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

• 批准号: 8399647
• 负责人: LUO LU
• 金额: $ 32.83万
• 依托单位: LUNDQUIST INSTITUTE FOR BIOMEDICAL INNOVATION AT HARBOR-UCLA MEDICAL CENTER
• 依托单位国家: 美国
• 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/8399647
• 关键词: 3' 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

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. PUBLIC HEALTH RELEVANCE: In this application, we propose to investigate significant questions concerning how corneal hypoxia in physiological and pathological conditions affect human corneal limbal stem cell differentiation and epithelial cell replacement. We will determine whether the Polo-like kinase 3 (Plk3) pathway is responsible for regulating activities of downstream hypoxia-induced determinative factors that are critical for corneal epithelial self- renewal and for improving the wound healing process in normal and disease stages. .

描述(申请人提供)：这个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(CE)细胞在生理和病理条件下如何响应低氧应激的分子机制。我们的初步数据显示，在人CE细胞中，低氧(1%O2)激活Polo-like kinase3(Plk3)级联，使一组调节细胞命运的重要决定因素如Hif-1、P53、c-Jun/AP-1和H2AX磷酸化。因此，根据Plk3活性增加的幅度，对低氧应激的更大反应会导致CE细胞凋亡。相比之下，人角膜LS/P细胞耐缺氧诱导的凋亡，因为缺氧抑制了Plk3的表达，而不能诱导这些细胞中Hif-1、P53、c-Jun/AP-1和H2AX的磷酸化。我们揭示了低氧刺激microRNA(MiRNA)表达谱的显著变化。这些miRNAs特异性地针对Plk3mRNA的3‘非翻译区(3’UTR)，以抑制低氧诱导的角膜LS/P细胞中的Plk3信号转导，而不是在CE细胞中。我们的中心假设是，人类角膜暴露在低氧条件下，激活了角膜LS/P和CE细胞中两个不同的过程，包括：1)激活Plk3介导的信号通路，进而增加P53的磷酸化和c-Jun/AP-1和H2AX的激活，导致CE细胞凋亡；2)激活miRNAs的特异性表达，抑制Plk3的表达，下调下游靶基因的表达，导致低氧耐受，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)明确低氧诱导的Plk3激活如何影响AP-1、P53和H2AX，并与其相互作用。低氧诱导的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Plk3是否可以直接激活P53、AP-1和H2AX，以及低氧诱导的ATM/ATR/Chk1/2激活如何导致CE细胞中Plk3的激活。2)探讨Plk3在低氧诱导的角膜LS/P细胞中的下调作用。低氧通过诱导高水平的Plk3特异性miRNAs抑制Plk3的表达。我们将确定低氧诱导的角膜LS/P细胞在低氧条件下的miRNA谱，哪些对低氧敏感的miRNAs抑制了LS/P细胞中的Plk3信号通路，以及这些miRNAs如何与Plk3mRNA相互作用影响其稳定性。3)探讨低氧诱导的Plk3活化在角膜上皮损伤修复中的作用。缺氧通过调节Plk3信号通路对角膜LS/P细胞分化和CE细胞凋亡的影响将整合在这一目标中。我们研究了Plk3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Plk3-/-小鼠LS/P细胞和角膜中Plk3活性的变化如何影响低氧诱导的伤口愈合过程。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低氧诱导的LS/P和CE细胞在角膜上皮自我更新和伤口愈合中的作用的理解。 公共卫生相关性：在这项应用中，我们建议调查有关生理和病理条件下的角膜缺氧如何影响人角膜缘干细胞分化和上皮细胞替换的重要问题。我们将确定Polo-like kinase3(Plk3)通路是否负责调节下游低氧诱导的决定因子的活性，这些决定因子对角膜上皮自我更新和改善正常和疾病阶段的伤口愈合过程至关重要。。