Ah Receptor Action and Apoptosis

描述（由申请人提供）：肝脏稳态是通过去除患病和受损的肝细胞并对其进行协调替代以维持恒定的肝细胞质量来实现的。肝硬化、病毒性肝炎和毒性药物作用都可以触发肝脏中的细胞凋亡，作为去除不需要的细胞的手段，并且Fas“死亡受体”途径包括发生这种情况的主要生理机制。芳烃受体（AhR）是一种配体激活的转录因子，已知其调节细胞凋亡和增殖过程，而AhR配体2，3，7，8-四氯二苯并对二恶英（TCDD）是一类已知影响这些过程的化合物的原型。我们的长期目标是从机制上了解AhR如何通过调节细胞生长和细胞死亡来促进组织稳态。我们的假设，支持的初步证据表明，AhR活性敏感的肝细胞Fas配体（FasL）诱导的凋亡，可能是通过调节蛋白质的表达，促进细胞死亡程序。一个可能的候选者是AhR调节的酶N-肉豆蔻酰转移酶2（NMT 2），因为Bid蛋白的N-肉豆蔻酰化对其促进FasL诱导的细胞凋亡的活性至关重要。本研究的目的是在体外和体内研究Fas介导的肝细胞凋亡中AhR的功能。目的1将检查是否增加的敏感性Fas介导的acutotis依赖于经典的转录活性的AhR，或涉及一个非经典的机制。这些研究将检查表达AhR分子的AhR阴性BP 8肝癌细胞中FasL诱导的细胞凋亡的严重程度，所述AhR分子具有特异性破坏AhR转录活性的靶向突变。在目的2中，我们将确定肝细胞对Fas介导的细胞凋亡的AhR依赖性易感性是否完全是由于NMT 2促进Bid活性的作用。目的3将研究AhR在Fas介导的离体原代肝细胞和体内肝脏细胞凋亡中的作用。这些研究将使用腺病毒基因转移策略来表达蛋白质，或使用小干扰RNA来抑制体内培养的肝细胞和肝脏中的靶基因表达，以获得对AhR和Fas介导的肝细胞凋亡之间的功能关系的机制理解。

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.