The Roles of Autophagy in Limbal/Corneal Epithelia.

自噬在角膜缘/角膜上皮中的作用。

• 批准号: 10400950
• 负责人: ROBERT M LAVKER
• 金额: $ 48.08万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10400950
• 关键词: 3-Dimensional; Affect; Aging; Aniridia; Anterior; Attenuated; Autophagocytosis; BCL2 gene; Basal Cell; Biochemical; Biological; Biological Models; Biology; Catabolic Process; Cell Maintenance; Cell Nucleus; Cell Proliferation; Cells; Cellular Assay; Chemical Burns; Clinical; Complement; Comprehension; Conjunctival Pterygium; Connexin 43; Cornea; Corneal Diseases; Defect; Diabetes Mellitus; Disease; Environment; Epithelial; Epithelial Cells; Etiology; Excision; Eye; Family; Film; Fuchs' Endothelial Dystrophy; Genes; Goals; Homeostasis; Human; Impairment; Injury; Investigation; Keratin; Keratopathy; Knock-in Mouse; Knowledge; Label; Lead; Lighting; Lysosomes; Maintenance; Measures; Mediating; MicroRNAs; Microscopy; Modality; Modeling; Molecular; Monitor; Mucin 1 protein; Mus; Nuclear; Organelles; Patients; Pharmacology; Physiological; Plasminogen Activator Inhibitor 2; Play; Proteins; Receptor Protein-Tyrosine Kinases; Recurrence; Regulation; Reporting; Rest; Role; Signal Transduction; Starvation; Stevens-Johnson Syndrome; Stream; Stress; Structure; Surface; Techniques; Testing; Tissues; Transplantation; Vesicle; YY1 Transcription Factor; base; biomarker evaluation; clinical application; corneal epithelial wound healing; corneal epithelium; diabetic; diabetic patient; epithelial stem cell; gain of function; improved; in vivo; insight; involucrin; knock-down; limbal; loss of function; mouse model; negative affect; notch protein; novel; novel strategies; ocular surface; preservation; response; stem; stem cell biomarkers; stem cell homeostasis; stem cell proliferation; stem cells; wound; wound healing

PROJECT SUMMARY/ABSTRACT The anterior surface of the eye functions as a barrier to the external environment, protects the delicate underlying structures from injury, supports a tear film and maintains transparency. These functions are achieved, in part, through the corneal and limbal epithelia, which must maintain proper proliferation and differentiation. Autophagy, a catabolic process by which cells adapt to intrinsic and extrinsic stress-related situations, has been shown to also regulate proliferation and differentiation. Despite the many advances made in our understanding of corneal and limbal epithelial biology, autophagy remains largely unstudied. We have recently reported that: (i) under resting conditions, autophagy is greater in limbal epithelial basal compared with corneal epithelial basal cells; (ii) autophagy contributes to limbal epithelial proliferative capacity; and (iii) autophagy enables proper activation of transit amplifying (TA) cells following wounding. We also have preliminary evidence that: (i) NUS1 is a novel upstream regulator of autophagy in the limbal epithelium; (ii) blocking autophagy impairs corneal epithelial differentiation and the removal of nuclear material (nucelophagy); and (iii) the receptor tyrosine kinase, EphA2, positively affects corneal epithelial differentiation via regulating end stage autophagy. These observations lead us to hypothesize that autophagy is required to maintain limbal epithelial stem and TA cell homeostasis as well as insure proper corneal epithelial differentiation. We propose to test this hypothesis by focusing on how autophagy: (i) affects limbal epithelial stem cell proliferation; (ii) regulates differentiation in the corneal epithelium; and (iii) is regulated by EphA2 in the corneal epithelium. To accomplish these goals, we will capitalize on our ability to conduct gain- and loss-of-function studies of autophagy-related genes and their key up-stream regulators in complimentary model systems that include cultured human limbal (HLEKs) and corneal (HCEKs) epithelial cells, 3-D raft cultures of limbal and corneal epithelia, and mouse models of impaired autophagy and EphA2 signaling. We will assess the functional consequences of such modulations with a combination of morphogenetic (including Structured Illumination Microscopy and TEM), biochemical, molecular biological, cell biological and physiological approaches. By focusing on autophagy, our proposal represents a new approach to study the regulation of: (i) limbal stem and TA cell proliferation; and (ii) corneal epithelial differentiation and nucelophagy. Such knowledge has clinical applicability as sustaining proliferation is necessary for the proper expansion of cultured HLEKs used in ex vivo transplantation as well as in wound healing. The impact of autophagy and nucleophagy on corneal epithelial differentiation has translational relevance since aberrant differentiation is a feature of diseases of the corneal epithelium (e.g., aniridia, chemical burns) and modulation of autophagy-associated genes are potential treatment modalities, Understanding how autophagy is regulated in corneal epithelium is essential as this tissue is routinely subjected to stress, which requires a proper autophagic response to maintain homeostasis.

项目总结/摘要 眼睛的前表面起着与外部环境的屏障的作用，保护脆弱的眼睛。 它可以保护底层结构免受损伤，支撑泪膜并保持透明度。这些功能 部分通过角膜和角膜缘上皮细胞实现，其必须保持适当的增殖， 分化自噬是细胞适应内在和外在应激相关的一种分解代谢过程。 在某些情况下，已经显示也调节增殖和分化。尽管取得了许多进展 在我们对角膜和角膜缘上皮生物学的理解中，自噬仍然在很大程度上未被研究。我们有 最近报道：（i）在静息条件下，自噬在角膜缘上皮基底层中比在角膜缘上皮基底层中更大， 角膜上皮基底细胞;（ii）自噬有助于角膜缘上皮增殖能力;和（iii） 自噬能够在创伤后适当激活转运扩增（TA）细胞。我们也有 初步证据表明：（i）NUS 1是角膜缘上皮自噬的一种新型上游调节因子;（ii） 阻断自噬损害角膜上皮分化和核物质的去除（核吞噬）; 和（iii）受体酪氨酸激酶EphA 2通过调节 晚期自噬这些观察使我们假设自噬是维持角膜缘细胞的必要条件。 以及确保适当角膜上皮分化。我们提出 通过关注自噬如何：（i）影响角膜缘上皮干细胞增殖;（ii） 调节角膜上皮中的分化;和（iii）在角膜上皮中受EphA 2调节。到 为了实现这些目标，我们将利用我们的能力进行功能获得和丧失研究， 自噬相关基因及其关键的上游调节剂在互补模型系统，包括 培养的人角膜缘（HLEKs）和角膜（HCEKs）上皮细胞，角膜缘和角膜的3-D筏培养物， 上皮和受损的自噬和EphA 2信号传导的小鼠模型。我们将评估功能 这种调节与形态发生（包括结构化照明）组合的结果 显微镜和TEM）、生物化学、分子生物学、细胞生物学和生理学方法。通过 聚焦于自噬，我们的建议代表了一种新的方法来研究调节：（i）角膜缘干细胞， TA细胞增殖;和（ii）角膜上皮分化和核吞噬。这些知识具有临床 适用性，因为维持增殖对于离体使用的培养HLEK的适当扩增是必要的 移植以及伤口愈合。自噬和噬核对角膜上皮细胞的影响 由于异常分化是角膜疾病的特征， 上皮（例如，无虹膜，化学烧伤）和自噬相关基因的调节是潜在的 了解自噬如何在角膜上皮中调节是至关重要的，因为这 组织通常受到应激，这需要适当的自噬反应来维持体内平衡。