The Function of cFLIP in B Lymphocytes

cFLIP 在 B 淋巴细胞中的功能

• 批准号: 7531980
• 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 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7531980
• 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; Purpose; 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; day; design; embryonic stem cell; in utero; in vivo; interest; 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）：这项拟议研究的重点是了解 B 淋巴细胞中细胞 FLICE 样抑制蛋白 cFLIP 的体内功能。该项目源于我们对 FADD 介导的细胞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 是几种死亡受体共享的常见信号传导适配器，包括 Fas、肿瘤坏死因子受体 1 (TNF-R1) 和 TNF 相关凋亡诱导配体受体 (TRAIL-R)。 FADD的死亡结构域与死亡受体的死亡结构域结合，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方面发挥着重要作用。然而，它们在胚胎发育过程中是可有可无的。有趣的是，缺乏 FADD 或 Caspase 8 会导致小鼠早期胚胎死亡。使用可行的 FADD-/-->RAG-1-/- 嵌合体，我们发现当胚胎干细胞中不存在 FADD 时，B 细胞发育完全受阻。然而，我们发现，当使用 CD19Cre 在前 B 细胞中删除 FADD 时，B 细胞发育并未受到明显影响，这表明 FADD 在早期阶段和/或 B 谱系定型之前是必需的。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。在条件性 Caspase 8 缺陷小鼠中也观察到了类似的缺陷。这些研究帮助揭示了 B 细胞中涉及 FADD 和 Caspase 8 的二元信号传导的新范例。FADD 和 Caspase 8 如何调节 B 细胞中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双重功能的调节中发挥作用。 cFLIP 与 Caspase 8 同源，但缺乏 Caspase 活性。在初步研究中，我们发现cFLIP缺陷小鼠具有胚胎致死性，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细胞增殖不足，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，cFLIP 的体外过度表达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 TLR 刺激诱导的 FADD/Caspase 8 介导的 B 细胞增殖中发挥作用。我们建议（1）通过使用B细胞中缺乏cFLIP的条件突变小鼠来分析cFLIP的体内功能，以及（2）通过反向遗传学剖析cFLIP在B淋巴细胞中的多种功能。这项研究将有助于确定 cFLIP 调节的体内信号通路，这将有助于包括免疫性疾病和癌症在内的各种疾病的转化研究。在此应用中，我们描述了研究 cFLIP 蛋白的科学计划，该蛋白在发育、免疫系统功能和癌症中发挥着重要作用。研究结果将有助于开发有效的预防和治疗方法。