TET1 in alcoholic liver disease progression

项目摘要 慢性酒精滥用与异常的表观遗传修饰有关，这种修饰影响了 控制肝细胞死亡的影响因素对酒精性肝病的影响。其中一个因素是 5-羟甲基胞嘧啶(5HmC)，但目前关于长期饮酒的信息很少 影响5HmC对细胞死亡的调节。了解酒精如何影响5hmC的形成，从而 细胞死亡途径可能会产生治疗ALD的方法。在我们的初步研究中，我们 发现5hmC在喂食酒精饮食的大鼠和小鼠的肝脏中表达下调 在人类ALD组织中。我们进一步检测了参与产生 5hmC，包括ALD样本中的甲基胞嘧啶双加氧酶TET1、TET2和TET3。结果发现， 在人类和啮齿动物的ALD样本中，Tet1的表达显著下调。我们确定使用shRNA- Tet1基因敲除人肝细胞TET1可显著抑制5hmC的形成并促进细胞 死亡以及促凋亡基因hrk。耐人寻味的是，DNA甲基化抑制剂5- 氮胞苷可以取代TET1基因敲除对肝细胞死亡的影响，进一步表明 DNA甲基化在TET1介导的肝细胞死亡中的重要性。然后我们分析了TET1是如何向下- 通过使用TET1基因敲除小鼠，调节参与了ALD的进展。发现TET1基因的敲除 实质上引起肝纤维化，因此这是慢性肝损伤伤口愈合的结果。 因此，我们的中心假设是，酒精暴露会增加肝细胞死亡，从而促进ALD 通过抑制TET1介导的5hmC表观遗传学改变而进展。我们的长远目标是澄清 TET1调节ALD进程的潜在机制，并确定TET1是否为潜在的 ALD的治疗靶点。两个具体的目标被用来评估我们的假设。在目标1中，我们将研究 TET1如何在ALD进展中调节细胞死亡途径。我们将研究它的酶功能。 TET1通过TET1催化域、全长TET1、TET1催化调控细胞死亡途径 结构域死亡突变和TET1特异性抑制物。在目标2中，我们将研究TET1在ALD中的作用 进步。我们将评估TET1下调对野生型(WT)肝细胞死亡的影响。 和TET1基因敲除(KO)小鼠，挑战酒精流质饮食。以进一步确定肝脏特异性 TET1在ALD进展中的作用，我们将通过白蛋白启动子建立肝脏特异性TET1基因敲除小鼠 驱动的Cre小鼠和Floted的TET1小鼠。这一结果将极大地促进我们对 TET1调节肝细胞死亡的机制及提高我们对 ALD进展的病理生理机制。我们还预计，它将有一个广泛的 对TET1表达及其与肝细胞功能关系的理解的影响。

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.