Genomic crosstalk between carbachol, a muscarinic receptor agonist, and indacaterol, a long-acting, ß2-adrenoceptor agonist, in human airway epithelial cells
Many patients with chronic obstructive pulmonary disease (COPD) are susceptible to recurrent exacerbations. Long-acting, muscarinic receptor antagonists protect against exacerbations suggesting that endogenous acetylcholine (ACh) could be a pro-inflammatory mediator. This idea was explored by determining if carbachol (CCh), a stable ACh analog, was a genomic stimulus in BEAS-2B bronchial epithelial cells. The ability of CCh to interact with indacaterol (Ind), a long-acting, ß2-adrenoceptor agonists (LABA), was also assessed because sympathomimetic bronchodilators can promote the expression of adverse-effect genes in airway structural cells that are often regulated by the same transcription factors. Checkerboard assays using BEAS-2B cells expressing a cAMP response element luciferase reporter determined that CCh was a weak stimulus but interacted with Ind in a supra-additive manner. Likewise, mRNA-seq revealed that CCh regulated only 20 genes in BEAS-2B cells whereas Ind and Ind+CCh were powerful genomic stimuli affecting 869 and 1027 unique mRNAs, respectively. Of those, 39 genes were induced by Ind+CCh in a supra-additive manner; for the remainder, the interaction was either additive or infra-additive. Functional annotation of the Ind-regulated transcriptome identified transcription and signaling as dominant themes with gene ontology (GO) terms associated with inflammation and immune processes being highly represented. A comparable GO signature was obtained in the presence of CCh; however, the number, magnitude and duration of gene expression changes were markedly enhanced. If genomic interactions occur between a LABA and ACh in vivo, then this may lead to the expression of adverse-effect genes that, paradoxically, could maintain features of lung pathology in COPD. Overall design: Confluent BEAS-2B cells were treated for one, two, six and eighteen hours with either vehicle (NS), indacaterol (100 nM), carbachol (10 µM), mometasone (100 nM), or the combination of mometasone and indacaterol, mometasone and carbachol, indacaterol and carbachol, or all three in combination. Total RNA was extracted, quantified, processed into mRNA libraries, and sequenced across two high-throughput sequencing kits on a NextSeq 500 instrument (Illumina). Samples from 5 replicates were submitted for sequencing.