The contribution of gene expression changes to the adverse and therapeutic effects of -adrenoceptor agonists in asthma was investigated using human airway epithelial cells as a therapeutically relevant target. Operational model-fitting established that the long-acting-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., ,, ,, ,, ,) encode proteins with proinflammatory activity and are annotated by several, enriched gene ontology (GO) terms, including ,, and The general enriched GO term was also associated with indacaterol-induced genes, and many of those, including ,, and 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 -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-adrenoceptor agonists in asthma.

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(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 [β-adrenoceptor agonist orthostere (β2A)] found in several β-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 β-adrenoceptor expressed on BEAS-2B human airway epithelial cells. However, compared with the monofunctional parent compound, β2A, the K of GS-5759 for the β-adrenoceptor was 35-fold lower. Schild analysis determined that the affinities of the β-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 β2A. 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 β-adrenoceptor that enhances the affinity of β2A 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 β-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.

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 b 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 concentrationresponse curve data from cells subjected to fractional, b 2adrenoceptor 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.

Chronic use of β2-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 β2adrenoceptor agonists, including formoterol, promote biased, β-arrestin 2 (βArr2)-dependent activation of the mitogen-activated protein (MAP) 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 down-stream 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 β2-adrenoceptor agonist biased towards βArr2, nor formoterol promoted ERK1/2 phosphorylation in BEAS-2B cells although β2-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 βArr2 signaling complex; instead, they indicate that β2adrenoceptor 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 β2-adrenoceptor-mediated signaling. SIGNIFICANCE STATEMENTIt has been proposed that the adverse-effects of β2-adrenoceptor agonist monotherapy in asthma are mediated by genomic mechanisms that occur principally in airway epithelial cells and are the result of β-arrestin 2-dependent activation of ERK1/2. This study shows that β2-adrenoceptor agonists, paradoxically, reduced ERK1/2 phosphorylation in airway epithelia by disrupting upstream Ras-C-Raf complex formation and inducing DUSP1. Moreover, these effects were PKA-dependent suggesting that β2-adrenoceptor agonists were not biased toward β-arrestin 2 and acted via canonical, cAMPdependent signaling.

Running Head: Genomic effects of an EP 4-receptor agonist in human airway epithelia

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Background and Purpose It has been proposed that genomic mechanisms contribute to adverse effects often experienced by asthmatic subjects who take regular, inhaled β2‐adrenoceptor agonists as a monotherapy. Moreover, data from preclinical models of asthma suggest that these gene expression changes are mediated by β‐arrestin‐2 rather than PKA. Herein, we tested this hypothesis by comparing the genomic effects of formoterol, a β2‐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 β‐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 β‐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 β‐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 β‐catalytic subunit (Cβ) 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‐Cβ.