The Role of HIF2-Induced Wnt5a in Small Intestine Regeneration after Radiation Injury

HIF2诱导的Wnt5a在放射损伤后小肠再生中的作用

• 批准号: 9905311
• 负责人: Carolina Jannet Garcia Garcia
• 金额: $ 3.3万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905311
• 关键词: Abdomen; Address; Alleles; Bacterial Translocation; Binding; Biological Models; Blood Vessels; Caenorhabditis elegans; Cell Lineage; Cell Survival; Cells; Cellular Metabolic Process; Cessation of life; Columnar Cell; Data; Dehydration; Electrolytes; Endothelium; Enterocytes; Enteroendocrine Cell; Epithelial; Epithelial Cells; Epithelium; Exposure to; Family; Functional disorder; Genes; Genetic Transcription; Goals; Grant; HIF1A gene; Heterogeneity; Homeostasis; Human; Hypoxia; Hypoxia Inducible Factor; Impairment; In Vitro; Injury; Intestines; Knock-out; LacZ Genes; Malabsorption Syndromes; Mechanics; Mediating; Molecular; Mucous Membrane; Mus; Natural regeneration; Nuclear Accidents; Organoids; Oxygen; Paneth Cells; Pathway interactions; Phenotype; Physiological; Play; Positioning Attribute; Predisposition; Procollagen-Proline Dioxygenase; Protein Isoforms; Proteins; Radiation; Radiation Dose Unit; Radiation Injuries; Radiation Protection; Radiation Tolerance; Radiation therapy; Reporter; Role; Secondary to; Secretory Cell; Sepsis; Small Intestines; Syndrome; System; Techniques; Terrorism; Testing; Therapeutic; Tissues; Toxic effect; WNT5A gene; angiogenesis; base; clinical predictors; colonic crypt; egg; experimental study; gastrointestinal; improved; in vivo; insight; intestinal barrier; intestinal crypt; intestinal injury; member; novel therapeutics; overexpression; prevent; promoter; radiation response; radiation-induced injury; radioprotected; radioresistant; response; self-renewal; sensor; stem cell niche; stem cell population; stem cells; transcriptome sequencing; transcriptomics; tumor

Project Summary/Abstract Radiation-induced gastrointestinal syndrome (RIGS) occurs when the small intestines are exposed to high doses of radiation. Radiation injury to the intestinal stem cell (ISC) and endothelial compartments impair intestinal regeneration, cause loss of epithelial integrity and mucosal barrier dysfunction. This in turn leads to malabsorption, dehydration, electrolyte imbalances, bacterial translocation, sepsis, and often death. Furthermore, radiation therapy for abdominal tumours is challenging because the small intestine is exquisitely radiosensitive. The intestine’s self-renewal ability and susceptibility to radiation derive from the rapid-cycling ISCs in the crypts. The crypt base columnar (CBC) cells give rise to all the intestinal cell lineages, which are broadly categorized as absorptive or secretory cells. Enteroendocrine cells, which are part of the secretory niche, have been shown to be cryptogenic and injury-inducible. There is also evidence that secretory progenitor cells can revert to CBCs when there is intestinal injury. Our preliminary data indicate that radiation induces the expression of markers associated to the secretory niche. However, the dynamics of the secretory niche plasticity and their relation to CBC cells in the context of radiation injury remain unclear. Moreover, there are no therapies to prevent, mitigate, or treat RIGS or even modest intestinal radiation injury. The EGLN family are cellular oxygen sensors that regulate cell survival and metabolism through the degradation hypoxia-inducible factors (HIFs). HIFs are known to induce tissue remodelling, increase epithelial integrity, stimulate intestinal angiogenesis, and promote stem cell survival, all of which are essential for response to radiation injury. Additionally, HIFs regulate genes required for intestinal barrier function. Our group has shown that stabilization of HIF2, but not HIF1, through inhibition of the EGLN proteins mitigates and protects against RIGS in mice. Yet, the mechanisms by which HIF2 confers this radioprotection remain poorly studied. To gain insight into this mechanism, we performed RNA-seq of HIF2-overexpressing intestinal organoids. We identified Wnt5a, a non-canonical WNT, as a direct transcriptional target of HIF2, but not HIF1. Interestingly, other groups have shown Wnt5a improves colonic crypt regeneration following mechanical injury. We also found that knock-out of Wnt5a decreased the clonogenic potential of ISCs. Thus, we hypothesize that HIF2 induces intestinal regeneration after radiation injury by inducing Wnt5a expression to promote ISC survival. We will examine this hypothesis in two aims. In aim 1 we will determine if HIF2 binds and activates the WNT5A promoter. We will also test if Wnt5a is necessary and sufficient for HIF2-mediated intestinal radioprotection both in vitro and in vivo. In aim 2 we will identify which ISC populations are radioprotected by the HIF2/Wnt5a axis. To do so, we will perform lineage-tracing experiments and single cell transcriptomic analyses. The objective of this proposal is to have sufficient mechanistic understanding of the role the EGLN-HIF2 pathway plays in intestinal regeneration following radiation injury. Insight into this mechanism could aid in new therapies for RIGS, which currently has no therapeutic option.

项目总结/摘要 当小肠暴露于高剂量时，会发生辐射诱导的胃肠综合征（RIGS） 辐射。肠干细胞（ISC）和内皮细胞的辐射损伤损害了肠功能， 再生，导致上皮完整性丧失和粘膜屏障功能障碍。这又导致 吸收不良、脱水、电解质失衡、细菌移位、脓毒症，并且常常死亡。 此外，腹部肿瘤的放射治疗是具有挑战性的，因为小肠是精致的， 对辐射敏感。肠道的自我更新能力和对辐射的敏感性来自快速循环 地下室的智能控制器。隐窝基底柱状（CBC）细胞产生所有的肠细胞谱系， 被广泛分类为吸收细胞或分泌细胞。肠内分泌细胞是分泌生态位的一部分， 已被证明是隐源性和损伤诱导性的。也有证据表明分泌祖细胞 当有肠道损伤时，可以恢复为CBC。我们的初步数据表明， 与分泌小生境相关的标记物的表达。然而，分泌生态位可塑性的动力学 在辐射损伤的背景下，它们与CBC细胞的关系仍不清楚。此外，没有任何治疗方法 以预防、减轻或治疗RIGS或甚至适度的肠辐射损伤。EGLN家族是细胞氧 通过降解缺氧诱导因子（HIF）调节细胞存活和代谢的传感器。 已知HIF诱导组织重塑，增加上皮完整性，刺激肠血管生成， 促进干细胞存活，所有这些都是对辐射损伤的反应所必需的。此外，HIFs调节 肠道屏障功能所需的基因。我们的研究小组已经表明，稳定HIF 2，而不是HIF 1， 通过抑制EGLN蛋白减轻并保护小鼠免受RIGS。然而， HIF 2赋予这种辐射保护的机制仍然研究得很少。为了深入了解这一机制，我们进行了 HIF 2过表达的肠类器官的RNA-seq。我们确定Wnt 5a，一个非典型的WNT，作为一个直接的 转录靶点是HIF 2，而不是HIF 1。有趣的是，其他研究小组已经表明Wnt 5a可以改善结肠隐窝， 机械损伤后的再生。我们还发现敲除Wnt 5a降低了克隆形成， ISCs的潜力。因此，我们假设HIF 2通过以下途径诱导辐射损伤后的肠再生： 诱导Wnt 5a表达以促进ISC存活。我们将从两个方面来检验这一假说。在aim 1中 我们将确定HIF 2是否结合并激活WNT 5A启动子。我们还将测试Wnt 5a是否必要， 足以在体外和体内提供HIF 2介导的肠辐射保护。在目标2中，我们将确定 群体通过HIF 2/Wnt 5a轴受到辐射保护。为此，我们将进行血统追踪实验 和单细胞转录组学分析。该提案的目的是要有足够的机械性 了解EGLN-HIF 2通路在辐射损伤后肠再生中的作用。 深入了解这种机制可以帮助RIGS的新疗法，目前没有治疗选择。