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

14-3-3o在角膜上皮发育和修复中的作用

• 批准号: 8321577
• 负责人: Qiutang Li
• 金额: $ 36万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321577
• 关键词: Affect; Age; Aging; Alleles; Anterior; Binding; Calcium; Cell Culture Techniques; Cell Cycle Arrest; Cell Differentiation process; Cell Proliferation; Cell surface; Cells; Complex; Cornea; Corneal Diseases; Corneal Injury; Corneal Opacity; Defect; Development; Differentiation Antigens; EGF gene; Embryo; Embryonic Development; Environment; Epidermal Growth Factor Receptor; Epithelial; Epithelial Cells; Epithelium; Eye; Eye Part; Failure; Family; Family member; Gene Activation; Gene Dosage; Gene Targeting; Genes; Genetic Crosses; Genetic Models; Heterozygote; Homeostasis; Individual; Inflammation; Injury; Invaded; Knock-out; Link; Lipids; MEKs; Mediating; Molecular; Molecular Genetics; Mus; Mutant Strains Mice; Mutation; Pathway interactions; Pattern; Phenotype; Phosphoproteins; Phosphoserine Motif; Phosphotransferases; Play; Prevention strategy; Process; Proliferating; Property; Protein Binding; Protein Family; Proteins; Receptor Signaling; Role; Series; Signal Pathway; Signal Transduction; Site; Stem cells; Testing; Tissues; Transgenic Mice; Tumor Suppressor Proteins; Undifferentiated; Vision; Vitamin A; Wound Healing; base; blind; corneal epithelium; design; eye dryness; in vivo; limbal; meibomian gland; mutant; neovascularization; notch protein; null mutation; prevent; progenitor; programs; receptor; receptor expression; repaired; research study; response to injury; self-renewal

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 14-3-3¿ in corneal epithelial differentiation. 14-3-3 is a family of proteins that bind phosphoproteins and regulate their subcellular localization. 14-3-3¿ 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, 14-3-3¿ 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 14-3-3¿ 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 14-3-3¿ 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 14-3-3¿, EGFR, and Notch1 (and its target genes) in corneal epithelia differentiation. These studies involve both cell culture and genetic cross using 14-3-3¿ mutant mice.

角膜上皮暴露在环境中使这一重要屏障容易受到影响 致伤。因此，角膜上皮具有显著的自我更新能力。这个自我 再生是由角膜缘干细胞储存库介导的，这些干细胞迁移到损伤部位 它们会增殖和分化，以取代受损的上皮。这项建议是针对 目的了解14-3-3在角膜上皮细胞分化中的作用。14-3-3是一个家庭 结合磷蛋白并调节其亚细胞定位的蛋白质。14-3-3？是 上皮特异性家族成员，并使用小鼠遗传模型证明了它的关键 双拷贝基因突变在角膜上皮细胞分化中的作用 胚胎中角膜上皮的发育。杂合突变允许正常 胚胎发育；然而，角膜上皮对损伤的反应 仍然是受损的。因此，14-3-3基因剂量区分角膜上皮发育 从胚胎学角度看，损伤后角膜上皮细胞分化。这一缺陷在 分化导致未分化的细胞生长，最终形成不透明的 伴有炎症和新生血管的角膜斑块。这块斑块形成了 随着杂合子小鼠年龄的增长，我们提供的证据表明，这与 由于眉毛腺有缺陷，不能分泌脂肪而引起的干眼症。 NOTCH1通过激活与角膜上皮细胞分化密切相关的基因，对角膜上皮细胞的分化起重要作用 分化(包括维生素A途径中的基因)以及细胞周期停滞，以及 我们发现14-3-3突变细胞不能激活Notch1。激活的Notch1的表达 恢复突变细胞的分化，从而恢复有缺陷的分化表型 由此形成的角膜斑块非常类似于组织特异性的 Notch1基因的敲除。我们将14-3-3与细胞表面EGF受体的表达联系起来 (EGFR)，从而与ERK信号通路有关，而ERK信号通路由EGF与其结合而激活 受体。这一点很重要，因为EGF信令抑制Notch1，并且该信令必须是 因Notch1表达和随后的角膜上皮分化而熄灭。我们 建议进一步研究14-3-3、EGFR和Notch1之间的联系(和 其靶基因)在角膜上皮细胞分化中起重要作用。这些研究涉及细胞培养和 用14-3-3突变小鼠进行遗传杂交。