Ah Receptor Action and Apoptosis

描述(由申请人提供)：肝脏动态平衡是通过去除病变和受损的肝细胞以及它们的协调替换来维持恒定的肝细胞质量来实现的。肝硬变、病毒性肝炎和毒性药物效应都可以触发肝脏细胞凋亡，作为清除多余细胞的一种手段，而Fas‘死亡受体’途径是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，调节细胞的凋亡和增殖过程，而AhR的配体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动态平衡。我们的假说得到了初步证据的支持，表明AhR活性使肝细胞对Fas配体(FasL)诱导的凋亡敏感，可能是通过调节促进细胞死亡程序的蛋白质的表达。一个可能的候选者是AhR调节的N-肉豆蔻酰基转移酶2(NMT2)，因为Bid蛋白的N-肉豆蔻酰化对于其促进FasL诱导的细胞凋亡至关重要。这项建议的目的是在体内外Fas介导的肝细胞凋亡的背景下研究AhR的功能。目的1将检验对Fas介导的细胞凋亡的敏感性增加是否依赖于AhR的经典转录活性，或者涉及非经典机制。这些研究将检测FasL诱导表达AhR分子的AhR阴性BP8肝癌细胞的凋亡的严重性，这些突变具有特定干扰AhR转录活性的靶向突变。在目标2中，我们将确定肝细胞对Fas介导的凋亡的依赖于AhR的敏感性是否完全归因于NMT2促进Bid活性的作用。目的3研究AhR在Fas介导的原代肝细胞和活体肝细胞凋亡中的作用。这些研究将使用腺病毒基因转移策略来表达蛋白质，或者使用小干扰RNA在培养的肝细胞和体内的肝脏中抑制靶基因的表达，以期从机制上了解AhR和Fas介导的肝细胞凋亡之间的功能关系。

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.