Targeting Foxm1 in pulmonary fibrosis

项目摘要。 肺纤维化的现有治疗方法没有显著改善生存率;迫切需要 新方法。环境和遗传因素的结合产生了肺泡上皮， 容易受伤。受损组织的修复失调导致肺泡上皮细胞增生和增殖， II型细胞（AECII）-所谓的“重编程”AECII，其是肺中的主要病理特征 纤维化的患者。重编程AECII在肺纤维化的发病机制中起关键作用， 产生TGF-β和促炎介质，导致肺成纤维细胞活化和肺成纤维细胞的募集， 巨噬细胞进一步去调节修复。我们的长期目标是剖析转录调控， 肺纤维化我们最近发现了一种新的促纤维化调节因子Foxm 1，它是一个家族成员， 叉头盒（Fox）转录因子。我们的初步数据表明Foxm 1在AECII中被诱导， 在人和小鼠肺的纤维化病变中，而不是在正常肺泡区域，表明Foxm 1可以 成为重新编程的AECII的标志。活化Foxm 1转录本在小鼠AECII中的转基因表达 加剧了放射性肺炎并导致严重的肺纤维化。有条件删除 来自AECII的Foxm 1减弱辐射诱导的肺纤维化。虽然这些数据表明， 通过Foxm 1介导的事件促进肺纤维化，下游信号通路由 AECII中的Foxm 1仍有待鉴定（客观）。我们将检验一个假设， AECII通过Foxm 1介导的成纤维细胞活化和募集促进肺纤维化 骨髓炎性细胞转化为纤维化病变。通过结合遗传学和药理学方法 在放射和博莱霉素诱导的小鼠肺纤维化模型中，所提出的研究将鉴定分子 Foxm 1在AECII中调节的机制，并确定增生性Foxm 1阳性AECII对 纤维化在目的1中，我们将确定Foxm 1阳性AECII在成纤维细胞活化中的作用。使用 具有Foxm 1功能获得和功能丧失的转基因小鼠，以及AECII的单细胞RNA-seq分析 从人IPF肺中分离，我们将鉴定Foxm 1靶基因。因为我们的初步数据显示 促纤维化和炎症介质在Foxm 1过表达的AECII中的表达，我们将研究 Foxm 1是否激活AECII中的骨桥蛋白和TGFβ1基因的转录，并刺激AECII中的 潜伏性TGFβ1蛋白，导致肺成纤维细胞活化。在目标2中，我们将检查Foxm 1是否激活 AECII中的CCL 2和CXCL 5基因，导致骨髓炎性细胞募集到纤维化肺中。在 目的3：利用本实验室新近发现的小分子Foxm 1抑制剂抑制AECII 重编程、肺部炎症和肺纤维化小鼠模型中的纤维化重塑。 完成我们的研究将（1）确定Foxm 1诱导肺动脉高压的新分子机制， 纤维化，和（2）测试新型Foxm 1抑制剂在肺纤维化中的功效。

PROJECT SUMMARY. Existing treatments for pulmonary fibrosis have not significantly improved survival; there is a critical need for new approaches. A combination of environmental and genetic factors creates an alveolar epithelium that is susceptible to injury. Deregulated repair of damaged tissue leads to the hyperplastic and proliferating alveolar type II cells (AECII) – the so called “re-programmed” AECII, which are main pathological features in the lungs of patients with fibrosis. Re-programmed AECIIs play a key role in the pathogenesis of pulmonary fibrosis, producing TGF- and pro-inflammatory mediators leading to activation of lung fibroblasts and recruitment of macrophages that further deregulate repair. Our long-term goal is to dissect transcriptional regulation of pulmonary fibrosis. We recently identified a novel pro-fibrotic regulator, Foxm1, a member of the family of Forkhead Box (Fox) transcription factors. Our preliminary data demonstrated that Foxm1 is induced in AECII within fibrotic lesions, but not in normal alveolar region of human and mouse lungs, indicating that Foxm1 can be a marker of re-programmed AECIIs. Transgenic expression of activated Foxm1 transcript in mouse AECII exacerbated radiation-induced pneumonitis and caused severe pulmonary fibrosis. Conditional deletion of Foxm1 from AECII attenuated radiation-induced lung fibrosis. While these data demonstrate that AECII promote pulmonary fibrosis through Foxm1-mediated events, the downstream signaling pathways regulated by Foxm1 in AECII remain to be identified (objective). We will test hypothesis that reprogrammed hyperplastic AECII promote pulmonary fibrosis through Foxm1-mediated activation of fibroblasts and recruitment of myeloid inflammatory cells into fibrotic lesions. By combining genetic and pharmacological approaches in radiation- and bleomycin-induced mouse models of lung fibrosis, the proposed studies will identify molecular mechanisms regulated by Foxm1 in AECII, and determine contribution of hyperplastic Foxm1-positive AECII to ling fibrogenesis. In Aim 1, we will determine the role of Foxm1-positive AECII in activation of fibroblasts. Using transgenic mice with Foxm1 gain-of-function and loss-of-function, and single cell RNA-seq analysis of AECII isolated from human IPF lungs, we will identify Foxm1 target genes. Since our preliminary data show increased expression of pro-fibrotic and inflammatory mediators in Foxm1-overexpressing AECII, we will examine whether Foxm1 activates transcription of Osteopontin and TGFβ1 genes in AECII, and stimulates activation of latent TGFβ1 protein, causing activation of lung fibroblasts. In Aim 2, we will examine whether Foxm1 activates CCL2 and CXCL5 genes in AECII, leading to recruitment of myeloid inflammatory cells into fibrotic lungs. In Aim 3, we will use novel small molecule Foxm1 inhibitor recently discovered in my laboratory to inhibit AECII re-programming, lung inflammation and fibrotic remodeling in murine models of pulmonary fibrosis. Completion of our studies will (1) identify novel molecular mechanisms whereby Foxm1 induces pulmonary fibrosis, and (2) test efficacy of novel Foxm1 inhibitors in lung fibrosis.