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TET1 in alcoholic liver disease progression

TET1 在酒精性肝病进展中的作用

基本信息

批准号：
10041989
负责人：
Chiung-Kuei Huang
金额：
$ 24.09万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-25 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10041989
关键词：
Affect Albumins Alcohol abuse Alcohol consumption Alcoholic Hepatitis Alcoholic Liver Cirrhosis Alcoholic Liver Diseases Alcohols Animals Apoptosis Apoptotic Asthma Azacitidine Catalytic Domain Cause of Death Cell Death Cessation of life Chronic Cirrhosis DNA Damage DNA Methylation DNA Sequence Data Development Diet Dioxygenases Disease Disease Progression Down-Regulation Enterobacteria phage P1 Cre recombinase Enzymes Epigenetic Process Ethanol Family Gene Expression Generations Genes Hepatic Hepatocyte Human In Vitro Knock-out Knockout Mice Knowledge Length Link Liquid substance Liver Liver Cirrhosis Liver Fibrosis LoxP-flanked allele Malignant Neoplasms Measures Mediating Modification Molecular Mus Mutation Necrosis Outcome Pathway interactions Patients Process Rattus Regulation Rodent Role Sampling Severities Therapeutic Tissues Vascular Diseases alcohol exposure chronic alcohol ingestion effective therapy improved inhibitor/antagonist knock-down liver injury member oxidation problem drinker promoter protein expression response small hairpin RNA targeted treatment therapeutic target wound healing

项目摘要

Project Summary Chronic alcohol abuse has been linked to abnormal epigenetic modifications that affect the progression of alcoholic liver disease (ALD) by influencing factors in controlling cell death in hepatocytes. One such factor is 5-hydroxymethylcytosine (5hmC), but there is currently little information as to how chronic alcohol consumption affects 5hmC's regulation of cell death. Understanding how alcohol impacts 5hmC formation and consequently cell death pathways will potentially yield therapeutic approaches towards ALD. In our preliminary studies, we found that 5hmC expression is down-regulated in the livers of rats and mice fed with an ethanol diet as well as in human ALD tissues. We further examined the expression levels of enzymes involved in the generation of 5hmC, which include methylcytosine dioxygenase TET1, TET2, and TET3 in ALD samples. It was found that TET1 is significantly down-regulated in human and rodent ALD samples. We determined that using shRNA- TET1 to knockdown TET1 in human hepatocytes significantly suppressed 5hmC formation and promoted cell death as well as the pro-apoptotic gene, HRK. Intriguingly, the treatment of the DNA methylation inhibitor, 5- Azacytidine, could replace the impact of TET1 knockdown on hepatocyte cell death, further suggesting the importance of DNA methylation in TET1-mediated hepatocyte cell death. We then analyzed how TET1 down- regulation is involved in ALD progression by using TET1 knockout mice. It was found that knockout of TET1 substantially elicited liver fibrosis, which is consequently the outcome of wound-healing in chronic liver damage. Thus, our central hypothesis is that ethanol exposure increases hepatocyte cell death to promote ALD progression by suppressing TET1-mediated 5hmC epigenetic changes. Our long-term objective is to clarify the underlying mechanisms by which TET1 modulates ALD progression, and determine if TET1 is a potential therapeutic target in ALD. Two specific aims propose to evaluate our hypothesis. In aim 1, we will examine how TET1 modulates cell death pathways in ALD progression. We will investigate the enzymatic function of TET1 in regulating cell death pathways by using TET1 catalytic domain, full length TET1, TET1 catalytic domain dead mutation, and TET1 specific inhibitor. In aim 2, we will examine the role of TET1 in ALD progression. We will evaluate the impact of TET1 down-regulation on hepatocyte cell death in wild-type (WT) and TET1 knockout (KO) mice challenged with an alcoholic liquid diet. To further determine the hepatic specific TET1 role in ALD progression, we will generate liver specific TET1 knockout mice by using albumin promoter driven Cre mouse and floxed TET1 mouse. The results will significantly advance our knowledge of the mechanisms by which TET1 modulates hepatocyte cell death and improve our understanding of pathophysiological mechanisms underlying ALD progression. We also anticipate that it will have a broad impact on the understanding of TET1 expression and its relationship to hepatocyte function in general.

项目摘要慢性酒精滥用与异常的表观遗传修饰有关，这些表观遗传修饰影响了酒精性肝病（ALD）通过影响因素控制肝细胞死亡。一个这样的因素是 5-羟甲基胞嘧啶（5 hmC），但目前很少有关于慢性酒精消费如何影响5 hmC对细胞死亡的调节。了解酒精如何影响5 hmC的形成，细胞死亡途径将潜在地产生针对ALD的治疗方法。在初步研究中，我们发现5 hmC表达在喂食乙醇饮食的大鼠和小鼠的肝脏中下调，在人类ALD组织中。我们进一步检测了参与产生细胞凋亡的酶的表达水平。 5 hmC，其包括ALD样品中的甲基胞嘧啶双加氧酶TET 1、TET 2和TET 3。结果发现 TET 1在人类和啮齿动物ALD样品中显著下调。我们确定使用shRNA- 在人肝细胞中敲低TET 1可显著抑制5 hmC的形成，并促进细胞增殖。死亡以及促凋亡基因HRK。有趣的是，DNA甲基化抑制剂，5- 氮杂胞苷，可以取代TET 1敲低对肝细胞死亡的影响，进一步表明， DNA甲基化在TET 1介导的肝细胞死亡中的重要性。然后我们分析了TET 1是如何下降的- 通过使用TET 1敲除小鼠，发现调节参与ALD进展。发现TET 1的敲除实质上引起肝纤维化，其因此是慢性肝损伤中伤口愈合的结果。因此，我们的中心假设是，乙醇暴露增加肝细胞死亡，促进ALD 通过抑制TET 1介导的5 hmC表观遗传学变化来进行进展。我们的长远目标是澄清 TET 1调节ALD进展的潜在机制，并确定TET 1是否是一种潜在的 ALD的治疗目标。两个具体的目标提出来评估我们的假设。在目标1中，我们将研究 TET 1如何调节ALD进展中的细胞死亡途径。我们将研究酶的功能，通过使用TET 1催化结构域、全长TET 1、TET 1催化结构域死亡突变和TET 1特异性抑制剂。在目标2中，我们将研究TET 1在ALD中的作用。进展我们将评估TET 1下调对野生型（WT）肝细胞死亡的影响。和用酒精性液体饮食攻击的TET 1敲除（KO）小鼠。为了进一步确定肝脏特异性 TET 1在ALD进展中的作用，我们将通过使用白蛋白启动子产生肝脏特异性TET 1敲除小鼠驱动的Cre小鼠和floxed TET 1小鼠。这些结果将大大提高我们对 TET 1调节肝细胞死亡的机制，并提高我们对 ALD进展的病理生理机制。我们还预计，对理解TET 1表达及其与一般肝细胞功能的关系的影响。