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.

项目概要 肝脏再生受损会加剧酒精性肝病 (ALD)。肝脏的细胞基础 再生尚不清楚，并且不同区域的肝细胞再生活性是否存在差异尚不清楚。 部分原因是命运图谱仅在少数肝细胞亚群上进行。肝脏被组织成 肝细胞表达不同代谢酶的区域。系统地比较再生 为了研究这些不同肝细胞亚群的活性，我们开发了 12 种新的 CreER 菌株。谱系追踪 在正常稳态期间，来自门静脉周围区域 1 和周围中心区域 3 的细胞数量收缩， 而来自中小叶区 2 的细胞数量则有所增加。因此，肝脏不同区域的肝细胞表现出 周转率和2区的差异是稳态期间新肝细胞的重要来源。因为 我们假设，2 区可能代表免受中心周围和门静脉周围肝损伤的储备人群 这些细胞也会优先在遭受酒精等轻度慢性损伤的肝脏中重新生长。我们的 初步的 scRNA-seq 和体内 CRISPR 筛选确定了两个关键区域 2 特定基因，它们调节 2区肝细胞增殖和存活：Igfbp2和Hamp2。这两种分泌因子均受到抑制 在人类 NAFLD 和 ALD 中，这表明其在疾病中的功能重要性。 Igfbp2 通过 mTOR 运作 Ccnd1 促进 2 区肝细胞增殖。 Hamp1 和 Hamp2 编码铁调素， 通过抑制铁转运蛋白来负向调节铁的吸收，从而保护身体免受铁过载的影响。 ALD 患者通过抑制铁调素积累肝铁。游离铁增强反应性 肝脏中产生氧气（ROS），导致酒精性肝损伤。我们假设 ALD 通过抑制 Igfbp2 和 Hamp1/2 会加速发病机制，这涉及 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 2 区细胞在 ETOH 环境下重新填充肝脏的程度。了解如何区域 2 细胞可能参与 ETOH 后的再生，我们将使用损失和增益扰乱两个关键区域 2 基因 的函数方法。在目标 2 中，我们将询问 Igfbp2 是否必要且足以调节频率或 在 ETOH 的背景下重新填充 2 区细胞的活动。在目标 3 中，我们将询问 Hamp1 和 Hamp2 是否 对于调节 2 区细胞的频率或重新增殖活动是必要且充分的。在这方面取得成功 该项目将首次定义响应 ETOH 的再生细胞基础，使我们能够专注于 关键亚群，并确定两个关键区域 2 特定基因在 ALD 中的重要性。