Transcriptomic Analysis Reveals Different Sensitivities of Genes to EP4 Receptor Agonist Stimulation in BEAS‐2B Human Bronchial Epithelial Cells

Abstract

IntroductionProstanoid EP4‐receptors are highly expressed on human airway epithelial cells (AECs) and typically mediate canonical, Gs‐cAMP signaling. EP4‐receptor agonists exhibit bronchodilator and anti‐inflammatory activity and could represent novel therapeutic candidates to treat obstructive lung diseases. We have reported previously that long‐acting β2‐adrenoceptor agonists (LABAs) promote significant gene expression changes in human AECs, which could contribute to their clinical efficacy. In this study, we have determined the EP4‐receptor‐regulated transcriptome in BEAS‐2B cells using the selective agonist, ONO‐AE1‐329, using the LABA, vilanterol, as a comparator.MethodsBEAS‐2B cells were treated for 2h with vehicle, and two concentrations of ONO‐AE1‐329 (1nM, ONO1; 1000nM, ONO1000). A maximally effective concentration of vilanterol (100nM, Vil100) was included to define maximum responses. Gene expression changes were determined by RNA sequencing. Simulated E/[A] curves were generated from ONO1 and ONO1000 data assuming a unity Hill coefficient for each gene expression change (≥3‐fold; FDR≤5%). BEAS‐2B cells transfected with a cAMP response element (CRE) luciferase reporter were used to determine the affinity (KA) of ONO‐AE1‐329 for the EP4‐receptor. This was estimated by operational model fitting of E/[A] curve data before and after fractional receptor depletion using siRNA mediated PTGER4 gene silencing. Occupancy‐response relationships for ONO‐AE1‐329‐induced genes were constructed using the simulated E/[A] curves and the KA value determined in CRE reporter cells.ResultsTranscriptomic changes produced by Vil100 and ONO1000 were highly correlated suggesting that the β2‐adrenoceptor and the EP4‐receptor shared a common mechanism of action. Vilanterol was a full agonist on all induced genes whereas ONO‐AE1‐329 was a partial agonist with intrinsic activity values that varied between genes. The KA of ONO‐AE1‐329 was found to be consistent with a previous radioligand binding study. ONO‐AE1‐329‐induced genes differed in sensitivity by ~18‐fold with NR4A3 and PDE4B representing transcripts at the extremes of this sensitivity spectrum. Accordingly, receptor occupancy‐response relationships varied from almost linear (where response is directly proportional to occupancy; e.g., NR4A3) to markedly hyperbolic (indicative of a significant receptor reserve; e.g., PDE4B) (Fig. 1).ConclusionsDifferences in the sensitivities of genes to ONO‐AE1‐329 indicate that the EP4‐receptor interprets equivalent degrees of receptor occupancy differently. This may be explained by ‐ a) regulation by multiple cAMP‐induced transcription factors, and/or b) variability in promoter context and co‐factor binding which may differentially affect the ability of transcription factors to interact favorably with DNA.Support or Funding InformationCIHR‐ PJT 152904Receptor occupancyresponse relationships for ONO‐AE1‐329 induced genesFigure 1