Hedgehog signaling in lung growth and injury

描述(申请人提供)：在发育过程中，肺间充质会形成非上皮性结构，而在成熟的肺中，间充质会对扰动做出反应，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、肺高压、癌症和支气管肺发育不良。在我们对常驻间充质细胞如何随着时间的推移在肺中发挥作用的知识方面存在着显著的差距。主要的挑战包括需要更多的标记物来描述不同功能的间充质细胞亚型，准确识别作为常驻祖细胞或干细胞的间充质细胞，以及识别间充质细胞中可以作为治疗靶点的新的信号通路或其他分子。我们工作的广泛目标是识别成纤维细胞亚群的新标记，特别是干细胞/祖细胞，这些细胞可能被用来识别和治疗处于疾病状态的这些细胞。Sonic hedgehog(Shh)是一种信号分子，参与肺发育过程中间充质的增殖和分化。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的产生在很大程度上因出生而停止，但我们发现成人肺中仍有一群间充质细胞对Shh或类似的Hedgehog(HH)配体产生反应。在Gli1-LacZ报告基因小鼠中可以识别这些细胞(Gli1基因既是HH信号通路的组成部分，也是HH信号的转录靶点)。HH反应细胞在呼吸道和肺血管外膜以及内脏胸膜中有独特的定位。通过共定位研究，我们发现这些细胞主要是COL1+的SMA成纤维细胞。根据我们的数据和其他组关于间充质干细胞/祖细胞的最新研究结果，我们假设Gli1-LacZ+细胞是具有祖细胞/干细胞能力的一个重要的间充质细胞群。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增和贡献不同细胞系的能力；(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报告小鼠品系和来自这些小鼠的细胞来完成，并将对肺部生物学和疾病产生广泛的影响。 公共卫生相关性：肺间充质细胞负责胶原和其他结构蛋白的合成，血管和呼吸道的收缩活动，损伤后的修复过程，以及不适应的瘢痕形成。尽管间充质细胞被认为存在于多种功能不同的亚型中，包括它们的分化、更新和干细胞行为的能力，这些功能在哮喘、肺高压和肺纤维化等疾病中是至关重要的，但我们对这些亚型的了解仍然不足，部分原因是缺乏明确的标记物来识别它们。我们发现，肺间充质细胞的一个独特的子集可以通过激活Hedgehog信号通路(在胚胎发育背景下研究最多的途径)来识别，并假设这些细胞在成人中具有重要的增殖和祖细胞功能，这些功能与许多疾病状态相关，可以作为治疗的目标；在R21奖的申请中，我们建议开发程序和进行实验，在体外和体内分离和表征这些细胞。

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.