Defining molecular signals that influence airway basal cell renewal and growth

项目摘要 哺乳动物气道基底细胞（basalcells，BCs）是气道假复层上皮的常驻干细胞 了解它们是如何被调节和维持的，对于了解它们的异常 在疾病如COPD、囊性纤维化和癌症中的生长，以及用于开发用于 利用生物细胞用于再生医学。河马信号通路已经成为一种 BC增长的重要调节剂。Hippo信号传导控制转录调节因子雅普和 TAZ（雅普/TAZ），通过限制成熟分化的气道细胞中的核雅普/TAZ定位。核雅普 活动已被证明可促进气道中的BC生长，并已被证明在以下方面发挥重要作用： 其他组织和器官中的各种干细胞群。雅普参与维持气道BC命运 通过与基底细胞特异性转录因子p63直接结合。气道基底干细胞 肺损伤修复的重要作用，但调节其生长和自我更新的信号很差 明白我们的研究以及其他研究表明，转录调节因子雅普的核活性 在气道基底细胞生长中起着重要作用，在这项提议中，我们的目的是了解这些作用， 在这种情况下调节雅普活性。在这项提议中，我们的目标是使用遗传小鼠模型， 在气道基底细胞中条件性敲除Hippo通路调节激酶LATS 1和LATS 2， 了解Hippo不活动和随后的雅普核活动如何通过其 与p63的相互作用我们的初步数据表明，在Krt 5阳性基底细胞中LATS 1/2缺失导致 基底细胞增生和形成不同的基底细胞群。那些靠近地下室的 细胞膜具有最高水平的核p63，由此这种表达在Krt 5+细胞中减少或丧失 在增生性病变中更像管腔。我们的研究进一步表明，BCs中的雅普活性受到 信号，如FGF 10，来自基底膜的基质细胞，我们假设， 这些信号影响雅普与p63的相互作用以指导BC自我更新转录程序。我们的目标 揭示新的生物化学途径，描述来自基底膜的信号是如何转导的， 通过BC受体酪氨酸激酶增加YAP-p63的功能活性和BC的自我更新和生长。 具体地说，我们提出：1）确定LATS 1/2缺失和随后的雅普激活是否在气道基底节中起作用， 细胞通过YAP-p63相互作用影响BC维持; 2）来自基底膜的信号如何 可能促进这些雅普/p63相互作用; 3）如果YAP-p63复合物差异调节基因表达 促进BC的命运。最终，这项研究将为BC的自我更新和扩张提供分子见解。 这可能为预防BC在疾病中的扩张提供指导，或指导新的扩张方法。 用于再生治疗的离体BC。

PROJECT SUMMARY Basal cells (BCs) of mammalian airways are resident stem cells of the pseudostratified epithelium of the airways and understanding how they are regulated and maintained will be important for understanding their aberrant growth in disease, such as COPD, cystic fibrosis and cancer, as well as for developing novel methods for harnessing BCs for the purposes of regenerative medicine. The Hippo signaling pathway has emerged as an important regulator of BC growth. Hippo signaling controls the activity of the transcriptional regulators YAP and TAZ (YAP/TAZ) by restricting nuclear YAP/TAZ localization in mature differentiated airway cells. Nuclear YAP activity has been shown to promote BC growth in the airways and has been shown to play important roles in a variety of stem cell populations in other tissues and organs. YAP is implicated in maintaining the airway BC fate through direct association with the basal cell specific transcription factor, p63. Airway basal stem cells play important roles lung injury repair, but the signals that regulate their growth and self-renewal are poorly understood. Our studies, as well as others, indicate that the nuclear activity of the transcriptional regulator YAP plays important roles in airway basal cell growth and in this proposal, we aim to understand the roles and regulation of YAP activity in this context. In this proposal we aim to use genetic mouse models which contain conditional knockouts of Hippo pathway regulating kinases, LATS1 and LATS2, in airway basal cells to understand how Hippo inactivity and subsequent YAP nuclear activity influences BC maintenance through its interaction with p63. Our preliminary data suggest that LATS1/2 deletion in Krt5 positive basal cells leads to basal cell hyperplasia and the formation of distinct populations of basal cells. Those adjacent to the basement membrane have the highest levels of nuclear p63, whereby this expression is reduced or lost in the Krt5+ cells that are more luminal in the hyperplastic lesion. Our studies further suggest that YAP activity in BCs is influenced by signals, such as FGF10, that come from stromal cells in the basement membrane, and we hypothesize that these signals influence YAP interactions with p63 to guide a BC self-renewal transcriptional program. We aim to unveil novel biochemical pathways that describe how signals from the basement membrane are transduced through BC receptor tyrosine kinases to increase YAP-p63 functional activity and BC self-renewal and growth. Specifically, we propose: 1) to determine if LATS1/2-depletion and subsequent YAP activation in airway basal cells influences BC maintenance through YAP-p63 interactions; 2) how signals from the basement membrane may promote these YAP/p63 interactions; and 3) if YAP-p63 complexes differentially regulate gene expression to promote BC fate. Ultimately this study will offer molecular insight into how BC self-renewal and expansion is regulated, which may offer directions for preventing BC expansion in disease or guide new methods to expand BCs ex vivo for regenerative therapy.