The Function of cFLIP in B Lymphocytes

描述（由申请方提供）：本拟定研究的重点是了解细胞FLICE样抑制蛋白cFLIP在B淋巴细胞中的体内功能。这个项目是从我们长期以来对FADD介导的凋亡和增殖信号机制的兴趣发展而来的。我们和其他人以前已经证明，FADD是一个共同的信号衔接子共享的几个死亡受体，包括Fas，肿瘤坏死因子受体1（TNF-R1），和TNF相关的凋亡诱导配体受体（TRAIL-R）。FADD的死亡结构域与死亡受体的死亡结构域结合，并且FADD的死亡效应结构域与胱天蛋白酶8的死亡效应结构域缔合。死亡受体诱导的细胞凋亡在抑制自身免疫和恶性肿瘤中起重要作用，但在胚胎发育过程中是不稳定的。有趣的是，缺乏FADD或Caspase 8导致小鼠早期胚胎死亡。使用活的FADD-/-->RAG-1-/-嵌合体，我们已经表明，当胚胎干细胞中FADD缺失时，B细胞发育被完全阻断。然而，我们发现当使用CD 19 Cre在前B细胞中缺失FADD时，B细胞发育没有明显影响，表明FADD在早期阶段和/或在B谱系定型之前是必需的。令人惊讶的是，FADD缺陷型B细胞不仅在凋亡方面有缺陷，而且在Toll样受体（TLR）诱导的增殖方面也受损。在条件性Caspase 8缺陷小鼠中也观察到类似的缺陷。这些研究有助于揭示B细胞中涉及FADD和Caspase 8的二元信号传导的新范例。FADD和半胱天冬酶8如何调节B细胞中的两种不同的信号传导（凋亡和增殖）尚不清楚。我们推测cFLIP可以与FADD结合，可能在B细胞中FADD和Caspase 8的双重功能调节中发挥作用。cFLIP与Caspase 8同源，但缺乏Caspase活性。在初步研究中，我们发现cFLIP缺陷小鼠是胚胎致死的，其表型与缺乏FADD或Caspase 8的小鼠相似。此外，我们发现，杂合cFLIP T细胞是低增殖的，而转基因cFLIP过表达导致T细胞过度增殖。有报道表明cFLIP抑制原代B细胞的凋亡。然而，体外cFLIP的过表达也显示出增强或抑制细胞凋亡。目前尚不清楚cFLIP是否在TLR刺激诱导的FADD/Caspase 8介导的B细胞增殖中发挥作用。我们提出（1）利用B细胞中cFLIP缺失的条件突变小鼠来分析cFLIP的体内功能;（2）利用反向遗传学来剖析cFLIP在B淋巴细胞中的多种功能。该研究将有助于确定cFLIP调控的体内信号通路，这将有助于包括免疫疾病和癌症在内的各种疾病的翻译研究。在本申请中，我们描述了一项研究cFLIP蛋白的科学计划，该蛋白在发育，免疫系统功能和癌症中起着至关重要的作用。研究结果将有助于制定有效的预防和治疗方法。

DESCRIPTION (provided by applicant): The focus of this proposed study is to understand the in vivo function of the cellular FLICE-like inhibitory protein, cFLIP, in B lymphocytes. This project has evolved from our longstanding interest in the FADD-mediated apoptotic and proliferation signaling mechanism. We and others have previously demonstrated that FADD is a common signaling adaptor shared by several death receptors including Fas, tumor necrosis factor receptor 1 (TNF-R1), and TNF-related apoptosis-inducing ligand receptors (TRAIL-R). The death domain of FADD binds to the death domain of death receptors, and the death effector domain of FADD associates with the death effector domains of Caspase 8. Death receptor-induced apoptosis plays an important role in suppressing autoimmunity and malignancy; however, they are dispensable during embryonic development. Interestingly, a lack of FADD or Caspase 8 results in early embryonic lethality in mice. Using viable FADD-/-->RAG-1-/- chimeras, we have showed that B cell development is completely blocked when FADD is absent in embryonic stem cells. However, we found that B cell development was not obviously affected when FADD is deleted in pre-B cells using CD19Cre, indicating FADD is essential at earlier stages and/or prior to B lineage commitment. Surprisingly, FADD-deficient B cells are not only defective in apoptosis but also impaired in Toll-like receptor (TLR)-induced proliferation. Similar defects have been observed in conditional Caspase 8-deficient mice. These studies have helped reveal a new paradigm of binary signaling in B cells involving FADD and Caspase 8. How FADD and Caspase 8 regulate two distinct signaling, apoptosis and proliferation in B cells is not well understood. We hypothesize that cFLIP, which can bind to FADD, may play a role in the regulation of the dual function of FADD and Caspase 8 in B cells. cFLIP is homologous to Caspase 8 but lack Caspase activity. In preliminary studies, we showed that cFLIP-deficient mice are embryonic lethal, a phenotype similar to that of mice lacking FADD or Caspase 8. In addition, we found that heterozygous cFLIP T cells are hypoproliferative, whereas transgenic cFLIP overexpression leads to T cell hyperproliferation. There are reports indicating that cFLIP inhibits apoptosis in primary B cells. However, in vitro overexpression of cFLIP was also shown to either enhance or inhibit apoptosis. It is not clear whether cFLIP play a role in FADD/Caspase 8-mediated B cell proliferation induced by TLR stimulation. We propose (1) to analyze the in vivo function of cFLIP by using conditional mutant mice lacking cFLIP in B cells, and (2) to dissect the multiple functions of cFLIP in B lymphocyte by reverse genetics. This study will help identify the in vivo signaling pathways regulated by cFLIP, which would be useful for translational research of various diseases including immune disorders and cancer. In this application, we describe a scientific plan to study the cFLIP protein which plays an essential role in development, immune system functions, and cancer. The results will facilitate the development of effective preventive and therapeutic approaches.