The Role of Magnesium in B-Cell Signaling

镁在 B 细胞信号传导中的作用

• 批准号: 6918504
• 负责人: CARSTEN SCHMITZ
• 金额: $ 10.77万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6918504
• 关键词: B cell receptor; B lymphocyte; biological signal transduction; cell biology; cell line; flow cytometry; gene expression; growth media; homeostasis; immunoprecipitation; leukocyte activation /transformation; magnesium ion; membrane channels; microarray technology; physiology; polymerase chain reaction; protein structure function; yeast two hybrid system

DESCRIPTION (provided by applicant): As opposed to Ca2+, the role of change in intracellular Mg2+-concentrations, [Mg2+]i, in lymphocyte activation remains unclear. Yet Mg2+ is the most abundant intracellular cation, and an essential cofactor for many cellular enzymes, as well as a critical regulator of cell growth and differentiation. Several studies undertaken in B-lymphocytes document an increase in [Mg2+]i following an increase in [Ca2+]i in response to receptor activation or ionophore treatment. A main difficulty in studying the relevance of [Mg2+]i in B-cell activation has been the very poor understanding of molecular mechanisms underlying Mg2+-homeostasis regulation in vertebrates. The Mg2+-permeable ion channel TRPM7, a member of the newly discovered TRPM-subfamily of cation channels, plays an essential role in Mg2+-homeostasis in DT40 B-lymphocytes in such that TRPM7 deficient DT40 B-cells become Mg2+-deficient, experience growth arrest within 24 hr and eventually die. Both, viability and proliferation of TRPM7 deficient DT40 B-cells are rescued by supplementation of Mg2+ to the growth media. Preliminary data in this grant proposal show furthermore the presence of Mrs2 in DT40 cells, the first mitochondrial Mg2+-transporter in B-cells. It was recently proven that reduced Mg2+-concentrations in Mrs2 knock-out mutant in yeast could be partially restored by complementing with the human form of Mrs2. Furthermore several studies in lymphocytes have shown that Mg2+ from intra-/extracellular sources is regulating phosphoinositide metabolism. For a better understanding of Mg2+-physiology and of Mg2+-dependent signaling events in immune cells, this grant proposes to further investigate the role of Mrs2 and TRPM7 in B-lymphocytes after receptor stimulation, and to analyze their involvement in Mg2+-homeostasis as well as Mg2+-dependent signaling events including PLCgamma activation, phosphoinositide metabolism and diacylglycerol production (DAG), following three lines of investigation: 1) What is the influence of Mrs2 and TRPM7 on intracellular Mg2+- and Ca2+-concentrations after B-cell receptor (BCR) stimulation in DT40 B-lymphocytes? 2) What is the role of Mrs2 and TRPM7 in activation of phosphoinositide metabolism after BCR stimulation? 3) How is gene expression regulated in TRPM7 and Mrs2 deficient DT40 B-cells under different Mg2+-concentrations in the growth media after BCR stimulation?

描述（由申请方提供）：与Ca 2+相反，细胞内Mg 2+浓度（[Mg 2 +]i）变化在淋巴细胞活化中的作用尚不清楚。 然而，Mg 2+是最丰富的细胞内阳离子，是许多细胞酶的必需辅因子，也是细胞生长和分化的关键调节因子。 在B淋巴细胞中进行的几项研究记录了响应于受体活化或离子载体处理的[Ca 2 +]i增加后[Mg 2 +]i增加。 研究[Mg 2 +]i在B细胞活化中的相关性的主要困难是对脊椎动物中Mg 2+稳态调节的分子机制的理解非常差。 Mg 2+渗透性离子通道TRPM 7是新发现的阳离子通道TRPM亚家族的成员，在DT 40 B淋巴细胞中的Mg 2+稳态中起重要作用，使得TRPM 7缺陷型DT 40 B细胞变得Mg 2+缺陷，在24小时内经历生长停滞并最终死亡。通过向生长培养基中补充Mg 2+来拯救TRPM 7缺陷型DT 40 B细胞的活力和增殖。在这个拨款提案中的初步数据进一步显示了Mrs 2在DT 40细胞中的存在，这是B细胞中的第一个线粒体Mg 2+转运蛋白。 最近证明，在酵母中Mrs 2敲除突变体中降低的Mg 2+浓度可以通过与人形式的Mrs 2互补而部分恢复。 此外，在淋巴细胞中的几项研究表明，来自细胞内/细胞外来源的Mg 2+调节磷酸肌醇代谢。 为了更好地理解免疫细胞中的Mg 2+生理学和Mg 2+依赖性信号传导事件，本授权提出进一步研究受体刺激后Mrs 2和TRPM 7在B淋巴细胞中的作用，并分析它们参与Mg 2+稳态以及Mg 2+依赖性信号传导事件，包括PLC γ激活，磷酸肌醇代谢和二酰基甘油产生（DAG），以下三条研究路线：1）在DT 40 B淋巴细胞中，在B细胞受体（BCR）刺激后，Mrs 2和TRPM 7对细胞内Mg 2 +-和Ca 2 +-浓度的影响是什么？2)Mrs 2和TRPM 7在BCR刺激后激活磷酸肌醇代谢中的作用是什么？3)在BCR刺激后，在生长培养基中不同Mg 2+浓度下，TRPM 7和Mrs 2缺陷型DT 40 B细胞中的基因表达是如何调节的？