Calcium Regulation of NF-kB Activation in Lymphocytes

淋巴细胞中 NF-kB 激活的钙调节

• 批准号: 9352513
• 负责人: BRUCE D FREEDMAN
• 金额: $ 57.6万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-23 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9352513
• 关键词: Address; Affinity; Antigen Receptors; Antigens; Avidity; Biochemical; Biochemical Genetics; CD28 Antigens; CD28 gene; CD8B1 gene; Calcium; Calcium Signaling; Calcium ion; Cells; Competence; Custom; Data; Defect; Development; Disease; Exhibits; Frequencies; Future; Gene Expression; Generations; Genes; Genetic Transcription; Image; Immune; Immune response; Immunity; Individual; Life; Ligation; Link; Lymphocyte; Mass Spectrum Analysis; Measurement; Mediating; Modification; Molecular; Mus; NF-kappa B; Nature; Nuclear; Pattern; Perfusion; Phenotype; Phosphorylation; Physiologic pulse; Play; Proteins; Public Health; Publications; Regulation; Reporter; Role; STIM1 gene; Signal Transduction; Specificity; System; T-Lymphocyte; TNF gene; Time; Transcriptional Regulation; Variant; Width; base; design; in vivo; innovation; insight; live cell imaging; novel; p65; prevent; progenitor; programs; receptor; therapeutic target; thymocyte; tool; transcriptome sequencing

PROJECT SUMMARY Antigen receptor-induced calcium (Ca2+) signals regulate lymphocyte development and effector functions, but major gaps persist in our understanding of the dynamics of these signals, their transcriptional consequences and how cell fates are governed by distinct receptor-induced Ca2+ waveforms. Supporting data in this application and a pending publication reveal entirely novel and indispensable mechanisms by which Ca2+ specifically tunes TCR, but not TNF, induced NF-κB p65 and c-Rel activation. Our findings that p65 and c-Rel phosphorylation may serve as Ca2+ dependent checkpoints provide a framework for understanding how distinct patterns of Ca2+ signaling in vivo control the transcriptional programs that drive lymphocyte development and function. Central to TCR-induced Ca2+ entry are the ER transmembrane Ca2+ sensing STIM1 and STIM2 proteins, and the STIM-activated Ca2+ channel CRAC/Orai. Mice whose T cell progenitors lack STIM1/2 exhibit a selective defect in nTreg induction and the immune phenotype of these mice is virtually indistinguishable from that of mice with a c-Rel deficiency. Moreover, as nTreg development is driven by CD28 costimulation in conjunction with high affinity/avidity TCR ligation, and we have previously shown that CD28 biases this deterministic signal toward high amplitude Ca2+ spikes over a range of antigen receptor ligation avidities, we hypothesize that CD28 tuning of TCR induced Ca2+ dynamics generates a quantitatively unique waveform that drives c-Rel-dependent nTreg generation. Based on our pending publication and new supporting data, we hypothesize that TCR induced Ca2+-dependent phosphorylation of individual p65 and c-Rel Ser/Thr residues is regulated by distinct Ca2+ thresholds and waveforms, thereby providing a mechanism by which quantitatively distinct Ca2+ signals differentially regulate NF-κB-dependent gene expression controlling lymphocyte development and functions. To address this hypothesis, we will: 1) Define the nature and function of Ca2+- regulated phosphorylation of p65 and c-Rel, 2) determine how Ca2+ controls NF-κB-driven transcriptional specificity, and 3) define the Ca2+ dependent mechanisms of thymic nTreg development. We will use a custom built “Ca2+ clamp” perfusion system to impose variations in the Ca2+ amplitude, pulse width, frequency, and total input to define how Ca2+ waveforms control sequential checkpoints in NF-B activation and long-term imaging measurements utilizing a novel genetically encoded Ca2+ indicator (GCaMP6f) to determine how antigen-induced Ca2+ signals contribute to the regulation of nTreg development. The combined expertise of the team, the systems and approaches, and the quantitative tools developed for these studies will provide a new and detailed mechanistic understanding of how calcium dependent phosphorylation regulates transcriptional specificity of NF-B in T cells and will provide insights into strategies and therapeutic targets for enhancing insufficient, suppressing auto-reactive, or redirecting inappropriate immune responses.

项目摘要 抗原受体诱导的钙（Ca 2+）信号调节淋巴细胞发育和效应功能，但 在我们对这些信号的动力学及其转录后果的理解上， 以及细胞命运如何受不同受体诱导的Ca 2+波形的支配。支持数据在此 申请和待发表揭示了完全新颖和不可或缺的机制，其中Ca 2 + 特异性调节TCR而非TNF诱导的NF-κB p65和c-Rel活化。我们发现p65和c-Rel 磷酸化可以作为Ca 2+依赖的检查点，为理解不同的 体内Ca 2+信号传导模式控制驱动淋巴细胞发育的转录程序， 功能TCR诱导的Ca 2+内流的中心是ER跨膜Ca 2+感应STIM 1和STIM 2 蛋白质，和STIM激活的Ca 2+通道CRAC/奥赖。T细胞祖细胞缺乏STIM 1/2的小鼠表现出 nTreg诱导的选择性缺陷和这些小鼠的免疫表型几乎无法区分 与c-Rel缺乏的小鼠相比。此外，由于nTreg的发育是由CD 28共刺激驱动的， 结合高亲和力/亲合力TCR连接，我们以前已经表明，CD 28偏向于这种结合。 在抗原受体连接亲合力范围内，确定性信号朝向高幅度Ca 2+尖峰，我们 假设TCR诱导Ca 2+动力学的CD 28调节产生定量独特的波形， 驱动c-Rel依赖性nTreg生成。根据我们即将发表的文章和新的支持数据，我们 假设TCR诱导单个p65和c-Rel Ser/Thr残基的Ca 2+依赖性磷酸化， 由不同的Ca 2+阈值和波形调节，从而提供了一种机制， 不同的Ca 2+信号差异调节NF-κ B依赖的基因表达控制淋巴细胞 发展和功能。为了解决这一假设，我们将：1）定义Ca 2+的性质和功能， 调节p65和c-Rel的磷酸化，2）确定Ca 2+如何控制NF-κ B驱动的转录 特异性，和3）定义胸腺nTreg发育的Ca 2+依赖性机制。我们将使用一个自定义 建立了“Ca 2+钳”灌注系统，以施加Ca 2+振幅、脉冲宽度、频率和 总输入，以定义Ca 2+波形如何控制NF-κ B B激活和长期 利用一种新的遗传编码的Ca 2+指示剂（GCaMP 6 f）进行成像测量，以确定 抗原诱导的Ca 2+信号有助于调节nTreg发育。联合专家的专业知识 团队，系统和方法，以及为这些研究开发的定量工具将提供一个新的 以及对钙依赖性磷酸化如何调节转录的详细机制的理解 NF-κ B B在T细胞中的特异性，并将提供对增强免疫应答的策略和治疗靶点的见解。 不足，抑制自身反应，或重新引导不适当的免疫反应。