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

描述(申请人提供)：上皮分化已被广泛研究，该过程可分为两个一般步骤：从驻留干细胞产生增殖的前体细胞，以及随后阻止和分化这些前体细胞。转录因子TCF3在激活因子和抑制因子之间的振荡是维持常驻干细胞种群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场，然后Notch途径触发它们的细胞周期停滞和分化。因此，当Notch1发生突变时，角膜、眉板腺和毛囊中的祖细胞无法分化，它们会扩散到组织范围之外，导致病理性功能丧失。在增殖的祖细胞中Notch1信号的启动时间对于确定准确的细胞数量和组织拓扑结构至关重要。对Notch1信令是如何在这一过程中启动的洞察才刚刚出现。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场。细胞接触抑制信号的突变会导致祖细胞超出正常组织范围的生长(如Notch1突变)，组织功能丧失，最终导致癌症。但是，Notch1信号的启动如何与这种细胞间的接触联系起来呢？钙粘附素启动的连接复合体不仅介导上皮细胞的顶端极性，它们还启动被称为HIPPO的激酶级联反应，使一系列转录因子磷酸化，调节细胞周期和分化程序。这种磷酸化为14-3-3提供了一个结合部位，而14-3-3又以非活性的形式将这些因子隔离在细胞质中。最近的研究结果表明，细胞接触抑制激酶级联通路的突变会导致Notch 1活性的丧失，同样，我们也发现该通路中14-3-3S的突变也会导致Notch1表达的丧失。综上所述，这些结果表明，随着祖细胞在组织中的扩张，它们最终的细胞接触信号14-3-3S依赖于Notch1的表达，从而发出停滞和分化的信号。为了支持这一假设，我们在小鼠中发现14-3-3S突变导致增殖的祖细胞积累，并在角膜、毛囊和导管结构中与Notch 1的表型无法区分，例如在眉板腺中的那些。这种14-3-3s或Notch1的丢失会导致严重的角膜缺陷，导管阻塞导致末端ascini的丢失，以及异常毛囊导致脱发。重要的是，我们证明了激活的Notch1的表达挽救了14-3-3S突变上皮祖细胞在培养中的分化缺陷。在这里，我们提出了一系列研究，旨在进一步建立14-3-3S和Notch1通路在角膜、眉板腺和毛囊上皮分化中的联系，并建立小鼠模型系统，作为未来R01提案的基础，研究14-3-3S和Notch1调节上皮分化起始的通路的分子细节。 与公共健康相关：作为眼睛最前面的部分，角膜的完整性对于视力是不可或缺的，全世界有超过4500万人双眼失明，另有1.35亿人因丧失角膜透明度而双眼视力严重受损，这一事实就是明证。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细胞动态平衡中的重要作用。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.