Signaling pathways in lung stem cell differentiation

描述(申请人提供)：肺修复和再生由沿肺轴分布的不同干/祖细胞群进行，包括细支气管会(Clara)细胞、肺泡II型上皮细胞和细支气管肺泡干细胞(BASCs)。这些上皮细胞类型在日常生活中也起着促进气体交换和维持组织动态平衡的作用。损伤后，信号指示干细胞产生受影响谱系的后代，但这些信号的分子特征仍然有限。我们已经建立了一种3D共培养实验，在单细胞水平上询问BASC分化的调节。与BASCs共培养的肺内皮细胞支持细支气管和肺泡细胞分化。最近，我们已经证明，调节共培养环境可以改变BASC的分化结果。我们发现，BMP4处理激活了内皮细胞中钙调神经磷酸酶依赖的转录因子NFATc1，以诱导血栓反应蛋白-1(TSP1)的表达，这是肺泡系特异性分化所必需的，也是充分的。体内肺泡损伤修复需要TSP1。因此，我们发现了一个新的信号轴，它运行在BASCs和内皮细胞之间，控制分化和损伤修复。我们现在试图进一步研究TSP1上游和下游的BMP-NFATc1-TSP1通路，并鉴定来自肺内皮细胞的调节BASC分化的额外信号。在目标1中，我们将定义BASCs中调节分化的分子。我们将检测候选TSP1受体在3D共培养和肺泡损伤样本中的表达。来自体内TSP1操纵的RNA-SEQ将以一种公正的方式识别TSP1依赖的分化调节因子，并在3D共培养中进行功能测试。在目标2中，我们将进一步阐明肺内皮细胞产生的影响肺泡分化的分泌因子。首先，我们将在BASC/内皮细胞共培养中测试候选的BMP4-NFATc1靶向R-Spindin2，这是我们最近发现的一种Wnt通路配体，是对BMP4处理的反应而诱导的。将使用谱系追踪的方法在体内测试BASCs中的R-Spin2途径。我们还将进行RNA-SEQ和NFATc1-CHIP-SEQ相结合，以确定肺内皮细胞中更多的BMP4靶点。我们的实验旨在确定多能肺干细胞是如何被指示产生特定血统的后代的。这项工作将为发现干细胞生态位中的作用机制和加强肺部疾病的肺损伤修复的治疗靶点提供机会。

DESCRIPTION (provided by applicant): Lung repair and regeneration is carried out by different stem/progenitor cell populations distributed along the pulmonary axis, including the bronchiolar club (Clara) cells, the alveolar type II epithelial cells, and bronchioalveolar stem cels (BASCs). These epithelial cell types also function on a daily basis to facilitate gas exchange and maintain tissue homeostasis. After injury, signals instruct the stem cell to produce progeny of the affected lineage, but the molecular characterization of these signals remains limited. We have developed a 3D co-culture assay to interrogate the regulation of BASC differentiation at the single cell level. Lung endothelial cells co-cultured with BASCs support bronchiolar and alveolar cell differentiation. Recently, we have shown that modulating the co-culture environment alters BASC differentiation outcomes. We found that BMP4 treatment activates the calcineurin-dependent transcription factor NFATc1 in endothelial cells to induce expression of Thrombospondin-1 (Tsp1), which was necessary and sufficient for alveolar lineage-specific differentiation. TSP1 was required for alveolar lung injury repair in vivo. Thus, we have uncovered a new signaling axis that operates between BASCs and endothelial cells to control differentiation and injury repair. We now seek to further characterize the BMP-NFATc1-TSP1 pathway up and down stream of TSP1, and to identify additional signals from lung endothelial cells that regulate BASC differentiation. In Aim 1, we will define the molecules in BASCs that regulate differentiation. We will examine expression of candidate TSP1 receptors in 3D co-cultures and alveolar lung injury samples. TSP1-dependent mediators of differentiation will be identified in an unbiased fashion by RNA-seq from in vivo Tsp1-manipulation and functionally tested in 3D co-cultures. In Aim 2, we will further delineate the secreted factors produced by lung endothelial cells that influence alveolar differentiation. Initially we will test the candidat BMP4-NFATc1 target R-spondin2, a Wnt pathway ligand that we recently found was induced in response to BMP4 treatment, in BASC/endothelial cell co-cultures. A lineage tracing approach will be used to test the R-spondin2 pathway in BASCs in vivo. We will also perform RNA-Seq coupled with NFATc1-ChIP-Seq to identify additional BMP4 targets in lung endothelial cells. Our experiments aim to define how a multipotent lung stem cell is instructed to produce lineage-specific progeny. This work will provide an opportunity for the discovery of mechanisms operating in the stem cell niche and therapeutic targets for enhanced lung injury repair in lung disease.