The role of extrusion in controlling epithelial homeostasis

描述(申请人提供)：挤出在控制上皮细胞动态平衡中的作用项目摘要/摘要上皮为其包裹的器官提供了一种保护屏障；然而，构成该屏障的细胞通过细胞死亡(凋亡)和分裂不断更新。令人惊讶的是，关于是什么触发细胞自然分裂或死亡，或者细胞死亡和分裂的数量如何在动态平衡中保持平衡，人们知之甚少。然而，保持这种平衡对于维持屏障功能和预防疾病至关重要：如果死亡率较高，可能会导致哮喘等屏障功能疾病，如果分裂比率较高，则会产生实体瘤。通过我们对细胞在死亡时如何维持屏障的研究， 我们相信，我们已经发现了一种新的机制，即上皮细胞如何维持内环境平衡的细胞数量，我们在这项提议中进一步研究了这一机制。我们之前发现，当上皮内的细胞死亡时，它会向邻近细胞发出信号，让其形成收缩的肌球蛋白RNG，将其挤出。为此，垂死的细胞释放脂类鞘氨醇1-磷酸(S1P)，通过向邻近细胞中的S1P受体2(S1P2)发出信号来激活挤出。最近，我们发现在成年结肠组织和发育中的斑马鱼表皮中，在细胞高度拥挤的部位，上皮细胞似乎在死亡之前挤出。在实验过度拥挤的情况下，活细胞也会在我们设计的密室中挤出。虽然稳态和过度拥挤诱导的排泄都使用我们为凋亡细胞排泄定义的相同的S1P-S1P2途径，但活细胞排泄途径是由拉伸激活的通道Piezo 1激活的。重要的是，我们发现破坏拉伸激活的信号或S1P-S1P2途径都会导致斑马鱼细胞培养和发育中的细胞团。因此，我们提出了一个模型，在该模型中，底层组织为上皮细胞提供了空间限制，因此任何细胞分裂和/或迁移都会导致过度拥挤的应变，从而导致活细胞挤出。挤压细胞在促进周围细胞存活和增殖的同时，由于缺乏生存信号而死亡。我们将通过1)在细胞培养模型中研究挤压是否促进细胞死亡，以及2)测试S1P2斑马鱼突变体中的细胞团是否由于缺乏 细胞挤出，增殖增加，或两者兼而有之。为了确定是什么激活了挤出，我们将3)确定Piezo 1拉伸激活的通道和钙电流如何介导S1P触发挤出，以及4)识别细胞-细胞或细胞基质蛋白质，这些蛋白质对于决定哪些细胞在均匀的上皮拥挤压力下挤出至关重要。这项拟议的研究具有创新性，因为它探索了一种新的上皮细胞如何维持内稳态细胞数量的机制，并使用了多学科方法，其中包括我们开发的用于研究上皮细胞的斑马鱼表皮模型，以及我们与生物工程师共同开发的细胞过度拥挤装置。这一建议具有重要的医学意义，因为上皮内稳态调节失调可能是大多数实体肿瘤形成和屏障功能疾病的核心。

DESCRIPTION (provided by applicant): The role of extrusion in controlling epithelial homeostasis Project Summary/Abstract Epithelia provide a protective barrier for the organs they encase; yet cells comprising this barrier are constantly renewed via cell death (apoptosis) and division. Surprisingly little is known about what triggers cells to naturally divide or die or how he number of cells dying and dividing are balanced during homeostasis. However, maintaining this balance is critical for maintaining barrier function and preventing diseases: if the death rate is higher, barrier function diseases such as asthma may result and if the division rate is higher, solid tumors will result. Through our investigations of how cells maintain a barrier when they die, we believe we have discovered a novel mechanism for how epithelia maintain homeostatic cell numbers, which we further investigate in this proposal. We previously discovered that when a cell dies within an epithelium, it signals its neighboring cells to form a contractile actomyosin rng to extrude it out. To do so, the dying cell emits the lipid Sphingosine 1-Phosphate (S1P), which activates extrusion by signaling the S1P receptor 2 (S1P2) in neighboring cells. More recently, we have found that in adult colon tissue and in developing zebrafish epidermis, epithelial cells appear to extrude prior to dying at sites of high cell crowding. Live cells also extrude in respons to experimental overcrowding using a chamber we devised. While both homeostatic and overcrowding-induced extrusion use the same S1P-S1P2 pathway we defined for apoptotic cell extrusion, the live cell extrusion pathway is activated by the stretch-activated channel Piezo 1. Importantly, we found that disrupting either stretch-activated signaling or the S1P-S1P2 pathway leads to cellular masses in both cell culture and developing zebrafish. Thus, we propose a model where the underlying tissue provides a space limit on epithelia so that any cell divisions and/or migrations cause overcrowding strain, which induces live cells to extrude. Extruding cells then die due to lack of survival signaling while promoting surrounding to survive and proliferate. We will test this hypothesis in Aims 1 and 2 by 1) investigating if extrusion promotes cell death in a cell culture model and 2) testing if cell masses in S1P2 zebrafish mutants result from lack of cell extrusion, increased proliferation, or both. To define what activates extrusion, we will 3) determine how Piezo 1 stretch-activated channels and calcium currents mediate S1P to trigger extrusion and 4) identify cell- cell or cell-matrix proteins critical for determining which cells extrude in response to uniform epithelial crowding forces. The proposed research is innovative because it investigates a novel mechanism for how epithelial cells maintain homeostatic cell numbers and uses multidisciplinary approaches, which include a zebrafish epidermal model we have developed to study epithelia and a cell overcrowding device we have developed with a bioengineer. The proposal is medically significant because misregulation of epithelial homeostasis is likely at the heart of most solid tumor formation and barrier function diseases.