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：
8539630
负责人：
LUO LU
金额：
$ 31.19万
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 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8539630
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）：该项目的总体目标是确定人角膜缘干/祖细胞（LS/P）和角膜上皮（CE）细胞在生理和病理条件下如何响应缺氧应激的分子机制。我们的初步数据显示，在人类 CE 细胞中，缺氧 (1% O2) 会激活 Polo 样激酶 3 (Plk3) 级联，从而磷酸化一组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 活性增加幅度的对缺氧应激的更大反应导致 CE 细胞凋亡。相比之下，人角膜 LS/P 细胞对缺氧诱导的细胞凋亡具有抵抗力，因为缺氧会抑制 Plk3 表达，并且不能诱导这些细胞中 Hif-1、p53、c-Jun/AP-1 和 H2AX 的磷酸化。我们发现缺氧会刺激 microRNA (miRNA) 表达谱的显着变化。这些 miRNA 特异性靶向 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 细胞中则不然。我们的中心假设是，人类角膜暴露于缺氧条件下会激活角膜 LS/P 和 CE 细胞中的两个不同过程，包括：1) 激活 Plk3 介导的信号通路，进而增加 p53 磷酸化和 c-Jun/AP 的激活-1和H2AX导致CE细胞凋亡； 2) 激活 miRNA 的特异性表达，抑制 Plk3 表达，下调下游靶标，从而产生缺氧耐受性并触发角膜 LS/P 细胞的分化。为了确定分子机制，我们提出了三个目标：1) 定义缺氧诱导的 Plk3 激活如何影响 AP-1、p53 和 H2AX 并与之相互作用。缺氧诱导的 p53 和 c-Jun 磷酸化与细胞凋亡直接相关。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/Chk1/2 激活如何导致 CE 细胞中的 Plk3 激活。 2) 研究缺氧诱导的角膜LS/P细胞中Plk3如何下调。缺氧通过诱导高水平的 Plk3 特异性 miRNA 来抑制 Plk3 表达。我们将确定缺氧条件下角膜LS/P细胞中缺氧诱导的miRNA谱，哪些缺氧敏感的miRNA抑制LS/P细胞中的Plk3信号通路，以及这些miRNA如何与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。该目标将整合缺氧通过调节 Plk3 信号通路对角膜 LS/P 细胞分化和 CE 细胞凋亡的影响。我们研究了改变 Plk3 活性对缺氧诱导的 LS/P 细胞分化和 CE 细胞凋亡的影响，以及改变 LS/P 细胞和 Plk3-/ 角膜中的 Plk3 活性如何影响缺氧诱导的伤口愈合过程延迟。 - 老鼠。通过实现联合研究的目标，我们将提供新的机制来促进我们对缺氧对角膜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。