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

描述（由申请人提供）：我们假设气道疾病和气道化生的病理性上皮重塑是由损伤后通常控制气道上皮再生的信号级联失调引起的。这些解除调控的级联反应可以靶向逆转气道疾病的病理性气道上皮变化。此外，适当调节这些级联可用于再生医学方法，以促进正常上皮修复。Hippo通路对多器官系统的上皮修复至关重要，其异常激活可导致化生和癌症。然而，这种信号在肺中的作用仍未得到充分的研究。我们建议研究Yap1转录共激活因子在肺中的作用，因为Yap1是Hippo信号传导的一个重要且易于操作的下游效应物。值得注意的是，在鳞状细胞肺癌中发现了Yap1和Notch基因改变。这是一个有趣的发现，因为Notch信号已被证明可以调节负责上皮细胞再生的气道干细胞，并且Notch信号也被证明与其他器官系统中的Hippo信号相互作用。总之，这些观察结果表明，Hippo和Notch通路将在气道再生和引起气道上皮化生过程中相互作用。我们的初步数据显示，气道上皮中Yap1的基因激活导致上皮增生，类似于鳞状化生的早期变化序列。我们还发现，Yap1的表达在气道损伤时被诱导，然后随着气道上皮再生的进行而减弱。因此，我们假设Hippo通路是气道上皮再生所必需的，并且气道基底干细胞中这一信号级联的解除将导致鳞状化生。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)研究其与Notch通路的关系，来确定Hippo信号在气道上皮修复和重塑中的机制作用。具体来说，在Aim 1中，我们将使用多西环素诱导的Yap1激活形式在气道基底干细胞中遗传和可逆地激活Yap1，并测试Yap1激活在小鼠和人气道上皮中的体外作用。在Aim 2中，我们将从气道基底干细胞中基因删除Yap1，然后用二氧化硫刺激小鼠引发再生反应，以确定Yap1是否是气道上皮损伤修复所必需的。我们还将在小鼠和人类离体气道上皮中确定Yap1缺失的后果。在Aim 3中，我们将调节小鼠和人类气道祖细胞中的Notch和Yap1，以确定一种信号通路如何影响另一种信号通路。这些信号级联的状态将通过定义Hippo和Notch通路下游靶点的存在或缺失，在人类鳞状化生和发育不良的样本中进行检查。

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.