Hedgehog signaling in lung growth and injury

描述（由申请人提供）：在发育过程中，肺间质产生非上皮结构，而在成熟的肺中，间质对扰动作出反应以维持体内平衡。间充质细胞（如成纤维细胞、肌成纤维细胞和平滑肌细胞）的功能或功能障碍与许多肺部疾病密切相关，包括肺气肿、哮喘、纤维化、肺动脉高压、癌症和支气管肺发育不良。我们对常驻间充质细胞如何随时间在肺中发挥作用的了解有很大的差距。主要的挑战包括需要额外的标记来描述功能不同的间充质细胞亚型，精确鉴定作为常驻祖细胞或干细胞的间充质细胞，以及鉴定新的信号通路或间充质细胞中可以治疗靶向的其他分子。我们工作的总体目标是鉴定成纤维细胞亚群的新标记，特别是干细胞/祖细胞，可用于鉴定和治疗这些处于疾病状态的细胞。Shh是一种在肺发育过程中参与间质增殖和分化的信号分子。在肺中，Shh由上皮细胞产生，并在分支形态发生所需的间质中诱导关键反应。虽然Shh的产生在出生后基本停止，但我们发现在成人肺中仍有大量间充质细胞对Shh或类似的Hedgehog （Hh）配体有反应。这些细胞在Gli1- lacz报告基因小鼠中是可识别的（Gli1基因既是Hh信号通路的一个组成部分，也是Hh信号通路的一个转录靶点）。hh反应细胞在气道外膜、肺血管和内脏胸膜中有独特的位置。通过共定位研究，我们发现这些细胞主要是col1+SMA-成纤维细胞。基于我们的数据和最近其他组间充质干细胞/祖细胞的结果，我们假设Gli1-lacZ+细胞是具有祖细胞/干细胞能力的重要间充质细胞群。为了验证这一假设，我们提出了两个广泛的目标：(1)在不同条件下对hh反应细胞进行遗传命运图谱，以确定它们在肺中扩展和促进不同细胞系的能力；(2)开发分离和培养这些细胞的技术，并在体外表征它们的行为。该项目将通过仔细分析各种hh报告小鼠菌株和来自这些小鼠的细胞来完成，并且应该对肺部生物学和疾病具有广泛的意义。

DESCRIPTION (provided by applicant): During development, the lung mesenchyme gives rise to non-epithelial structures, while in the mature lung the mesenchyme responds to perturbations to maintain homeostasis. The function, or malfunction, of mesenchymal cells (e.g., fibroblasts, myofibroblasts and smooth muscle cells) is intimately involved with numerous lung diseases, including emphysema, asthma, fibrosis, pulmonary hypertension, cancer and bronchopulmonary dysplasia. There are significant gaps in our knowledge of how resident mesenchymal cells function over time in the lung. Major challenges include the need for additional markers that delineate functionally distinct subtypes of mesenchymal cells, the precise identification of mesenchymal cells that function as resident progenitor or stem cells, and the identification of new signaling pathways or other molecules in mesenchymal cells that can be therapeutically targeted. The broad goal of our work is to identify novel markers of fibroblast subsets, in particular stem/progenitor cells that might be used to identify and therapeutically target these cells in disease states. Sonic hedgehog (Shh) is a signaling molecule involved in mesenchyme proliferation and differentiation during lung development. In lung, Shh is produced by epithelium and induces critical responses in mesenchyme required for branching morphogenesis. While Shh production largely ceases by birth, we find there remains a population of mesenchymal cells in adult lung responding to Shh or a similar Hedgehog (Hh) ligand. These cells are identifiable in Gli1-lacZ reporter mice (the Gli1 gene is both a component of the Hh signaling pathway and a transcriptional target of Hh signaling). The Hh-responding cells have a distinctive location in the adventitia of airways and pulmonary vessels, and in the visceral pleura. By colocalization studies we find that the cells are largely col1+SMA- fibroblasts. Based on our data and recent results on mesenchymal stem/progenitor cells from other groups, we hypothesize that the Gli1-lacZ+ cells are an important population of mesenchymal cells with progenitor/stem cell capability. To test this hypothesis we propose two broad aims: (1) to genetically fate-map the Hh-responding cells under various conditions to determine their ability to expand and contribute to different cell lineages in the lung, and (2) to develop techniques to isolate and culture these cells, and characterize their behavior in vitro. The project will be accomplished by careful analysis of various Hh-reporter mouse strains and cells derived from these mice, and should have broad implications for lung biology and disease. PUBLIC HEALTH RELEVANCE: Lung mesenchymal cells are responsible for synthesis of collagen and other structural proteins, the contractile activity of vessels and airways, repair processes after injury, and maladaptive scarring. Although mesenchymal cells are thought to exist in multiple subtypes that differ in their functions, including their capacity for differentiation, renewal and stem cell behavior, and these functions are critical in diseases such as asthma, pulmonary hypertension and lung fibrosis, our understanding of these subtypes remains inadequate, in part because of a lack of definitive markers by which to identify them. We found that a distinct subset of lung mesenchymal cells is identifiable by activation of the Hedgehog signaling pathway (a pathway that has been most studied in the context of embryonic development), and hypothesize that these cells have important proliferative and progenitor functions in the adult that are relevant to many disease states and that could be targeted for therapeutic benefit; in this application for an R21 award we propose to develop procedures and perform experiments to isolate and characterize these cells in vitro and in vivo.