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

项目总结/摘要 COPD是美国第四大死亡原因;然而，我们并不完全了解COPD的发病机制。 COPD和缺乏疾病改善疗法。叉头盒蛋白P1（FoxP1）是一种转录抑制因子， 参与肺上皮发育。来自英国生物库、ECLIPSE和COPDGene的最新数据 队列暗示FoxP1是气流受限的重要预测因子。然而，FoxP1在 COPD的发病机制尚未研究。 初步工作表明，在肺中FoxP1蛋白减少，而FoxP1 mRNA增加， 与对照组相比，患有COPD的人和小鼠。具体来说，我们发现暴露在香烟烟雾中 导致E3连接酶FBXO 24泛素化FoxP1，导致其在肺上皮细胞中的蛋白酶体降解 体外细胞FoxP1蛋白的缺失增加了FoxP1 mRNA的表达，因为FoxP1抑制了 自己的推动者。出乎意料的是，FoxP1蛋白的缺失增加了未折叠蛋白反应（UPR）的活性。 以及肺上皮细胞中UPR凋亡诱导剂C/EBP同源蛋白（CHOP）的水平。 对公开可用的FoxP1 ChIP-seq数据的分析表明FoxP1结合位点的显著富集 在人胚胎干细胞和HepG2细胞中的关键UPR基因和CHOP的启动子中。此外，本发明还 计算研究确定了UPR和CHOP DNA序列中FoxP1的高概率结合位点 发起人。在体内，CHOP的缺失减少了肺中的细胞凋亡和肺气肿。最后，诱导性缺失 通过鼻内给予表达Cre的腺病毒以增加FoxP1小鼠的吸烟量 吸烟引起的肺气肿。因此，我们假设香烟烟雾导致FBXO 24泛素化， 并降解肺上皮中的FoxP1蛋白，从而增加关键UPR基因的启动子活性， CHOP和诱导细胞凋亡和肺气肿。 在这个奖项，我们将：（1）测试的假设，FoxP1结合并抑制启动子的 通过CUT & RUN和荧光素酶报告基因测定，以及作图， 通过启动子诱变研究FoxP1结合位点;（2）测试缺失FoxP1基因在小鼠中表达的假设。 肺上皮细胞将增加香烟烟雾诱导的肺中UPR活性、CHOP和细胞凋亡 上皮，并增加肺气肿;和（3）测试的假设，删除FBXO 24基因将防止 香烟烟雾诱导FoxP1蛋白降解，降低香烟烟雾诱导的UPR活性、CHOP 肺上皮细胞凋亡，减少肺气肿。本提案将调查机制 对于FoxP1和UPR之间的新联系，证明这种机制在体内的功能影响， 在临床前模型中测试抵消FBXO 24作为COPD治疗策略的功效。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， 改善COPD病情的能力。

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.