The Role of Magnesium in B-Cell Signaling

描述（由申请人提供）：与Ca2+相反，细胞内Mg2+-浓度[Mg2+]i的变化在淋巴细胞活化中的作用尚不清楚。然而，Mg2+是最丰富的细胞内阳离子，是许多细胞酶的重要辅助因子，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表明，在受体激活或离子载体处理下，[Ca2+]i增加后，[Mg2+]i也增加。研究[Mg2+]i在b细胞活化中的相关性的一个主要困难是对脊椎动物中Mg2+稳态调节的分子机制了解非常少。Mg2+渗透离子通道TRPM7是新发现的阳离子通道trpm亚家族的成员，在DT40 b淋巴细胞的Mg2+稳态中起重要作用，使TRPM7缺陷的DT40 b细胞在24小时内发生Mg2+缺陷，生长停滞并最终死亡。在生长培养基中添加Mg2+可以挽救TRPM7缺失的DT40 b细胞的活力和增殖。这项拨款申请的初步数据进一步表明，Mrs2存在于DT40细胞中，这是b细胞中第一个线粒体Mg2+转运体。最近的研究证明，酵母中Mrs2敲除突变体中减少的Mg2+浓度可以通过与人形式的Mrs2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/细胞外来源的Mg2+调节磷酸肌苷代谢。为了更好地了解免疫细胞中Mg2+生理和Mg2+依赖的信号事件，本基金拟进一步研究Mrs2和TRPM7在受体刺激后的b淋巴细胞中的作用，并分析它们参与Mg2+稳态和Mg2+依赖的信号事件，包括PLCgamma激活、磷酸肌肽代谢和二酰基甘油生成（DAG），以下三条研究线：1) Mrs2和TRPM7在b细胞受体（BCR）刺激后对DT40 b淋巴细胞内Mg2+-和Ca2+浓度的影响是什么？2) Mrs2和TRPM7在BCR刺激后磷酸化肌肽代谢激活中的作用是什么？3) BCR刺激后，生长培养基中不同Mg2+浓度下TRPM7和Mrs2缺陷DT40 b细胞的基因表达是如何调控的？

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?