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.
This proposal seeks to generate new knowledge regarding the pathogenesis and treatment of Chronic Obstructive Pulmonary Disease, the fourth leading cause of death in the US. Specifically, we propose to investigate how a mediator called FoxP1 helps maintain protein quality control in cells that line the airways of the lung after exposure to cigarette smoke, and determine if increasing FoxP1 protein levels in the lung is a viable treatment strategy for COPD in preclinical models. The new knowledge gained may lend itself to therapeutics that lessen the burden of COPD; therefore, this proposal is aligned with the objectives of the COPD National Action Plan.
