Defining the role of Hippo signaling in airway injury repair and remodeling

描述(申请人提供)：我们假设，呼吸道疾病和气道化生中的病理性上皮重构是由于损伤后通常控制气道上皮再生的信号级联反应的放松调节所致。然后，这些解除调节的级联反应可以被靶向逆转呼吸道疾病中的病理性呼吸道上皮变化。此外，适当地调节这些级联反应可以用于再生医学的方法，以促进正常的上皮修复。河马途径对于多器官系统的上皮修复是必不可少的，它的异常激活被认为会导致化生和癌症。然而，该信号在很大程度上仍未在肺中被探索。我们建议研究YAP1转录共激活因子在肺中的作用，因为YAP1是一种VITA，易于操纵河马信号的下游效应因子。值得注意的是，在鳞状细胞肺癌中已经发现了YAP1和Notch基因改变。这很有趣，因为Notch信号已经被证明调节负责上皮再生的呼吸道干细胞，并且Notch信号也被证明在其他器官系统中与河马信号相互作用。总而言之，这些观察表明，河马和Notch通路在呼吸道再生过程中将相互作用，并导致呼吸道上皮化生。我们的初步数据显示，YAP1在呼吸道上皮细胞中的基因激活导致了类似于与鳞状化生相关的早期变化序列的上皮增生。我们还表明，YAP1的表达在呼吸道损伤时被诱导，然后随着呼吸道上皮再生的进行而消退。因此，我们假设HIPPO通路是呼吸道上皮再生所必需的，并且该信号级联在气道基底干细胞中的失控将导致鳞状化生。本研究旨在通过(1)在小鼠和人类模型系统中调节气道基底干细胞中YAP1的表达，以及(2)通过检测其与Notch通路的关系，来确定Hippo信号在呼吸道上皮修复和重塑中的机制作用。具体地说，在目标1中，我们将使用多西环素诱导的YAP1激活形式，从基因上和可逆地激活呼吸道基础干细胞中的YAP1，并在体外测试YAP1在小鼠和人类呼吸道上皮细胞中的激活作用。在目标2中，我们将从呼吸道基础干细胞中从基因上删除YAP1，然后用二氧化硫攻击小鼠以诱导再生反应，以确定YAP1是否对呼吸道上皮损伤修复是必要的。我们还将在体外确定YAP1在小鼠和人类呼吸道上皮细胞中缺失的后果。在目标3中，我们将调节小鼠和人类呼吸道祖细胞中的Notch和YAP1，以确定一种信号通路如何影响另一种信号通路。然后，通过定义河马和Notch通路下游靶点的存在与否，在人类鳞状化生和异型增生的样本中检查这些信号级联的状态。

DESCRIPTION (provided by applicant): We hypothesize that pathologic epithelial remodeling in airways disease and airway metaplasia is caused by a deregulation of the signaling cascades that normally govern airway epithelial regeneration after injury. These deregulated cascades can then be targeted to reverse the pathologic airway epithelial changes in airway diseases. In addition, appropriately modulating these cascades could be used in a regenerative medicine approach in order to promote normal epithelial repair. The Hippo pathway is essential for epithelial repair in multiple organ systems and its aberrant activation is known to result in metaplasia and cancer. However, the signaling remains largely unexplored in the lung. We propose to study the role of the Yap1 transcriptional co-activator in the lung since Yap1 is a vita and easy to manipulate downstream effector of Hippo signaling. Of note, Yap1 and Notch genetic alterations have been identified in squamous cell lung cancer. This is of interest because Notch signaling has been shown to regulate the airway stem cells that are responsible for epithelial regeneration and Notch signaling has also been shown to interact with Hippo signaling in other organ systems. In aggregate, these observations suggest that the Hippo and Notch pathways will interact during airway regeneration and in causing airway epithelial metaplasia. Our preliminary data show that genetic activation of Yap1 in airway epithelium causes an epithelial hyperplasia resembling the early sequence of changes associated with squamous metaplasia. We also show that Yap1 expression is induced upon airway injury and then subsides as airway epithelial regeneration proceeds. We therefore hypothesize that the Hippo pathway is required for airway epithelial regeneration and that a deregulation of this signaling cascade in airway basal stem cells will cause squamous metaplasia. This proposal aims to define the mechanistic role of Hippo signaling in airway epithelial repair and remodeling by (1) modulating Yap1 expression in airway basal stem cells in both murine and human model systems and (2) by examining its relationship to the Notch pathway. Specifically, in Aim 1 we will genetically and reversibly activate Yap1 in airway basal stem cells using a doxycycline-inducible activated form of Yap1 and also test the role of Yap1 activation in both mouse and human airway epithelium ex vivo. In Aim 2, we will genetically delete Yap1 from airway basal stem cells and then challenge mice with sulfur dioxide to elicit a regenerative response to determine whether Yap1 is necessary for airway epithelial injury repair. We will also determine the consequences of Yap1 loss in mouse and human airway epithelium ex vivo. In Aim 3, we will modulate Notch and Yap1 in mouse and human airway progenitor cells to determine how one signaling pathway affects the other. The state of these signaling cascades will then be examined in samples of human squamous metaplasia and dysplasia by defining the presence or absence of downstream targets of the Hippo and Notch pathways.