The Role of Magnesium in B-Cell Signaling

镁在 B 细胞信号传导中的作用

• 批准号: 7062134
• 负责人: CARSTEN SCHMITZ
• 金额: $ 11.03万
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7062134
• 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(由申请人提供)：与钙离子相反，细胞内镁离子浓度的变化在淋巴细胞激活中的作用尚不清楚。然而，镁离子是细胞内最丰富的阳离子，是许多细胞酶的重要辅因子，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证明，随着受体激活或离子载体治疗而导致[Ca~(2+)]i升高，[Mg~(2+)]_i增加。研究[Mg2+]i在B细胞激活中的相关性的一个主要困难是对脊椎动物体内镁离子稳态调节的分子机制了解很少。镁离子通透性离子通道TRPM7是新近发现的TRPM阳离子通道亚家族成员之一，它在DT40 B淋巴细胞的镁离子平衡中起着重要作用，使TRPM7缺乏的DT40 B细胞出现镁离子缺乏，24小时内生长停止，最终死亡。在生长介质中添加镁离子可以挽救TRPM7缺陷的DT40B细胞的活性和增殖。这项拨款提案中的初步数据进一步表明，在DT40细胞中存在MRS-2，这是B细胞中第一个线粒体镁离子转运体。最近已经证明，酵母中mrs2基因敲除突变体中降低的镁离子浓度可以通过与mrs2的人类形式互补而部分恢复。此外，在淋巴细胞中的一些研究表明，来自细胞内外的镁离子正在调节磷脂酰肌醇的代谢。为了更好地了解免疫细胞中的镁生理学和镁依赖的信号事件，这项研究建议进一步研究受体刺激后MRS和TRPM7在B淋巴细胞中的作用，并分析它们在镁稳态以及镁依赖的信号事件中的作用，包括PLC伽马激活、磷脂酰肌醇代谢和二酰甘油产生(DAG)。1)在B细胞受体(BCR)刺激后，MRS2和TRPM7对DT40 B淋巴细胞内的镁离子和钙离子浓度有何影响？2)在BCR刺激后，MRS2和TRPM7在激活肌醇磷脂代谢中起什么作用？3)在BCR刺激后的培养液中，不同浓度的镁离子对TRPM7和TRPM2缺陷的DT40 B细胞的基因表达有何调节作用？