Molecular mechanisms of pulmonary disease in Birt-Hogg-Dube syndrome

项目摘要/摘要 Bert-Hogg-Dubé(BHD)综合征是由卵泡蛋白(Flcn)基因的胚系突变引起的。囊性肺 伴有气胸的疾病是BHD的主要临床表现，早在34周就有报道称囊性病变。 早在14岁时就有妊娠和气胸。BHD囊性肺疾病的机制是 理解不足，代表着严重的知识差距。我们在小鼠肺中特异性地删除了Flcn 并发现出生后早期肺泡扩大和成人肺泡扩大的显著表型。 类似于BHD患者肺部病理的囊性病变。Flcn定位于溶酶体，在溶酶体中是一个关键 MTORC1的激活子，提示mTORC1信号的异常是FLCN影响的一个潜在机制 在肺里。我们还发现，间充质转录因子TCF21是一种新的候选基因。 FLCN物理相互作用蛋白，TCF21在FLCN缺乏的细胞和组织中下调。因此， 我们假设间充质失活导致肺泡发育的年龄相关性缺陷。 通过两个平行的效应器：mTORC1和TCF21实现动态平衡。这一假设将在两个目标上得到检验。 目的1：研究大鼠肺间充质中Flcn基因缺失对肺泡发育和动态平衡的影响。 活着。我们将检验这一工作假说，即间充质FLCN是肺泡生长所必需的，并且 以依赖年龄的方式维持生活。我们的方法将包括肺内时间和空间上的Flcn缺失 间充质细胞。小鼠的BHD样肺损害将在病理、细胞和 分子水平，包括肺间充质祖细胞的定量和定性分析。 目的2：探讨肺间充质细胞内Flcn缺乏导致肺损伤的机制。 体外和体内的肺泡发育和动态平衡缺陷。(A)确定外国联如何监管 间充质mTORC1活性及其对肺泡发育和维持的影响。我们会 验证Flcn缺陷肺间充质细胞mTORC1活性失调的工作假说 而肺间充质中mTORC1活性的下调将在我们的肺泡缺陷中表现出来。 小鼠肺间充质中Flcn失活。(B)确定FLCN-TCF21互动如何影响 肺泡的生长和维持。我们将测试FLCN与TCF21直接相互作用的工作假设 调节肺间充质中依赖TCF21的靶点，而缺乏Flcn会损害肺间质的稳定性和 TCF21的定位，并影响肺泡发育和动态平衡。 我们的长期目标是阐明BHD肺部疾病的发病机制。我们预计这将是 导致范式转换的发现，表明肺间充质与囊性肺的发病机制有关 并为新的预防和治疗策略提供了基础。

Project Summary/Abstract Bert-Hogg-Dubé (BHD) syndrome is caused by germline mutations in the folliculin (FLCN) gene. Cystic lung disease with pneumothorax is a major clinical manifestation of BHD, with cysts reported as early as 34 weeks’ gestation and pneumothorax as early as age 14. The mechanisms underlying cystic lung disease in BHD are poorly understood, representing a critical knowledge gap. We deleted Flcn specifically in mouse lung mesenchyme, and discovered striking phenotypes of early postnatal alveolar enlargement and adult pulmonary cysts that resemble the lung pathology in BHD patients. FLCN localizes to the lysosome, where it is a key activator of mTORC1, suggesting that abnormal mTORC1 signaling is a potential mechanism for Flcn’s impact in the lung. We have also discovered that Tcf21, a mesenchymal transcription factor, is a novel candidate of Flcn physical interacting protein, and that Tcf21 is downregulated in Flcn-deficient cells and tissues. Therefore, we hypothesize that mesenchymal inactivation of Flcn leads to age-dependent defects in alveolar development and homeostasis via two parallel effectors: mTORC1 and Tcf21. This hypothesis will be tested in two Aims. Aim 1: To determine how loss of Flcn in lung mesenchyme impacts alveolar development and homeostasis in vivo. We will test the working hypothesis that mesenchymal Flcn is required for alveolar growth and maintenance in an age-dependent manner. Our approaches will include temporal-spatial Flcn deletion in lung mesenchymal cells. The BHD-like lung lesions in mice will be characterized at pathologic, cellular, and molecular levels, including quantitative and qualitative analyses of lung mesenchymal progenitor cells. Aim 2: To determine the mechanisms through which Flcn-deficiency in lung mesenchymal cells leads to defective alveolar development and homeostasis, in vitro and in vivo. (A) To determine how Flcn regulates mesenchymal mTORC1 activity and how mTORC1 impacts alveolar development and maintenance. We will test the working hypotheses that Flcn-deficient lung mesenchymal cells have dysregulation of mTORC1 activity and that downregulation of mTORC1 activity in lung mesenchyme will phenocopy the alveolar defects in our mice with Flcn inactivation in lung mesenchyme. (B) To determine how the Flcn-TCF21 interaction impacts alveolar growth and maintenance. We will test the working hypotheses that Flcn directly interacts with Tcf21 to regulate Tcf21-dependent targets in lung mesenchyme, and that the absence of Flcn impairs the stability and localization of Tcf21, and affects alveolar development and homeostasis. Our long-term goal is to elucidate the pathogenic mechanisms of lung disease in BHD. We expect that this will lead to paradigm-shifting discoveries implicating the lung mesenchyme in the pathogenesis of cystic lung disease and providing the foundation for novel preventative and therapeutic strategies.