Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.
J Mol Biol. 2010 Mar 5;396(4):858-69. doi: 10.1016/j.jmb.2010.01.014. Epub 2010 Jan 11.
TRPM7 is a Ca(2)(+)-permeant and Mg(2)(+)-permeant ion channel in possession of its own kinase domain. In a previous study, we showed that overexpression of the channel-kinase in HEK-293 cells produced cell rounding and loss of adhesion, which was dependent on the Ca(2+)-dependent protease m-calpain. The TRPM7-elicited change in cell morphology was channel-dependent and occurred without any significant increase in cytosolic Ca(2+). Here we demonstrate that overexpression of TRPM7 increased levels of cellular reactive oxygen species (ROS) and nitric oxide, causing the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Application of inhibitors of p38 MAPK and JNK blocked TRPM7-induced cell rounding and activation of m-calpain, without affecting the phosphorylation state of the protease. Overexpression of TRPM7 increased intracellular Mg(2+); however, when the concentration of either external Ca(2+) or Mg(2+) was increased to favor the permeation of one divalent cation over the other, a similar increase in cell rounding and calpain activity was detected, indicating that TRPM7-mediated activation of m-calpain is not dependent on the nature of the divalent conducted by the channel. Application of inhibitors of nitric oxide synthase and mitochondrial-derived ROS reduced TRPM7-induced increases in nitric oxide and ROS production, blocked the change in cell morphology, and reduced cellular calpain activity. Collectively, our data reveal that excessive TRPM7 channel activity causes oxidative and nitrosative stresses, producing cell rounding mediated by p38 MAPK/JNK-dependent activation of m-calpain.
TRPM7 是一种 Ca(2)(+)和 Mg(2)(+)可通透的离子通道，拥有自身的激酶结构域。在之前的研究中，我们发现，在 HEK-293 细胞中过表达该通道-激酶会导致细胞变圆和失去黏附，这依赖于 Ca(2+)-依赖性蛋白酶 m-calpain。TRPM7 引起的细胞形态变化是通道依赖性的，并且在细胞溶质 Ca(2+)没有明显增加的情况下发生。在这里，我们证明了过表达 TRPM7 会增加细胞内活性氧（ROS）和一氧化氮（NO）的水平，导致 p38 丝裂原活化蛋白激酶（MAPK）和 c-Jun N 端激酶（JNK）的激活。应用 p38 MAPK 和 JNK 的抑制剂阻断了 TRPM7 诱导的细胞变圆和 m-calpain 的激活，而不影响蛋白酶的磷酸化状态。过表达 TRPM7 增加了细胞内的 Mg(2+)；然而，当外部 Ca(2+)或 Mg(2+)的浓度增加到有利于一种二价阳离子的通透性超过另一种时，会检测到类似的细胞变圆和 calpain 活性增加，表明 TRPM7 介导的 m-calpain 激活不依赖于通道所传导的二价阳离子的性质。一氧化氮合酶和线粒体来源的 ROS 抑制剂的应用降低了 TRPM7 诱导的 NO 和 ROS 产生的增加，阻断了细胞形态的变化，并降低了细胞 calpain 活性。总的来说，我们的数据表明，TRPM7 通道活性的过度增加会导致氧化和硝化应激，产生由 p38 MAPK/JNK 依赖性 m-calpain 激活介导的细胞变圆。
