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及其协调替代来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细胞凋亡，作为清除不需要细胞的一种手段，Fas“死亡受体”途径包含了发生这种情况的主要生理机制。芳烃受体（AhR）是一种已知的调节细胞凋亡和增殖过程的配体激活转录因子，而AhR配体2,3,7,8-四氯二苯并-对二恶英（TCDD）是一类已知影响细胞凋亡和增殖过程的化合物的原型。我们的长期目标是了解AhR如何通过调节细胞生长和细胞死亡来促进组织稳态的机制。我们的假设得到了初步证据的支持，表明AhR活性可能通过调节促进细胞死亡程序的蛋白质表达，使肝细胞对Fas配体（FasL）诱导的凋亡敏感。一个合理的候选是ahr调节的n -肉豆蔻酰基转移酶2 (NMT2)，因为Bid蛋白的n -肉豆蔻酰基化对其促进fasl诱导的细胞凋亡的活性至关重要。本课题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将研究对fas介导的凋亡的敏感性升高是否取决于AhR的经典转录活性，还是涉及非经典机制。这些研究将检测fasl诱导的AhR阴性BP8肝癌细胞凋亡的严重程度，这些细胞表达AhR分子，具有特异性破坏AhR转录活性的靶向突变。在Aim 2中，我们将确定肝细胞对fas介导的凋亡的ahr依赖性敏感性是否完全归因于NMT2促进Bid活性的作用。目的3将研究AhR在分离的原代肝细胞和体内肝脏中fas介导的细胞凋亡中的作用。本研究将采用腺病毒基因转移策略，在培养的肝细胞和体内肝脏中表达蛋白，或使用小干扰rna抑制靶基因的表达，以获得AhR和fas介导的肝细胞凋亡之间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.