Determining how chronic ETOH influences the regenerative activities of hepatocyte subpopulations

确定慢性 ETOH 如何影响肝细胞亚群的再生活动

• 批准号: 10297361
• 负责人: Hao Zhu
• 金额: $ 54.81万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10297361
• 关键词: Alcoholic Liver Diseases; Alcohols; CRISPR screen; Cell Proliferation; Cell physiology; Cells; Chronic; Contracts; Data; Disease; Enzymes; Ethanol; Exhibits; Exposure to; FRAP1 gene; Frequencies; Genes; Genetic Transcription; Hepatic; Hepatocyte; Homeostasis; Human; Impairment; Injury; Iron; Iron Overload; Iron Uptake Inhibition; Label; Liver; Liver Regeneration; Lobular; Metabolic; Modeling; Morbidity - disease rate; Natural regeneration; Pathogenesis; Patients; Population; Production; Proliferating; Reactive Oxygen Species; Regulation; Source; Time; Tissues; gain of function; healing; hepcidin; in vivo; liver cell proliferation; liver injury; mortality; non-alcoholic fatty liver disease; overexpression; regenerative; response; single-cell RNA sequencing; success; tissue repair

PROJECT SUMMARY Alcoholic liver disease (ALD) is exacerbated by impaired liver regeneration. The cellular basis of liver regeneration is unclear and whether hepatocytes in different zones differ in regenerative activity is unclear, in part because fate-mapping has only been performed on a few hepatocyte subsets. The liver is organized into zones in which hepatocytes express different metabolic enzymes. To systematically compare the regenerative activities of these distinct subsets of hepatocytes, we developed twelve new CreER strains. Lineage tracing during normal homeostasis showed that cells from periportal zone 1 and pericentral zone 3 contracted in number, while cells from mid-lobular zone 2 expanded in number. Hepatocytes in different regions of the liver thus exhibit differences in turnover and zone 2 is an important source of new hepatocytes during homeostasis. Because zone 2 may represent a reserve population sheltered from pericentral and periportal liver injuries, we hypothesize that these cells also preferentially repopulate livers exposed to modest chronic injuries such as alcohol. Our preliminary scRNA-seq and in vivo CRISPR screens identified two critical zone 2 specific genes that regulate zone 2 hepatocyte proliferation and survival: Igfbp2 and Hamp2. Both of these secreted factors are suppressed in NAFLD and ALD in humans, which suggests functional importance in disease. Igfbp2 operates through mTOR and Ccnd1 to promote zone 2 hepatocyte proliferation. Hamp1 and Hamp2 encode for hepcidins, which negatively regulate iron uptake by inhibition of iron transporters and thus protects the body from iron overload. Patients with ALD accumulate hepatic iron through suppression of hepcidins. Free iron enhances reactive oxygen species (ROS) production in the liver, leading to alcohol-induced liver injury. We hypothesize that ALD pathogenesis is accelerated through the suppression of Igfbp2 and Hamp1/2, and that this involves changes in the number or function of zone 2 hepatocytes. In Aim 1, we will first use our CreER models to systematically determine the extent to which zone 2 cells repopulate the liver in the setting of ETOH. To understand how zone 2 cell might be involved in regeneration after ETOH, we will perturb two critical zone 2 genes using loss and gain of function approaches. In Aim 2, we will ask if Igfbp2 is necessary and sufficient to regulate the frequency or repopulating activities of zone 2 cells in the context of ETOH. In Aim 3, we will ask if Hamp1 and Hamp2 are necessary and sufficient to regulate the frequency or repopulating activities of zone 2 cells. Success in this project will for the first time define the cellular basis of regeneration in response to ETOH, allow us to focus on critical subpopulations, and determine the importance of two critical zone 2 specific genes in ALD.

项目摘要 酒精性肝病（ALD）是由受损的肝脏再生加剧。肝脏的细胞基础 再生不清楚，不同区域的肝细胞再生活性是否不同也不清楚， 部分原因是仅对少数肝细胞亚群进行了命运映射。肝脏组织成 肝细胞表达不同代谢酶的区域。为了系统地比较 这些不同的肝细胞亚群的活性，我们开发了12个新的CreER菌株。谱系追踪 在正常体内平衡期间显示来自门静脉周围区1和中央周围区3的细胞在数量上收缩， 而来自小叶中带2的细胞在数量上扩增。因此，肝脏不同区域的肝细胞表现出 周转和区2的差异是体内平衡期间新肝细胞的重要来源。因为 2区可能代表了一个保护区，不受中心周围和门静脉周围肝损伤的影响，我们假设 这些细胞也优先在暴露于酒精等中度慢性损伤的肝脏中重新繁殖。我们 初步的scRNA-seq和体内CRISPR筛选鉴定了两个关键区2特异性基因， 2区肝细胞增殖和存活：Igfbp 2和Hamp 2。这两种分泌因子都受到抑制 在人类的NAFLD和ALD中，这表明在疾病中的功能重要性。Igfbp 2通过mTOR发挥作用 和Ccnd 1促进2区肝细胞增殖。Hamp 1和Hamp 2编码hepcidin， 通过抑制铁转运蛋白来负调节铁的摄取，从而保护身体免受铁过载。 ALD患者通过抑制hepcidins积累肝脏铁。游离铁增强活性 在肝脏中产生氧物种（ROS），导致酒精诱导的肝损伤。我们假设ALD 通过抑制Igfbp 2和Hamp 1/2加速发病，这涉及到 2区肝细胞的数量或功能。在目标1中，我们将首先使用CreER模型系统地 确定在ETOH的情况下2区细胞重新填充肝脏的程度。要了解区域如何 2细胞可能参与ETOH后的再生，我们将使用丢失和获得干扰两个关键区域2基因， 功能的方法。在目标2中，我们将询问Igfbp 2是否必要且足以调节频率， 在ETOH的情况下，2区细胞的再增殖活动。在目标3中，我们将询问Hamp 1和Hamp 2是否 这是调节2区细胞的频率或重新增殖活动所必需的和足够的。胜任这 该项目将首次定义ETOH再生的细胞基础，使我们能够专注于 关键亚群，并确定两个关键区2特异性基因在ALD中的重要性。