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: 10458730
• 负责人: Hao Zhu
• 金额: $ 54.84万
• 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/10458730
• 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.

项目总结 酒精性肝病(ALD)会因肝脏再生受损而加重。肝脏的细胞学基础 再生尚不清楚，不同区域的肝细胞再生活性是否不同也不清楚。 部分原因是命运映射只在少数几个肝细胞亚群上执行。肝脏被组织成 肝细胞表达不同代谢酶的区域。系统地比较可再生能源 为了提高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。世系溯源 在正常内稳态期间，来自门静脉周围区1和中心周区3的细胞数量收缩， 而小叶中部2区的细胞数量增多。因此，肝脏不同区域的肝细胞 周转率和区域2的差异是动态平衡期间新肝细胞的重要来源。因为 我们推测，2区可能代表了保护中心周围和门静脉周围肝脏损伤的储备人群。 这些细胞还优先重新填充暴露在酒精等轻度慢性损伤中的肝脏。我们的 初步scRNA-seq和体内CRISPR筛选发现了两个关键的第2区特异性基因，它们调节 第2区肝细胞增殖与存活：IGFBP2和HAMP2。这两种分泌因子都被抑制了 在人类的NAFLD和ALD中，这表明了功能在疾病中的重要性。IGFBP2通过mTOR运行 Ccnd1促进2区肝细胞增殖。Hamp1和Hamp2编码海普西丁，它 通过抑制铁转运蛋白来负向调节铁的摄取，从而保护身体免受铁超载的影响。 ALD患者通过抑制海普西丁积聚肝铁。游离铁增强活性 肝脏中产生的氧物种(ROS)，导致酒精引起的肝损伤。我们假设ALD 通过抑制IGFBP2和Hamp1/2来加速发病，这涉及到 2区肝细胞的数量或功能。在目标1中，我们将首先使用我们的Creer模型来系统地 在无水乙醇的设置下，确定2区细胞重新填充肝脏的程度。要了解区域如何 2细胞可能参与乙醇脱氢后的再生，我们将利用丢失和获得来扰乱两个关键区2基因。 函数方法的一部分。在目标2中，我们将询问IGFBP2是否必要且足以调节频率或 在乙醇的背景下重新填充第2区细胞的活动。在目标3中，我们将询问Hamp1和Hamp2是否 对于调节第2区细胞的频率或再填充活动是必要的和充分的。在这方面取得了成功 项目将首次定义再生的细胞基础，以响应Etoh，使我们能够专注于 关键亚群，并确定两个关键区2特定基因在ALD中的重要性。