especially effective in patients with severe COPD.

Concurrent agonism of adenosine A2B and glucocorticoid receptors in human airway epithelial cells cooperatively induces genes with anti-inflammatory potential: a novel approach to treat chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a neutrophilic inflammatory disorder that is weakly responsive to glucocorticoids. Identification of ways to enhance the anti-inflammatory activity of glucocorticoids is, therefore, a major research objective. Adenosine receptor agonists that target the A2B-receptor subtype are efficacious in several cell-based assays and preclinical models of inflammation. Accordingly, the present study was designed to determine if a selective A2B-receptor agonist, 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulphanyl]acetamide (Bay 60-6583), and a glucocorticoid, dexamethasone, in combination display putative anti-inflammatory activity that is superior to either drug alone. In BEAS-2B human airway epithelial cells stably transfected with cAMP-response element (CRE) and glucocorticoid response element (GRE) reporter constructs, Bay 60-6583 promoted CRE-dependent transcription and enhanced GRE-dependent transcription by an adenosine A2B-receptor-mediated mechanism that was associated with cAMP formation and abolished by an inhibitor of cAMP-dependent protein kinase. Analysis of the concentration-response relationship that described the enhancement of GRE-dependent transcription showed that Bay 60-6583 increased the magnitude of response without affecting the potency of dexamethasone. Bay 60-6583 and dexamethasone also induced a panel of genes that, collectively, could have benefit in COPD. These were categorized into genes that were induced in a positive cooperative manner (RGS2, p57(kip2)), an additive manner (TTP, BRL-1), or by Bay 60-6583 (CD200, CRISPLD2, SOCS3) or dexamethasone (GILZ) only. Thus, the gene induction "fingerprints" produced by Bay 60-6583 and dexamethasone, alone and in combination, were distinct. Collectively, through their actions on gene expression, an adenosine A2B-receptor agonist and a glucocorticoid administered together may have utility in the treatment of inflammatory disorders that respond suboptimally to glucocorticoids as a monotherapy.

Prostaglandin E2 represses interleukin 1 beta-induced inflammatory mediator output from pregnant human myometrial cells through the EP2 and EP4 receptors.

Inflammatory mediators, including prostaglandins, cytokines, and chemokines, are strongly implicated in the mechanism of human labor, though their precise roles remain unknown. Here we demonstrate that interleukin 1 beta (IL-1beta) significantly increased the expression and release of interleukin-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), and granulocyte macrophage colony-stimulating factor (CSF2) by primary human myometrial cells. However, this effect was repressed by prostaglandin E(2) (PGE(2)). As PGE(2) can activate four distinct PGE(2) receptors (EP(1), EP(2), EP(3), and EP(4)) to elicit various responses, we sought to define the EP receptor(s) responsible for this repression. Using selective EP receptor agonists and a selective EP(4) antagonist, we show that PGE(2) mediates the repression of IL-1beta-induced release of CXCL8, CCL2, and CSF2 via activation of the EP(2) and EP(4) receptors. The use of siRNA gene-specific knockdown further confirmed a role for both receptors. Real-time RT-PCR demonstrated that EP(2) was the most highly expressed of all four EP receptors at the mRNA level in human myometrial cells, and immunocytochemistry showed that EP(2) protein is abundantly present throughout the cells. Interestingly, PGE(2) does not appear to reduce mRNA expression of CXCL8, CCL2, and CSF2. Our results demonstrate that PGE(2) can elicit anti-inflammatory responses via activation of the EP(2) and EP(4) receptors in lower segment term pregnant human myometrial cells. Further elucidation of the EP receptor-mediated signaling pathways in the pregnant human uterus may be beneficial for optimizing the maintenance of pregnancy, induction of labor or indeed treatment of preterm labor.

α and ß catalytic subunits of cAMP-dependent protein kinase regulate formoterol-induced inflammatory gene expression changes in human bronchial epithelial cells.

BACKGROUND AND PURPOSE: It has been proposed that genomic mechanisms contribute to adverse effects often experienced by asthmatic subjects who take regular, inhaled ß2 -adrenoceptor agonists as a monotherapy. Moreover, data from preclinical models of asthma suggest that these gene expression changes are mediated by ß-arrestin-2 rather than PKA. Herein, we tested this hypothesis by comparing the genomic effects of formoterol, a ß2 -adrenoceptor agonist, with forskolin in human primary bronchial epithelial cells (HBEC). EXPERIMENTAL APPROACH: Gene expression changes were determined by RNA-sequencing. Gene silencing and genome editing were employed to explore the roles of ß-arrestin-2 and PKA. KEY RESULTS: The formoterol-regulated transcriptome in HBEC treated concurrently with TNFα was defined by 1480 unique gene expression changes. TNFα-induced transcripts modulated by formoterol were annotated with enriched gene ontology terms related to inflammation and proliferation, notably "GO:0070374~positive regulation of ERK1 and ERK2 cascade," which is an apparent ß-arrestin-2 target. However, expression of the formoterol- and forskolin-regulated transcriptomes were highly rank-order correlated and the effects of formoterol on TNFα-induced inflammatory genes were abolished by an inhibitor of PKA. Furthermore, formoterol-induced gene expression changes in BEAS-2B bronchial epithelial cell clones deficient in ß-arrestin-2 were comparable with those expressed by their parental counterparts. Contrariwise, gene expression was partially inhibited in clones lacking the α-catalytic subunit (Cα) of PKA and abolished following the additional knockdown of the ß-catalytic subunit (Cß) paralogue. CONCLUSIONS: The effects of formoterol on inflammatory gene expression in airway epithelia are mediated by PKA and involve the cooperation of PKA-Cα and PKA-Cß.

Evidence for a second receptor for prostacyclin on human airway epithelial cells that mediates inhibition of CXCL9 and CXCL10 release.

Herein we provide evidence for the coexpression of two distinct prostacyclin (PGI(2)) 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 PGI(2) 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, PGI(2), taprostene, and iloprost activated both reporters whereas 15-deoxy-TIC was inert. Conversely, 15-deoxy-TIC, PGI(2), 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 PGI(2), 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 "IP(2)" subtype.

Inflammatory stimuli inhibit glucocorticoid-dependent transactivation in human pulmonary epithelial cells: rescue by long-acting beta2-adrenoceptor agonists.

By repressing inflammatory gene expression, glucocorticoids are the most effective treatment for chronic inflammatory diseases such as asthma. However, in some patients with severe disease, or who smoke or suffer from chronic obstructive pulmonary disease, glucocorticoids are poorly effective. Although many investigators focus on defects in the repression of inflammatory gene expression, glucocorticoids also induce (transactivate) the expression of numerous genes to elicit anti-inflammatory effects. Using human bronchial epithelial (BEAS-2B) and pulmonary (A549) cells, we show that cytokines [tumor necrosis factor α (TNFα) and interleukin 1ß], mitogens [fetal calf serum (FCS) and phorbol ester], cigarette smoke, and a G(q)-linked G protein-coupled receptor agonist attenuate simple glucocorticoid response element (GRE)-dependent transcription. With TNFα and FCS, this effect was not overcome by increasing concentrations of dexamethasone, budesonide, or fluticasone propionate. Thus, the maximal ability of the glucocorticoid to promote GRE-dependent transcription was reduced, and this was shown additionally for the glucocorticoid-induced gene p57(KIP2). The long-acting ß(2)-adrenoceptor agonists (LABAs) formoterol fumarate and salmeterol xinafoate enhanced simple GRE-dependent transcription to a level that could not be achieved by glucocorticoid alone. In the presence of TNFα or FCS, which repressed glucocorticoid responsiveness, these LABAs restored glucocorticoid-dependent transcription to levels that were achieved by glucocorticoid alone. Given the existence of genes, such as p57(KIP2), which may mediate anti-inflammatory actions of glucocorticoids, we propose that repression of transactivation represents a mechanism for glucocorticoid resistance and for understanding the clinical benefit of LABAs as an add-on therapy in asthma and chronic obstructive pulmonary disease.

Salbutamol delays human eosinophil apoptosis via a cAMP-dependent mechanism.

Eosinophils play a major role in asthma. One described mechanism leading to the impaired clearance of these cells from the lung is the delay in their programmed cell death (apoptosis). ß(2)-Adrenoceptor agonists have been shown to prolong survival and delay apoptosis of eosinophils. The aim of the present study was to evaluate the mechanisms, especially the role of cAMP pathway, in the prolongation of human eosinophil survival by a selective ß(2)-agonist salbutamol. Isolated human peripheral blood eosinophils were cultured in the absence or presence of a ß(2)-agonist salbutamol and the indicated antagonists/inhibitors under sterile conditions. Apoptosis was measured by using the relative DNA fragmentation assay and Annexin-V binding. Salbutamol prolonged survival of human eosinophils and it was inhibited by a ß-receptor antagonist propranolol and mimicked by cell-permeant cAMP analogues dibutyryl- and 8-bromo-cAMP. Pharmacological inhibitors of adenylyl cyclase (SQ-22,536) and protein kinase A (Rp-8-CPT-cAMPS) antagonized the effects of salbutamol. The survival-prolonging action of salbutamol was potentiated by a phosphodiesterase inhibitor rolipram (EC(50) for the salbutamol effect was 13.6 ± 4.0 and 8.1 ± 3.1 nM in the absence and presence of rolipram, respectively; p=0.0142, n=10). In contrast, inhibition of Ca(2+)-activated K(+)-channels by apamin, charybdotoxin, iberiotoxin or paxilline did not affect the ability of salbutamol to prolong eosinophil survival. Taken together, the present results suggest that salbutamol at clinically relevant concentrations decreases apoptosis in human eosinophils by activating the cannonical ß(2)-receptor-adenylyl cyclase-cAMP-protein kinase A pathway.

Selective prostacyclin receptor agonism augments glucocorticoid-induced gene expression in human bronchial epithelial cells.

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 p57(kip2)) 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.