Role of FBXO24 mediated ubiquitination of FoxP1 protein in the pathogenesis and treatment of COPD

项目摘要/摘要 慢性阻塞性肺病是美国第四大死因；然而，我们并不完全了解 慢性阻塞性肺病，缺乏治疗疾病的方法。叉头盒蛋白P1(FoxP1)是一种转录抑制因子 参与肺上皮细胞发育。来自英国生物库、ECLIPSE和COPDgene的最新数据 队列研究表明，FoxP1是气流受限的重要预测因子。然而，FoxP1在 COPD的发病机制尚不清楚。 初步研究表明，大鼠肺组织中FoxP1蛋白表达减少，而FoxP1基因表达增加 将患有COPD的人和小鼠与对照组进行比较。具体地说，我们发现暴露在香烟烟雾中 导致E3连接酶FBXO24泛素化FoxP1，导致其在肺上皮细胞中的蛋白酶体降解 体外培养的细胞。由此导致的FoxP1蛋白的丢失增加了FoxP1的mRNA，因为已知FoxP1可以抑制 它自己的推动者。出乎意料的是，FoxP1蛋白的丢失增加了未折叠蛋白反应(UPR)的活性 以及肺上皮细胞中UPR的凋亡诱导蛋白C/EBP同源蛋白(CHOP)的水平。 对公开可用的FoxP1芯片序列数据的分析表明，FoxP1结合位点显著丰富 在人类胚胎干细胞和HepG2细胞中关键的UPR基因和CHOP的启动子中。此外， 计算研究确定了UPR和CHOP DNA序列中FoxP1的高概率结合位点 推动者。在体内，CHOP的缺失可以减少肺组织中的细胞凋亡和肺气肿。最后，可诱导删除 FoxP1小鼠鼻腔注射Cre重组腺病毒后吸烟增加 吸烟引起的肺气肿。因此，我们假设香烟烟雾导致FBXO24无处不在 并降解肺上皮细胞中的FoxP1蛋白，从而增加关键UPR基因的启动子活性和 砍掉并诱导细胞凋亡和肺气肿。 在这个奖项中，我们将：(1)测试FoxP1结合并抑制FoxP1启动子的假设 肺上皮细胞关键UPR基因和CHOP的原位杂交和荧光素酶报告分析及图谱分析 通过启动子突变研究Foxp1结合位点；(2)验证FoxP1基因在 肺上皮细胞会增加吸烟引起的肺内UPR活性、CHOP和细胞凋亡。 上皮细胞，增加肺气肿；以及(3)检验删除FBXO24基因将防止 香烟烟雾诱导FoxP1蛋白降解并降低香烟烟雾诱导的UPR活性 和肺上皮细胞的凋亡，减少肺气肿。这项提案将调查这一机制 对于FoxP1和UPR之间的新联系，证明了这一机制在体内的功能影响，以及 在临床前模型中测试抗FBXO24作为COPD治疗策略的有效性。这项建议 反映了我们开发新的基于机制的疗法的长期目标，这种疗法可能具有前所未有的 慢性阻塞性肺疾病患者的疾病改正能力。

PROJECT SUMMARY/ABSTRACT COPD is the fourth leading cause of death in the US; however, we do not fully understand the pathogenesis of COPD and lack disease-modifying therapies. Forkhead box protein P1 (FoxP1) is a transcriptional repressor that participates in lung epithelial development. Recent data from the UK Biobank, ECLIPSE, and COPDGene cohorts implicate FoxP1 as an important predictor of airflow limitation. However, a role for FoxP1 in the pathogenesis of COPD remains unexamined. Preliminary work suggests that FoxP1 protein is reduced while FoxP1 mRNA is increased in the lungs of humans and mice with COPD compared with controls. Specifically, we find that exposure to cigarette smoke causes the E3 ligase FBXO24 to ubiquitinate FoxP1, resulting in its proteasomal degradation in lung epithelial cells in vitro. The resulting loss of FoxP1 protein increases FoxP1 mRNA because FoxP1 is known to repress its own promoter. Unexpectedly, loss of FoxP1 protein increases activity of the unfolded protein response (UPR) as well as levels of the UPR’s apoptosis inducer C/EBP-homologous protein (CHOP) in lung epithelial cells. Analyses of publicly available FoxP1 ChIP-seq data demonstrates significant enrichment for FoxP1 binding sites in the promoters of key UPR genes and CHOP in human embryonic stem cells and HepG2 cells. Further, computational studies identify high probability binding sites for FoxP1 in the DNA sequence of UPR and CHOP promoters. In vivo, deletion of CHOP reduces apoptosis and emphysema in the lung. Finally, inducible deletion of FoxP1 by intranasal administration of Cre expressing adenovirus to floxed FoxP1 mice increased cigarette smoke induced emphysema. Therefore, we hypothesize that cigarette smoke causes FBXO24 to ubiquitinate and degrade FoxP1 protein in the lung epithelium thereby increasing promotor activity for key UPR genes and CHOP and inducing apoptosis and emphysema. During this award, we will: (1) Test the hypothesis that FoxP1 binds to and suppresses the promoters of key UPR genes and CHOP in lung epithelial cells by CUT&RUN and luciferase reporter assays, as well as map FoxP1 binding sites via promoter mutagenesis studies; (2) Test the hypothesis that deleting the FoxP1 gene in the lung epithelium will increase cigarette smoke induced UPR activity, CHOP, and apoptosis in the lung epithelium, and increase emphysema; and (3) Test the hypothesis that deleting the FBXO24 gene will prevent cigarette smoke induced degradation of FoxP1 protein and reduce cigarette smoke induced UPR activity, CHOP and apoptosis in the lung epithelium, and decrease emphysema. This proposal will investigate the mechanism for a novel link between FoxP1 and the UPR, demonstrate the functional impact of this mechanism in vivo, and test the efficacy of counteracting FBXO24 as a therapeutic strategy for COPD in preclinical models. This proposal reflects our long-term objective of developing new mechanism-based therapies that may have unprecedented disease modifying ability in COPD.