The Function of cFLIP in B Lymphocytes

cFLIP 在 B 淋巴细胞中的功能

• 批准号: 7646458
• 负责人: JIANKE ZHANG
• 金额: $ 7.73万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7646458
• 关键词: Affect; Alternative Splicing; Apoptosis; Apoptotic; Autoimmunity; B Cell Proliferation; B-Cell Development; B-Lymphocytes; Binding; Caspase; Cell Death; Cell Proliferation; Cell physiology; Cessation of life; Chimera organism; Death Domain; Defect; Development; Disease; Effector Cell; Embryo; Embryonic Development; Funding; Gene Transfer; Gene Transfer Techniques; Genes; Grant; Immune System Diseases; Immune system; In Vitro; Knockout Mice; Malignant Neoplasms; Mediating; Messenger RNA; Molecular; Mouse Protein; Mus; Mutant Strains Mice; Pathway interactions; Phenotype; Play; Pregnancy; Preventive; Protein Deficiency; Protein Isoforms; Protein Overexpression; Proteins; Proteolytic Processing; Regulation; Reporting; Research Project Grants; Role; Signal Pathway; Signal Transduction; Staging; T-Lymphocyte; TNF-related apoptosis-inducing ligand; Testing; Therapeutic; Toll-like receptor 6; Toll-like receptors; Transgenic Organisms; Translational Research; Tumor Necrosis Factor Receptor; Yeasts; caspase-10; caspase-8; design; embryonic stem cell; in utero; in vivo; interest; overexpression; positional cloning; protein B; receptor; reconstitution; yeast two hybrid system

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.

描述（由申请人提供）：本研究的重点是了解细胞类flicp抑制蛋白（cFLIP）在B淋巴细胞中的体内功能。该项目源于我们对fadd介导的凋亡和增殖信号机制的长期兴趣。我们和其他人之前已经证明，FADD是几种死亡受体（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））共有的共同信号转接器。FADD的死亡结构域与死亡受体的死亡结构域结合，FADD的死亡效应结构域与Caspase 8的死亡效应结构域结合。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；然而，它们在胚胎发育过程中是必不可少的。有趣的是，缺乏FADD或Caspase 8会导致小鼠的早期胚胎死亡。通过使用存活的FADD-/- > rag1 -/-嵌合体，我们发现当胚胎干细胞中没有FADD时，B细胞的发育被完全阻断。然而，我们发现当使用CD19Cre在前B细胞中删除FADD时，B细胞的发育不受明显影响，这表明FADD在早期阶段和/或在B谱系承诺之前是必不可少的。令人惊讶的是，fadd缺陷的B细胞不仅在凋亡方面存在缺陷，而且在toll样受体（TLR）诱导的增殖方面也受到损害。在条件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式。FADD和Caspase 8如何调节B细胞的凋亡和增殖这两种不同的信号传导尚不清楚。我们推测可以结合FADD的cFLIP可能在B细胞中调控FADD和Caspase 8的双重功能中发挥作用。cFLIP与Caspase 8同源，但缺乏Caspase活性。在初步研究中，我们发现，缺乏cflp的小鼠是胚胎致死性的，这种表型与缺乏FADD或Caspase 8的小鼠相似。此外，我们发现杂合子的cFLIP T细胞增殖能力低，而转基因的cFLIP过表达导致T细胞过度增殖。有报道表明，cFLIP可抑制原代B细胞的凋亡。然而，体外过表达cFLIP也被证明可以增强或抑制细胞凋亡。目前尚不清楚cFLIP是否在FADD/Caspase 8介导的TLR刺激诱导的B细胞增殖中发挥作用。我们提出(1)利用B细胞中缺乏cFLIP的条件突变小鼠来分析cFLIP在体内的功能，(2)通过反向遗传学来解剖cFLIP在B淋巴细胞中的多种功能。本研究将有助于确定cFLIP调控的体内信号通路，为免疫疾病、癌症等多种疾病的转化研究提供依据。在本申请中，我们描述了一个科学计划来研究在发育、免疫系统功能和癌症中起重要作用的cFLIP蛋白。研究结果将有助于开发有效的预防和治疗方法。