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

描述（由申请人提供）：角膜上皮暴露于环境中使这一重要屏障容易受到损伤。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库介导的，这些干细胞迁移到损伤部位，在那里增殖和分化以取代受损的上皮。本研究旨在了解14-3-3 - 3c在角膜上皮分化中的作用。14-3-3是一个结合磷蛋白并调节其亚细胞定位的蛋白家族。14-3-3 - 3c是上皮特异性家族成员，通过小鼠遗传模型，我们证明了其在角膜上皮分化中的关键作用，该基因的两个拷贝的突变导致胚胎角膜上皮发育缺陷。杂合突变允许正常的胚胎发育；然而，角膜上皮分化对损伤的反应仍然受到损害。因此，14-3-3 - 3c基因剂量在胚胎学上区分了角膜上皮的发育和损伤后角膜上皮的分化。这种分化缺陷导致未分化细胞的生长，最终形成伴随炎症和新生血管形成的不透明角膜斑块。随着杂合小鼠年龄的增长，斑块会自发形成，我们提供的证据表明，这与睑板腺缺陷导致的干眼效应有关，睑板腺缺陷无法分泌脂质。Notch1通过激活对分化（包括维生素A通路中的基因）和细胞周期阻滞都很重要的基因，对角膜上皮分化至关重要，我们发现14-3-3 3c突变细胞不能激活Notch1。激活的Notch1的表达恢复了突变细胞的分化，因此，分化缺陷表型和由此产生的角膜斑块形成与Notch1基因的组织特异性敲除非常相似。我们将14-3-3 - 3c与EGF受体（EGFR）的细胞表面表达联系起来，从而与通过EGF与该受体结合而激活的Erk信号通路联系起来。这一点很重要，因为EGF信号会抑制Notch1，而Notch1的表达和随后的角膜上皮分化必须消除这一信号。我们建议进一步研究14-3-3 - 3c、EGFR和Notch1（及其靶基因）在角膜上皮分化中的联系。这些研究使用14-3-3 - 3c突变小鼠进行细胞培养和遗传杂交。

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.