Differential Effects of Corneal Hypoxia on Limbal Stem and Epithelial Cell Fates

角膜缺氧对角膜缘干和上皮细胞命运的不同影响

基本信息

批准号：
8916118
负责人：
LUO LU
金额：
$ 32.17万
依托单位：
LUNDQUIST INSTITUTE FOR BIOMEDICAL INNOVATION AT HARBOR-UCLA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8916118
关键词：
3&apos Untranslated Regions Abbreviations Affect Apoptosis Ataxia Telangiectasia Cell Differentiation process Cell Hypoxia Cell Proliferation Cell physiology Cells Cornea Corneal Injury Cytokine-Inducible Kinase Cytoplasmic Granules Data Disease Embryo Epithelial Cells FOS gene Fibroblasts Genetic Transcription Goals Histones Human Hypoxia Hypoxia Inducible Factor Impaired wound healing JUN gene MAPK8 gene Mediating Messenger RNA MicroRNAs Molecular Molecular Profiling Mus Mutate PTEN gene Pathway interactions Pattern Phosphorylation Phosphotransferases Physiological Process Proto-Oncogene Proteins Resistance Response Elements Role Signal Pathway Signal Transduction Staging Stem cells Stress Transcription Factor AP-1 Translations Tumor Suppressor Genes Variant Wound Healing activating transcription factor base corneal epithelium exposed human population improved in vivo limbal migration novel progenitor response self-renewal stem stem cell differentiation transcription factor

项目摘要

DESCRIPTION (provided by applicant): The overall objective of this project is to define the molecular mechanisms of how human corneal Limbal Stem/Progenitor (LS/P) and Corneal Epithelial (CE) cells respond to hypoxic stresses in physiological and pathological conditions. Our preliminary data show in human CE cells that hypoxia (1% O2) activates Polo-like kinase 3 (Plk3) cascades that phosphorylate a group of important determinants for regulating cell fates, such as Hif-1, p53, c-Jun/AP-1 and H2AX. Thus, larger responses to hypoxic stress based on the magnitude of increases in Plk3 activities result in CE cell apoptosis. By contrast, human corneal LS/P cells are resistant to hypoxia-induced apoptosis because hypoxia suppresses Plk3 expression and fails to induce phosphorylation of Hif-1, p53, c-Jun/AP-1 and H2AX in these cells. We reveal that hypoxia stimulates significant changes in microRNA (miRNA) expression profiles. These miRNAs specifically target the 3'-untranslated region (3' UTR) of Plk3 mRNA to suppress hypoxia-induced Plk3 signaling in corneal LS/P cells, but not in CE cells. Our central hypothesis is that exposure of human corneas to hypoxic conditions activates two distinct processes in corneal LS/P and CE cells including: 1) activation of a Plk3-mediated signaling pathway that in turn increases p53 phosphorylation and activations of c-Jun/AP-1 and H2AX resulting in CE cell apoptosis; and 2) activation of specific expressions of miRNAs that suppress Plk3 expression to down-regulate downstream targets resulting in hypoxic tolerance and to trigger differentiation of corneal LS/P cells. To identify the molecular mechanisms, we propose three aims: 1) To define how hypoxia-induced Plk3 activation affects and interacts with AP-1, p53 and H2AX. Hypoxia-induced p53 and c-Jun phosphorylation are directly relevant to apoptosis. We will determine whether hypoxia-induced Plk3 can directly activate p53, AP-1 and H2AX, and how hypoxia-induced ATM/ATR/Chk1/2 activation leads to Plk3 activation in CE cells. 2) To investigate how Plk3 is down-regulated in hypoxia-induced corneal LS/P cells. Hypoxia suppresses Plk3 expression through a novel mechanism by inducing high levels of Plk3-specific miRNAs. We will determine the hypoxia-induced miRNA profiles in corneal LS/P cells in hypoxic conditions, which of the hypoxia-sensitive miRNAs suppress the Plk3 signaling pathway in the LS/P cells, and how these miRNAs interact with Plk3 mRNA to affect its stability. 3) To determine roles of hypoxia-induced Plk3 activation in corneal epithelial wound healing. Effects of hypoxia on corneal LS/P cell differentiation and CE cell apoptosis through regulating the Plk3 signaling pathways will be integrated in this aim. We investigate the effects of altered Plk3 activities on hypoxia-induced LS/P cell differentiation and CE cell apoptosis, and how hypoxia-induced delay of the wound healing process is affected by altering Plk3 activity in LS/P cells and corneas of Plk3-/- mice. By achieving the goal of combined studies, we will provide novel mechanisms to advance our understanding of hypoxia-induced effects on LS/P and CE cell functions in corneal epithelial self-renewal and wound healing.

描述（由申请人提供）：该项目的总体目的是定义人角膜角膜膜茎/祖细胞（LS/P）和角膜上皮（CE）细胞如何应对生理和病理条件下的低氧应激的分子机制。我们的初步数据表明，在人CE细胞中，缺氧（1％O2）激活类似Polo样激酶3（PLK3）级联反应，该级联反应磷酸化一组用于调节细胞命运的重要决定因素，例如HIF-1，p53，c-jun/ap-1和h2ax。因此，基于PLK3活性增加的幅度，对低氧应激的反应更大导致CE细胞凋亡。相比之下，人角膜LS/P细胞对缺氧诱导的凋亡具有抗性，因为缺氧抑制了PLK3的表达，并且无法诱导这些细胞中HIF-1，p53，C-Jun/AP-1和H2AX的磷酸化。我们揭示缺氧刺激microRNA（miRNA）表达谱的显着变化。这些miRNA专门针对PLK3 mRNA的3'-非翻译区（3'UTR），以抑制角膜LS/P细胞中缺氧诱导的PLK3信号传导，但在CE细胞中却不是。我们的中心假设是，人角膜暴露于缺氧条件下激活角膜LS/P/P和CE细胞中的两个不同过程，包括：1）激活PLK3介导的信号传导途径，从而增加p53的磷酸化以及C-Jun/ap-1的激活以及CE细胞中CE细胞中导致的H2AX的激活； 2）激活miRNA的特定表达，抑制PLK3表达以下调下游靶标，从而导致低氧耐受性并触发角膜LS/P细胞的分化。为了识别分子机制，我们提出了三个目的：1）定义缺氧诱导的PLK3激活如何影响AP-1，p53和H2AX。缺氧诱导的p53和C-JUN磷酸化与凋亡直接相关。我们将确定缺氧诱导的PLK3是否可以直接激活p53，AP-1和H2AX，以及缺氧诱导的ATM/ATR/ATR/CHK1/2激活如何导致CE细胞中PLK3激活。 2）研究在缺氧诱导的角膜LS/P细胞中PLK3如何下调。低氧通过诱导高水平的PLK3特异性miRNA来抑制PLK3的表达。我们将在缺氧条件下确定低氧诱导的角膜LS/P细胞中的miRNA谱，哪种对低氧敏感的miRNA抑制了LS/P细胞中的PLK3信号通路，以及这些miRNA与PLK3 mRNA相互作用以影响其稳定性。 3）确定缺氧诱导的PLK3激活在角膜上皮伤口愈合中的作用。缺氧对调节PLK3信号通路的角膜LS/P细胞分化和CE细胞凋亡的影响将集成到此目标中。我们研究了PLK3活性改变对缺氧诱导的LS/P细胞分化和CE细胞凋亡的影响，以及缺氧引起的伤口愈合过程的延迟如何受到PLK3 - / - 小鼠的LS/P细胞和角膜的改变的影响。通过实现合并研究的目标，我们将提供新的机制，以促进我们对低氧诱导的对角膜上皮上皮自我更新和伤口愈合中LS/P和CE细胞功能的影响的理解。