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

Abstract

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.

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

“…Determination of mRNA Expression by RNA-Seq. RNA extracted from BEAS-2B cells (N = 4) and HBEC grown in submersion culture and at an ALI (both N = 5) was submitted to the Centre for Health Genomics and Informatics, University of Calgary, for sequencing as described previously (Joshi et al, 2019). Goodquality reads were mapped to the GRCh37/hg19 reference human genome, quantified using Kallisto (v0.42.4) and differential expression analysis performed at the gene level using the R package, Sleuth (v0.30.0).…”

“…RNA-seq of BEAS-2B cells determined that RNO (1PM; 2h) had little or no effect on the baseline expression of a panel of genes known to be induced by cAMP (BinMahfouz et al, 2015;Giembycz and Newton, 2014;Greer et al, 2013;Joshi et al, 2019;Yan et al, 2018). In contrast, ONO-AE1-329 (1nM and/or 1PM; 2h) significantly up-regulated these genes relative to vehicle matched for time, which was potentiated by RNO in an apparently synergistic manner.…”

“…in BEAS-2B cells and HBEC grown in submersion culture were also mined from prior microarray data sets (accession numbers: GSE27973 and GSE106710), which were generated with Affymetrix U133 plus 2.0 gene chips (Joshi et al, 2019;Proud et al, 2012;Yan et al, 2018).…”

“…In Gsα-dependent signaling, this can be achieved by "adding-on" a PDE inhibitor or by incorporating both pharmacophores in a bifunctional ligand. These approaches have been tested in proof-of-concept experiments with β 2 -adrenoceptor agonists and shown to be feasible (Forkuo et al, 2016;Joshi et al, 2019;Joshi et al, 2017). The extent to which ONO-AE1-329 displays biased agonism at its cognate receptor is unexplored.…”

“…Finally, the genomic impact of an EP 4 -receptor agonist (ONO-AE1-329) was assessed in each epithelial cell variant. To provide clinical insight, these determinations were made in the presence of a glucocorticoid (dexamethasone) and a PDE4 inhibitor (roflumilast N-oxide [RNO]), which exert profound genomic effects in subjects with respiratory diseases (Govoni et al, 2020;Yick et al, 2013) and often cooperate in an additive or synergistic manner with Gs-coupled receptor agonists (Greer et al, 2013;Joshi et al, 2019;Wilson et al, 2009). The LABAs, indacaterol, vilanterol and formoterol were used throughout as clinically-relevant comparators.…”