Role of 14-3-3o in development and repair of corneal epithelium

描述(由申请人提供)：角膜上皮暴露在环境中使这一重要的屏障容易受到伤害。因此，角膜上皮具有显著的自我更新能力。这种自我更新是由角膜缘干细胞的储存库介导的，这些干细胞迁移到损伤部位，在那里它们增殖和分化，以取代受损的上皮。本研究旨在了解14-3-3C在角膜上皮细胞分化中的作用。14-3-3是一个蛋白家族，能结合磷蛋白并调节其亚细胞定位。14-3-3C是上皮特异性家族成员，利用小鼠遗传模型，我们证明了它在角膜上皮细胞分化中的关键作用。该基因的两个拷贝的突变会导致胚胎中角膜上皮发育缺陷。杂合子突变允许正常的胚胎发育；然而，对损伤反应的角膜上皮分化仍然受损。因此，14-3-3C基因剂量区分了角膜上皮细胞胚胎发育和损伤后角膜上皮细胞分化。这种分化缺陷会导致未分化细胞的生长，最终形成不透明的角膜斑块，并伴随着炎症和新生血管。随着杂合子小鼠年龄的增长，这种斑块自发形成，我们提供的证据表明，这与眼干眼样效应有关，这种干眼症是由于眉板腺缺陷而导致的，无法分泌脂肪。NOTCH1通过激活对角膜上皮细胞分化至关重要的基因(包括维生素A途径中的基因)以及细胞周期停滞，是角膜上皮细胞分化所必需的，我们发现14-3-3C突变细胞无法激活Notch1。激活的Notch1的表达恢复了突变细胞的分化，因此，分化缺陷的表型和由此产生的角膜斑块的形成似乎与组织特异性的Notch1基因敲除非常相似。我们将14-3-3C与EGF受体(EGFR)的细胞表面表达联系起来，从而与ERK信号通路联系起来，ERK信号通路由EGF与该受体结合而激活。这一点很重要，因为EGF信号会抑制Notch1，而对于Notch1的表达和随后的角膜上皮分化，这种信号必须被抑制。我们建议进一步研究14-3-3C、EGFR和Notch1(及其靶基因)在角膜上皮细胞分化中的联系。这些研究既包括细胞培养，也包括使用14-3-3C突变小鼠进行遗传杂交。 与公众健康相关：作为眼睛最前面的部分，角膜的完整性对视力是不可或缺的。全世界有4500万人双眼失明，另有1.35亿人因角膜透明度丧失而双眼视力严重受损。我们提出的实验旨在阐明14-3-3C在角膜上皮细胞动态平衡和伤口愈合中的重要作用，并了解控制这一过程的分子网络。我们将研究14-3-3C突变小鼠的角膜上皮发育和伤口愈合过程、屏障完整性以及分子/遗传学特性。我们希望更好地了解角膜疾病的发展，为预防和治疗提供新的策略。

DESCRIPTION (provided by applicant): Exposure of the corneal epithelium to the environment leaves this important barrier susceptible to injury. Accordingly, the corneal epithelium has a remarkable self-renewal capacity. This self renewal is mediated by a reservoir of limbal stem cells which migrate to sites of injury where they proliferate and differentiate to replace the damaged epithelium. This proposal is directed toward understanding the role of 14-3-3C in corneal epithelial differentiation. 14-3-3 is a family of proteins that bind phosphoproteins and regulate their subcellular localization. 14-3-3C is the epithelial-specific family member, and using a mouse genetic model we demonstrate its crucial role in corneal epithelial differentiation Mutation of both copies of the gene leads to defective corneal epithelial development in the embryo. Heterozygous mutation allows for normal embryonic development; however, the corneal epithelial differentiation in response to injury remains impaired. Thus, 14-3-3C gene dosage distinguishes corneal epithelial development embryologically from corneal epithelial differentiation following injury. This defect in differentiation leads to outgrowth of undifferentiated cells which eventually form an opaque corneal plaque with associated inflammation and neovascularization. This plaque forms spontaneously as the heterozygous mice age, and we provide evidence that this is linked to a dry eye-like effect resulting from a defective meibomian gland which fails to secrete lipid. Notch1 is essential for corneal epithelial differentiation via its activation of genes important for both differentiation (including genes in the vitamin A pathway) as well as cell cycle arrest, and we have found that 14-3-3C mutant cells fail to activate Notch1. Expression of activated Notch1 restores differentiation in mutant cells, and accordingly the defective differentiation phenotype and the resulting corneal plaque formation closely resemble that seem with tissue-specific knockout of the Notch1 gene. We link 14-3-3C to cell surface expression of the EGF receptor (EGFR) and thus to the Erk signaling pathway which is activated by binding of EGF to this receptor. This is important because EGF signaling represses Notch1, and this signaling must be extinguished for Notch1 expression and subsequent corneal epithelial differentiation. We propose studies to further characterize the linkage between 14-3-3C, EGFR, and Notch1 (and its target genes) in corneal epithelia differentiation. These studies involve both cell culture and genetic cross using 14-3-3C mutant mice. PUBLIC HEALTH RELEVANCE: The integrity of the cornea, the most anterior part of the eye, is indispensable for vision. 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 elucidate the essential role of 14-3-3C in corneal epithelial homeostasis and wound healing and to understand the molecular network that controls the process. We shall study the cornea epithelium development and wound healing process, barrier integrity, and the molecular/genetic properties of 14-3-3C mutant mice. We hope to better understand corneal disease development and provide new strategies for prevention and treatment.