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（CA2+）信号调节淋巴细胞发育和效应子功能，但 主要差距仍然存在我们对这些信号的动态的理解，以及它们的转录后果 以及细胞命运如何受不同接收器诱导的Ca2+波形的控制。支持数据 应用和未决的出版物揭示了CA2+的完全新颖和必不可少的机制 调谐TCR，但不是TNF诱导NF-κBp65和C-Rel激活。我们的发现P65和C-REL 磷酸化可以用作Ca2+依赖性检查点，提供了一个框架，以了解如何不同 体内Ca2+信号传导的模式控制驱动淋巴细胞发育和的转录程序 功能。 TCR诱导的Ca2+进入的中心是ER跨膜Ca2+传感STIM1和STIM2 蛋白质和刺激激活的Ca2+通道CRAC/ORAI。 T细胞祖细胞缺乏STIM1/2的小鼠 NTREG诱导和这些小鼠的免疫表型的选择性缺陷实际上是无法区分的 来自C-REL缺乏的小鼠。此外，随着NTREG的开发是由CD28共刺激驱动的 与高亲和力/亲和力TCR连接的结合，我们先前已经表明CD28偏向于此 在一系列抗原受体连接狂热范围内，针对高放大器Ca2+尖峰的确定性信号，我们 假设TCR诱导的Ca2+动力学的CD28调整会产生一个定量独特的波形 驱动依赖C-REL的NTREG生成。基于我们未决的出版物和新的支持数据，我们 假设TCR诱导的Ca2+依赖性的p65和C-Rel Ser/Thr残基的辉磷酸化为 由不同的Ca2+阈值和波形调节，从而提供了一种定量的机制 不同的Ca2+信号对控制淋巴细胞的NF-κB依赖基因表达的调节不同 发展和功能。为了解决这一假设，我们将：1）定义Ca2+ - 的性质和功能 p65和c-Rel的调节磷酸化，2）确定CA2+如何控制NF-κB驱动的转录。 特异性和3）定义胸腺NTREG发育的Ca2+依赖性机制。我们将使用自定义 构建的“ Ca2+夹具”灌注系统以在CA2+放大器，脉冲宽度，频率和 总输入以定义CA2+波形如何控制NF-B激活中的顺序检查点 利用新型遗传编码的Ca2+指标（GCAMP6F）的成像测量结果来确定如何 抗原诱导的Ca2+信号有助于调节NTREG发育。联合专家 团队，系统和方法以及为这些研究开发的定量工具将提供新的 以及详细的机械理解如何依赖钙的磷酸化调节转录 NF-B在T细胞中的特异性，并将为增强策略和治疗靶标提供见解 不足，抑制自动反应性或重定向不当免疫反应。