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.

项目摘要/摘要 眼睛的前表面充当外部环境的障碍，保护精致 受伤的基础结构支持催泪膜并保持透明度。这些功能是 在某种程度上通过角膜和边缘上皮来实现，必须保持适当的增殖和 分化。自噬，一种分解代谢过程，细胞适应固有和外在应力相关 情况已被证明还调节增殖和分化。尽管取得了许多进步 在我们对角膜和边缘上皮生物学的理解时，自噬基本上仍然没有研究。我们有 最近报道：（i）在静止条件下，自噬在缘缘上皮的基础上比 角膜上皮基底细胞； （ii）自噬有助于缘缘上皮增殖能力； （iii） 自噬可在受伤后适当激活过境扩增（TA）细胞。我们也有 初步证据表明：（i）NUS1是缘缘上皮中自噬的新型上游调节剂； （ii） 阻塞自噬会损害角膜上皮分化和去除核材料（核噬菌体）； （iii）受体酪氨酸激酶EPHA2通过调节对角膜上皮分化产生积极影响 末期自噬。这些观察结果导致我们假设需要自噬以保持边缘 上皮茎和TA细胞稳态，并确保适当的角膜上皮分化。我们建议 通过关注自噬的方式来检验这一假设：（i）影响边缘上皮干细胞增殖； （ii） 调节角膜上皮的分化； （iii）在角膜上皮中受Epha2的调节。到 实现这些目标，我们将利用我们进行获得和功能丧失研究的能力 自动噬相关基因及其关键的免费模型系统中的关键上流调节器，包括 培养的人类缘缘（HLEK）和角膜（HCEK）上皮细胞，缘和角膜的3-D筏培养物 上皮和自噬和EPHA2信号的小鼠模型。我们将评估功能 这种调制与形态发生的结合的后果（包括结构化照明 显微镜和TEM），生化，分子生物学，细胞生物学和生理方法。经过 我们的建议专注于自噬，代表了一种研究的新方法，以研究：（i）边缘茎和 TA细胞增殖； （ii）角膜上皮分化和成核。这样的知识有临床 适用性作为维持增殖是必不可少的 移植以及伤口愈合。自噬和亲核对角膜上皮的影响 差异化具有转化相关性，因为异常分化是角膜疾病的特征 上皮（例如，Aniridia，化学燃烧）和自噬相关基因的调节是潜在的 治疗方式，了解角膜上皮中如何调节自噬是必不可少的 组织通常会承受压力，这需要适当的自噬反应以维持稳态。