Genomic effects of an EP4-receptor agonist in airway epithelia

It has been proposed that inhaled EP4-receptor agonists could represent an alternative class of bronchodilators for the treatment of asthma that are as effective as ß2-adrenoceptor agonists. However, the genomic impact of such drugs is often overlooked despite being potentially deleterious to respiratory health. Herein, we used mRNA-seq to compare the genomic impact of ONO-AE1-329 (an EP4-receptor agonist) and vilanterol (a ß2-adrenoceptor agonist) in BEAS-2B human airway epithelial cells. We also determined if an increase in cAMP mediated by different GPCRs promoted distinct transcriptional signatures by expanding this enquiry to include the adenosine A2B- and I-prostanoid receptor agonists, Bay-60-6583 and taprostene, respectively. Maximally-effective concentrations of ONO-AE1-329 and vilanterol significantly regulated (q = 0.05; =1.5-/=0.67-fold) 232 and 320 genes, respectively of which 217 were shared. Spearman analysis showed these gene expression changes to be highly rank order correlated indicating that the functional overlap between the two interventions should be considerable. Unexpectedly, the genomic effects of ONO-AE1-329, vilanterol, Bay 60-6583 and taprostene were also highly rank order correlated. This finding raises the prospect that cAMP generated by any GPCR would initiate the same transcriptional program. Nevertheless, relative to vilanterol, ONO-AE1-329 typically behaved as a partial agonist with a potency and intrinsic activity that varied across transcripts. Collectively, this investigation demonstrated that each ONO-AE1-329-regulated gene differs in sensitivity to cAMP and is defined by a unique receptor occupancy-response relationship. Moreover, if this relatively modest genomic response in BEAS-2B cells is reproduced in vivo, then inhaled EP4-receptor agonists could represent a safer class of bronchodilators. Overall design: Confluent BEAS-2B cells were treated for two hours with vehicle, RNO (1 µM), a maximal concentration of ONO-AE1-329 (1 µM), two sub-maximal concentrations of ONO-AE1-329 (0.1 nM and 1 nM), a maximal concentration of Vilanterol (100 nM) and their combination with RNO (ONO0.1 + RNO, ONO1 + RNO, ONO1000 + RNO, Vil100 + RNO). 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 4 replicates were submitted for sequencing.

有研究提出，吸入式EP4受体激动剂可作为一类新型支气管扩张剂，其疗效与β2-肾上腺素受体激动剂相当，用于哮喘治疗。然而，此类药物对基因组的影响常被忽视，尽管其可能对呼吸系统健康存在潜在危害。本研究采用mRNA测序（mRNA-seq）技术，对比了ONO-AE1-329（一种EP4受体激动剂）与维兰特罗（vilanterol，β2-肾上腺素受体激动剂）在人支气管上皮BEAS-2B细胞中对基因组的影响。此外，为探究不同G蛋白偶联受体（G protein-coupled receptor, GPCR）介导的环磷酸腺苷（cyclic adenosine monophosphate, cAMP）升高是否会引发不同的转录特征，本研究进一步拓展实验，纳入了腺苷A2B受体激动剂Bay-60-6583与前列环素I受体激动剂taprostene。ONO-AE1-329与维兰特罗的最大有效浓度可显著调控232个和320个基因（q值=0.05；变化倍数≥1.5或≤0.67），其中二者共有217个调控基因。斯皮尔曼相关性分析显示，这两种干预手段的基因表达变化具有高度的秩相关一致性，表明二者的功能重叠程度可观。出乎意料的是，ONO-AE1-329、维兰特罗、Bay 60-6583与taprostene的基因组效应同样呈现高度秩相关。这一发现提示，由任意GPCR产生的cAMP或可启动相同的转录程序。不过相较于维兰特罗，ONO-AE1-329通常表现为部分激动剂，其效能与内在活性随转录本不同而存在差异。综上，本研究证实，每一个受ONO-AE1-329调控的基因对cAMP的敏感性各不相同，且由独特的受体占有率-应答关系所决定。此外，如果BEAS-2B细胞中这种相对温和的基因组反应在体内也能得到重现，那么吸入式EP4受体激动剂或许可成为一类更安全的支气管扩张剂。整体实验设计：将汇合状态的BEAS-2B细胞分别以溶剂对照、RNO（1 μM）、最大浓度ONO-AE1-329（1 μM）、两个亚最大浓度ONO-AE1-329（0.1 nM与1 nM）、最大浓度维兰特罗（100 nM），以及RNO与上述药物的联合给药组（ONO0.1 + RNO、ONO1 + RNO、ONO1000 + RNO、Vil100 + RNO）处理2小时。提取总RNA并进行定量，构建mRNA文库，随后在NextSeq 500测序仪（Illumina）上通过两套高通量测序试剂盒完成测序。所有样本均设置4次生物学重复并进行测序。