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: 9755738
• 负责人: Carolina Jannet Garcia Garcia
• 金额: $ 3.25万
• 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/9755738
• 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; 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; Stem cells; 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 crypt; member; novel therapeutics; overexpression; prevent; promoter; radiation response; radiation-induced injury; radioprotected; radioresistant; response; self-renewal; sensor; stem cell niche; stem cell population; 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.

项目概要/摘要 当小肠暴露于高剂量时会发生辐射诱发胃肠道综合征 (RIGS) 的辐射。肠干细胞 (ISC) 和内皮细胞的放射损伤会损害肠道 再生，导致上皮完整性丧失和粘膜屏障功能障碍。这反过来又导致 吸收不良、脱水、电解质失衡、细菌移位、败血症，甚至死亡。 此外，腹部肿瘤的放射治疗具有挑战性，因为小肠非常脆弱。 辐射敏感。肠道的自我更新能力和对辐射的敏感性源于快速循环 隐窝中的 ISC。隐窝基底柱状 (CBC) 细胞产生所有肠细胞谱系，它们是 大致分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， 已被证明具有隐源性和损伤诱导性。还有证据表明分泌祖细胞 当肠道损伤时可以恢复为 CBC。我们的初步数据表明辐射会引起 与分泌生态位相关的标记的表达。然而，分泌生态位可塑性的动态 它们与辐射损伤背景下的 CBC 细胞的关系仍不清楚。此外，目前尚无治疗方法 预防、减轻或治疗 RIGS 甚至轻微的肠道辐射损伤。 EGLN 家族是细胞氧 通过降解缺氧诱导因子（HIF）来调节细胞存活和代谢的传感器。 众所周知，HIF 可以诱导组织重塑、增加上皮完整性、刺激肠道血管生成，以及 促进干细胞存活，所有这些对于应对辐射损伤都是至关重要的。此外，HIF 还调节 肠道屏障功能所需的基因。我们的小组已经证明 HIF2 稳定，但 HIF1 不稳定， 通过抑制 EGLN 蛋白，可以减轻和保护小鼠免受 RIGS 的侵害。然而，该机制通过 HIF2 赋予这种辐射防护作用的具体机制仍不清楚。为了深入了解这一机制，我们进行了 HIF2 过表达肠道类器官的 RNA 测序。我们将 Wnt5a（一种非规范 WNT）确定为直接 HIF2 的转录靶标，但不是 HIF1。有趣的是，其他小组已经表明 Wnt5a 可以改善结肠隐窝 机械损伤后的再生。我们还发现 Wnt5a 的敲除降低了克隆形成 ISC 的潜力。因此，我们假设 HIF2 通过以下方式诱导辐射损伤后肠道再生： 诱导 Wnt5a 表达以促进 ISC 存活。我们将从两个目标来检验这个假设。目标1 我们将确定 HIF2 是否结合并激活 WNT5A 启动子。我们还将测试 Wnt5a 是否必要，以及 足以在体外和体内进行 HIF2 介导的肠道辐射防护。在目标 2 中，我们将确定哪个 ISC 人群受到 HIF2/Wnt5a 轴的辐射防护。为此，我们将进行谱系追踪实验 和单细胞转录组分析。该提案的目的是要有足够的机制 了解 EGLN-HIF2 通路在辐射损伤后肠道再生中的作用。 深入了解这一机制可能有助于开发 RIGS 的新疗法，目前 RIGS 还没有治疗选择。