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类角膜缘干细胞/祖细胞（LS/P）和角膜上皮细胞（CE）在生理和病理条件下对缺氧应激的反应的分子机制。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 （Plk3）级联反应，磷酸化一组重要的细胞命运调节决定因子，如Hif-1、p53、c-Jun/AP-1和H2AX。因此，基于Plk3活性增加幅度的低氧应激反应导致CE细胞凋亡。相比之下，人角膜LS/P细胞对缺氧诱导的凋亡具有抗性，因为缺氧抑制Plk3的表达，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，缺氧刺激了microRNA （miRNA）表达谱的显著变化。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细胞中没有作用。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-1和H2AX的激活，导致CE细胞凋亡；2)激活抑制Plk3表达的mirna特异性表达，下调下游靶标，导致缺氧耐受，触发角膜LS/P细胞分化。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Plk3在CE细胞中的激活。2)探讨Plk3在缺氧诱导的角膜LS/P细胞中的下调机制。缺氧通过诱导高水平Plk3特异性mirna的新机制抑制Plk3的表达。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)探讨缺氧诱导Plk3活化在角膜上皮创面愈合中的作用。缺氧通过调控Plk3信号通路对角膜LS/P细胞分化和CE细胞凋亡的影响将在本研究中得到整合。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Plk3-/-小鼠LS/P细胞和角膜中Plk3活性的改变如何影响缺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，以促进我们对缺氧诱导的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。

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.