14-3-3?? and epithelial differentiation in the eye and other tissues

描述（由申请人提供）：上皮分化已被广泛研究，并且该过程可分为两个一般步骤：从驻留干细胞产生增殖的祖细胞，以及随后这些祖细胞的停滞和分化。转录因子TCF 3在激活子和阻遏子之间的振荡是维持常驻干细胞群体和为这些干细胞产生祖细胞脉冲的关键。这些祖细胞增殖以填充组织区域，然后Notch途径触发它们的细胞周期停滞和分化。因此，当Notch1突变时，角膜、睑板腺和毛囊中的祖细胞不能分化，并且它们增殖超出组织区域，导致病理性功能丧失。Notch1信号在增殖祖细胞中启动的时间对于确定细胞的精确数量以及组织拓扑结构至关重要。深入了解Notch1信号是如何在这一过程中启动的现在才出现。当增殖的祖细胞接触时，通过细胞-细胞接触抑制建立上皮组织区。细胞接触抑制信号中的突变导致祖细胞生长超出正常组织区域（如Notch1突变），组织功能丧失并最终导致癌症。但是，Notch1信号的发生如何与这种细胞间接触联系起来？钙粘蛋白启动的连接复合物不仅介导上皮细胞中的顶端极性，它们还启动称为Hippo的激酶级联，其磷酸化一组调节细胞周期以及分化程序的转录因子。这种磷酸化作用为14 - 3 - 3提供了一个结合位点，而14 - 3 - 3又将这些因子以无活性形式隔离在细胞质中。最近的结果表明，细胞接触抑制激酶级联的组分中的突变导致Notch 1活性的丧失，并且同样地，我们发现该途径中的14 - 3 - 3s突变也导致Notch 1表达的丧失。总之，这样的结果意味着当祖细胞在组织中扩增时，它们最终的细胞接触发出Notch 1的14 - 3 - 3s依赖性表达的信号，从而发出停滞和分化的信号。为了支持这一假设，我们发现小鼠中14 - 3 - 3s的突变导致增殖祖细胞的积累，并且在角膜、毛囊和导管结构（如睑板腺中的那些）中形成与Notch 1的表型不可区分的表型。这种14 - 3 - 3s或Notch1的缺失会导致严重的角膜缺损、导管阻塞导致终末ascini缺失和毛囊异常导致脱发。重要的是，我们证明了激活Notch1的表达挽救了培养中14 - 3 - 3s突变上皮祖细胞的分化缺陷。在这里，我们提出了一系列的研究，旨在进一步建立14 - 3 - 3s和刻缺蛋白1途径之间的联系，上皮分化的角膜，睑板腺和毛囊和发展小鼠模型系统，可用作未来的R01建议的基础上，检查分子的细节，通过14 - 3 - 3s和刻缺蛋白1调节上皮分化的发病途径。 公共卫生相关性：角膜是眼睛的最前部，其完整性对于视力是不可或缺的，全世界有超过4500万人是双眼失明，另有1.35亿人由于角膜透明度的丧失而双眼视力严重受损。我们提出的实验旨在揭示14 - 3 - 3s和信号网络在控制角膜上皮稳态中的重要作用。我们的研究将阐明角膜上皮内瘤变和鳞状细胞癌等角膜疾病发生的分子机制，并为检测和治疗提供新的策略。

DESCRIPTION (provided by applicant): Epithelial differentiation has been widely studied, and the process can be divided into two general steps: generation of proliferating progenitors from resident stem cells, and the subsequent arrest and differentiation of these progenitors. Oscillation of the transcription factor TCF3 between activator and repressor is key to both maintaining a resident stem cell population and generating pulses of progenitor cells for these stem cells. These progenitors proliferate to fill the tissue field, and then the Notch pathway triggers their cell cycle arrest and differentiation. Accordingly when Notch1 is mutated, progenitors in the cornea, meibomian gland and hair follicles fail to differentiate and they proliferate beyond the tissue fields leading to pathologic loss of function. The timing of Notch1 signal initiation in proliferating progenitors is critical to define the precise number of cells and thus tissue topology. Insight into to how Notch1 signaling is initiated in this process is only now emerging. Epithelial tissue fields are established by cell-cell contact inhibition as proliferating progenitors come in contact. Mutations in cell contact inhibition signaling lead to progenitor outgrowth beyond normal tissue fields (as with Notch1 mutation), loss of tissue function and ultimately cancer. But, how might onset of Notch1 signaling be linked to such cell-cell contact? Cadherin-initiated junctional complexes not only mediate apical polarity in epithelial cells, they also initiate a kinase cascade known as Hippo which phosphorylates a set of transcription factors that regulate both the cell cycle as well as differentiation programs. This phosphorylation provides a binding site for 14-3-3, which in turn sequesters the factors in an inactive form in the cytoplasm. Recent results demonstrate that mutations in components of the cell contact inhibition kinase cascade lead to loss of Notch 1 activity, and likewise we have found that mutation of 14-3-3s in this pathway also leads to loss of Notch1 expression. Taken together, such results imply that as progenitors expand in tissues, their eventual cell contact signals 14-3-3s -dependent expression of Notch1 thereby signaling arrest and differentiation. In support of this hypothesis, we have found that mutation of 14-3-3s in mice leads to accumulation of proliferating progenitor cells and a phenotype indistinguishable from that of Notch 1 in the cornea, hair follicle and ductal structures such as those in the meibomian gland. This loss of 14-3-3s or Notch1 causes severe corneal defects, ductal obstruction leading to loss of terminal ascini and abnormal hair follicles leading to hair loss. Importantly, we demonstrate that expression of activated Notch1 rescues the differentiation defect in 14-3-3s mutant epithelial progenitors in culture. Here, we propose a series of studies designed to further establish linkage between 14-3-3s and the Notch1 pathway in epithelial differentiation in the cornea, meibomian gland and hair follicles and to development mouse model systems which can be used as a basis for a future R01 proposal examining the molecular details of the pathway through which 14-3-3s and Notch1 regulate the onset of epithelial differentiation. PUBLIC HEALTH RELEVANCE: The integrity of the cornea, the most anterior part of the eye, is indispensable for vision, as evident by the facts that more than forty-five million individuals worldwide are bilaterally blind and another 135 million have severely impaired vision in both eyes because of the loss of corneal transparency. Our proposed experiments aim to uncover the essential role of 14-3-3s and the signaling network in controlling the corneal epithelial homeostasis. Our studies will elucidate the molecular mechanism underlying the corneal disease development such as corneal intraepithelial neoplasia and squamous cell carcinoma; and provide new strategies for detection and treatment.