Ah Receptor Action and Apoptosis

Ah 受体的作用和细胞凋亡

• 批准号: 7054159
• 负责人: Cornelis Johan Elferink
• 金额: $ 31.52万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-14 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7054159
• 关键词: CD95 molecule; HTC cell; apoptosis; aromatic hydrocarbon receptor; cell growth regulation; enzyme activity; fatty acylation; flow cytometry; fluorescence microscopy; gel mobility shift assay; gene expression; genetically modified animals; green fluorescent proteins; homeostasis; laboratory mouse; liver function; myristates; polymerase chain reaction; protein protein interaction; protein structure function; small interfering RNA; tissue /cell culture; transcription factor; transfection

DESCRIPTION (PROVIDED BY APPLICANT): Liver homeostasis is achieved by the removal of diseased and damages hepatocytes and their coordinated replacement to maintain a constant liver cell mass. Cirrhosis, viral hepatitis and toxic drug effects can all trigger apoptosis in the liver as a means to remove the unwanted cells, and the Fas 'death receptor' pathway comprises a major physiological mechanism by which this is occurs. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to regulate both apoptotic and proliferative processes, and the AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is the prototype for a class of compounds known to affect these processes. Our long term goal is to understand mechanistically how the AhR contributes to tissue homeostasis by regulating cell growth and cell death. Our hypothesis, supported by the preliminary evidence, suggests that AhR activity sensitizes liver cells to Fas ligand (FasL) induced apoptosis, possibly by regulating expression of proteins that promote the cell death program. A plausible candidate is the AhR-regulated enzyme N-myristoyltransferase 2 (NMT2), because N-myristoylation of the Bid protein is critical for its activity in promoting FasL-induced apoptosis. The goal of this proposal is to study AhR function in the context of Fas-mediated liver apoptosis in vitro and in vivo. Aim 1 will examine whether the heightened susceptibility to Fas-mediated apoptotis depends on classical transcriptional activity by the AhR, or involves a non-classical mechanism. These studies will examine the severity of FasL-induced apoptosis in AhR-negative BP8 hepatoma cells expressing AhR molecules with targeted mutations that specifically disrupt AhR transcriptional activity. In Aim 2 we will determine whether the AhR-dependent susceptibility of hepatocytes to Fas-mediated apoptosis is due entirely to NMT2 action facilitating Bid activity. Aim 3 will examine the AhR's role in Fas-mediated apoptosis in isolated primary hepatocytes and in the liver in vivo. The studies will use an adenovirus gene transfer strategy to either express proteins, or use small interfering RNAs to suppress target gene expression in both cultured hepatic cells and the liver in vivo, in order to gain a mechanistic understanding of the functional relationship between the AhR and Fas-mediated hepatocyte apoptosis.

描述（由申请人提供）：肝动态平衡是通过清除患病和损伤肝细胞及其协调替代品来实现的，以维持恒定的肝细胞肿块。肝硬化，病毒性肝炎和有毒药物作用都可以引发肝脏中的凋亡，以作为去除不良细胞的一种手段，而FAS“死亡受体”途径包括发生这种情况的主要生理机制。芳基烃受体（AHR）是一种已知调节凋亡和增殖过程的配体激活转录因子，而AHR配体为2,3,7,8-四氯迪本佐-P-二恶英（TCDD），是对这些过程的化合物的原型。我们的长期目标是通过调节细胞生长和细胞死亡来机械理解AHR如何对组织稳态促进。我们的假设得到了初步证据的支持，表明AHR活性使肝细胞对FAS配体（FASL）诱导的细胞凋亡敏感，这可能是通过调节促进细胞死亡程序的蛋白质的表达。合理的候选者是AHR调节的酶N- myristoyltransferase 2（NMT2），因为BID蛋白的N- myristoylation对其在促进FASL诱导的细胞凋亡方面的活性至关重要。该建议的目的是在FAS介导的体外和体内研究AHR功能。 AIM 1将检查对FAS介导的凋亡的敏感性是否取决于AHR经典的转录活性，还是涉及非经典机制。这些研究将检查FASL诱导的AHR阴性BP8肝癌细胞中具有特异性破坏AHR转录活性的AHR分子的AHR肝癌细胞的严重程度。在AIM 2中，我们将确定肝细胞对FAS介导的细胞凋亡的易感性是否完全归因于NMT2作用促进竞标活性。 AIM 3将检查AHR在孤立的原发性肝细胞和体内肝脏中的FAS介导的凋亡中的作用。该研究将使用腺病毒基因转移策略来表达蛋白质，或者使用小型干扰RNA抑制培养的肝细胞和体内肝脏中的靶基因表达，以便对AHR和Fas介导的肝细胞凋亡之间的功能关系有一种机械理解。