Transcriptomic Analysis Reveals Different Sensitivities of Genes to EP ₄ Receptor Agonist Stimulation in BEAS‐2B Human Bronchial Epithelial Cells

Introduction Prostanoid EP 4 ‐receptors are highly expressed on human airway epithelial cells (AECs) and typically mediate canonical, Gs‐cAMP signaling. EP 4 ‐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 β 2 ‐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 EP 4 ‐receptor‐regulated transcriptome in BEAS‐2B cells using the selective agonist, ONO‐AE1‐329, using the LABA, vilanterol, as a comparator. Methods BEAS‐2B cells were treated for 2h with vehicle, and two concentrations of ONO‐AE1‐329 (1nM, ONO 1 ; 1000nM, ONO 1000 ). A maximally effective concentration of vilanterol (100nM, Vil 100 ) was included to define maximum responses. Gene expression changes were determined by RNA sequencing. Simulated E /[A] curves were generated from ONO 1 and ONO 1000 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 ( K A ) of ONO‐AE1‐329 for the EP 4 ‐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 K A value determined in CRE reporter cells. Results Transcriptomic changes produced by Vil 100 and ONO 1000 were highly correlated suggesting that the β 2 ‐adrenoceptor and the EP 4 ‐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 K A 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. ). Conclusions Differences in the sensitivities of genes to ONO‐AE1‐329 indicate that the EP 4 ‐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 Information CIHR‐ PJT 152904 Receptor occupancyresponse relationships for ONO‐AE1‐329 induced genes Figure 1

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