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

描述（由申请人提供）：本项目的总体目标是确定人角膜缘干细胞/祖细胞（LS/P）和角膜上皮细胞（CE）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）激活Polo样激酶3（Plk 3）级联，磷酸化一组重要的决定因素，调节细胞命运，如Hif-1，p53，c-Jun/AP-1和H2 AX。因此，基于Plk 3活性增加幅度的对缺氧应激的较大响应导致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抗性，因为缺氧抑制Plk 3表达，并且不能诱导这些细胞中Hif-1、p53、c-Jun/AP-1和H2 AX的磷酸化。我们发现，缺氧刺激microRNA（miRNA）表达谱的显着变化。这些miRNAs特异性靶向Plk 3 mRNA的3 '非翻译区（3' UTR），以抑制角膜LS/P细胞中低氧诱导的Plk 3信号传导，但不抑制CE细胞中的Plk 3信号传导。我们的中心假设是，人角膜暴露于缺氧条件激活角膜LS/P和CE细胞中的两个不同过程，包括：1）Plk 3介导的信号通路的激活，其进而增加p53磷酸化和c-Jun/AP-1和H2 AX的激活，导致CE细胞凋亡;和2）激活抑制Plk 3表达的miRNA的特异性表达以下调下游靶点，导致低氧耐受性并触发角膜LS/P细胞的分化。为了确定其分子机制，我们提出了三个目标：1）确定缺氧诱导的Plk 3活化如何影响AP-1、p53和H2 AX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。我们将确定低氧诱导的Plk 3是否可以直接激活p53，AP-1和H2 AX，以及低氧诱导的ATM/ATR/Chk 1/2激活如何导致Plk 3在CE细胞中的激活。2)探讨Plk 3在低氧诱导的角膜LS/P细胞中的表达下调。低氧通过诱导高水平Plk 3特异性miRNA抑制Plk 3表达我们将确定低氧条件下角膜LS/P细胞中低氧诱导的miRNA谱，哪些低氧敏感的miRNA抑制LS/P细胞中的Plk 3信号通路，以及这些miRNA如何与Plk 3 mRNA相互作用以影响其稳定性。3)探讨低氧诱导Plk 3活化在角膜上皮损伤愈合中的作用。低氧通过调控Plk 3信号通路对角膜LS/P细胞分化和CE细胞凋亡的影响将被整合到这一目标中。我们研究了Plk 3活性改变对低氧诱导的LS/P细胞分化和CE细胞凋亡的影响，以及如何通过改变Plk 3-/-小鼠的LS/P细胞和角膜中的Plk 3活性来影响低氧诱导的伤口愈合过程的延迟。通过实现联合研究的目标，我们将提供新的机制，以促进我们对角膜上皮自我更新和伤口愈合中低氧诱导的LS/P和CE细胞功能的影响的理解。

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.