EGR-mediated STIM1-PMCA expression and function in T cell subsets

T 细胞亚群中 EGR 介导的 STIM1-PMCA 表达和功能

• 批准号: 9229047
• 负责人: Jonathan A Soboloff
• 金额: $ 35.63万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9229047
• 关键词: Affect; Amino Acids; Autoimmunity; Biological Assay; CD8B1 gene; Ca(2+)-Transporting ATPase; Caenorhabditis elegans; Cardiovascular system; Cations; Cell membrane; Cell model; Cell physiology; Cells; Cellular Immunity; Cytotoxic T-Lymphocytes; Defect; Dependence; Development; Endoplasmic Reticulum; Event; Exhibits; Fluorescence Resonance Energy Transfer; Genes; Growth; Hour; Human; Immune; Immunologics; Investigation; Knock-in; Knock-in Mouse; Lymphocyte; Lymphocyte Activation; Lymphoproliferative Disorders; Maintenance; Malignant Neoplasms; Measures; Mediating; Mediator of activation protein; Molecular; Mus; Muscle Contraction; Mutation Analysis; NF-kappa B; Neoplasms; Neurologic; Pattern; Phenotype; Physiological; Process; Proteins; Pump; Regulatory T-Lymphocyte; Reporting; Role; STIM1 gene; Signal Transduction; Site; Synaptic Transmission; T-Cell Activation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; Time; Transcriptional Activation; Up-Regulation; Work; Zinc Fingers; base; cell type; cytokine; experimental study; immunological synapse; insight; intraepithelial; novel; nuclear factors of activated T-cells; receptor; receptor-mediated signaling; response; sensor; transcription factor

Store-operated Ca2+ entry (SOCE) is the process whereby loss of Ca2+ in the endoplasmic reticulum (ER) leads to Ca2+ entry into cells across the plasma membrane (PM). STIM1 and STIM2 are critical ER Ca2+ sensors that control the activation of Orai1, the store-operated Ca2+ channel. STIM1/2 are critical for T cell receptor (TCR)- mediated signaling and their deficiency in humans results in lymphoproliferative disorder and autoimmunity sug- gesting defects in T cell development and activation. In addition to ER Ca2+ release and SOCE, Ca2+ signals involve Ca2+ extrusion mechanisms. In lymphocytes, Plasma Membrane Ca2+/ATPase 4 (PMCA4) is the primary mediator of Ca2+ extrusion and is essential for normal function. Recently, we identified PMCA4 as a further target of STIM1 (but not STIM2) during lymphocyte activation. Unlike SOCE, STIM1-induced PMCA inhibition is initi- ated by Early Growth Response 1 (EGR1) and EGR4-mediated STIM1 upregulation. EGRs are ubiquitous and rapidly upregulated zinc finger transcription factors. The current proposal is based on the following hypothesis: TCR engagement stimulates EGR-mediated STIM1 upregulation in naïve T cells that initiates Ca2+ clearance inhibition, critical for the maintenance of NFAT/NF-κB activation and subsequent T cell activation. This work is organized into 3 aims. Aim 1 is to characterize EGR1/4-mediated control of T cell diversity and function. While EGR1KO mice exhibit previously described defects in early T cell development, preliminary findings re- ported here are the first to reveal a T cell phenotype in EGR4KO mice. We have observed altered progression through the double-negative stages, dysregulation of Ca2+ clearance during T cell activation and defective T cell expansion. These investigations will be expanded, both to include EGR1/4dKO mice and to include analysis of developmental, conventional and non-conventional subtypes. Aim 2 is to assess how STIM1∆597 knockin af- fects T cell activation. As established in our recent study, STIM1 truncation at amino acid 597 (STIM1∆597) eliminates PMCA association without affecting Orai1 activation13. Preliminary analysis of T cells obtained from a recently generated STIM1∆597 knock-in mouse reveals intriguing T cell subset-specific changes in Ca re- 2+ sponses. Implications to T cell activation will be determined by measuring transcription factor activation, cytokine expression patterns and T cell diversity. Aim 3 is to define the mechanisms whereby POST coordinates functional STIM1-PMCA4 interactions. We revealed that EGR-induced STIM1 upregulation causes decreased PMCA function within the immunological synapse during T cell activation. However, a third protein termed POST interacts with both proteins and regulates Ca2+ clearance during store depletion. Our preliminary findings provide new insight into how these interactions are coordinated during T cell activation and define their functional impli- cations. The dynamics of these interactions will be determined using mutational analysis, FRET and Ca2+ en- try/clearance assays. Collectively, these studies reveal a novel new concept for how Ca2+ signals contribute to events that occur beyond the seconds to minutes time periods encompassing a single Ca2+ transient.

钙库操纵的 Ca2+ 进入 (SOCE) 是导致内质网 (ER) 中 Ca2+ 损失的过程 Ca2+ 穿过质膜 (PM) 进入细胞。 STIM1 和 STIM2 是关键的 ER Ca2+ 传感器， 控制 Orai1 的激活，Orai1 是钙池操纵的 Ca2+ 通道。 STIM1/2 对于 T 细胞受体 (TCR) 至关重要 - 介导的信号传导及其在人类中的缺陷会导致淋巴增殖性疾病和自身免疫性疾病 T 细胞发育和激活的妊娠缺陷。除了 ER Ca2+ 释放和 SOCE 之外，Ca2+ 信号 涉及 Ca2+ 挤出机制。在淋巴细胞中，质膜 Ca2+/ATPase 4 (PMCA4) 是主要的 Ca2+ 排出的介质，对于正常功能至关重要。最近，我们确定 PMCA4 作为进一步的目标 STIM1（但不是 STIM2）在淋巴细胞激活过程中的变化。与 SOCE 不同，STIM1 诱导的 PMCA 抑制是起始的。 早期生长反应 1 (EGR1) 和 EGR4 介导的 STIM1 上调。 EGR 无处不在 快速上调锌指转录因子。当前的提议基于以下假设： TCR 参与刺激初始 T 细胞中 EGR 介导的 STIM1 上调，从而启动 Ca2+ 清除 抑制，对于维持 NFAT/NF-κB 激活和随后的 T 细胞激活至关重要。这部作品是 分为 3 个目标。目标 1 是表征 EGR1/4 介导的 T 细胞多样性和功能控制。 虽然 EGR1KO 小鼠在早期 T 细胞发育中表现出先前描述的缺陷，但初步发现重新 这里移植的是第一个揭示 EGR4KO 小鼠 T 细胞表型的细胞。我们观察到进展发生了变化 通过双阴性阶段，T 细胞激活过程中 Ca2+ 清除失调和缺陷 T 细胞 扩张。这些研究将扩大，既包括 EGR1/4dKO 小鼠，也包括对 发育亚型、常规亚型和非常规亚型。目标 2 是评估 STIM1Δ597 如何敲入 af- 影响T细胞活化。正如我们最近的研究所证实的，STIM1 在氨基酸 597 处截短 (STIM1Δ597) 消除 PMCA 关联而不影响 Orai1 激活13。对从 T 细胞获得的初步分析 最近生成的 STIM1Δ597 敲入小鼠揭示了 Ca re- 中 T 细胞亚群特异性的有趣变化 2+ 响应。对 T 细胞激活的影响将通过测量转录因子激活、细胞因子来确定 表达模式和 T 细胞多样性。目标 3 是定义 POST 协调机制 STIM1-PMCA4 功能性相互作用。我们发现 EGR 诱导的 STIM1 上调会导致 PMCA 在 T 细胞激活过程中在免疫突触内发挥作用。然而，第三种蛋白质称为 POST 与两种蛋白质相互作用并在储存耗尽期间调节 Ca2+ 清除。我们的初步调查结果表明 关于这些相互作用在 T 细胞激活过程中如何协调并定义其功能含义的新见解 阳离子。这些相互作用的动态将通过突变分析、FRET 和 Ca2+ en- 来确定。 尝试/清除分析。总的来说，这些研究揭示了 Ca2+ 信号如何促进 发生在几秒到几分钟时间段之外、包含单个 Ca2+ 瞬变的事件。