Sheikh-Hamad D, Di Mari J, Suki W N, Safirstein R, Watts B A, Rouse D
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 1998 Jan 16;273(3):1832-7. doi: 10.1074/jbc.273.3.1832.
In renal cells, hypertonicity induces genes for heat shock proteins (HSP70, alpha B-crystallin), as well as enzymes and transporters directly involved in the metabolism and transport of protective organic osmolytes. While heat shock proteins are induced by many stresses including osmotic stress, the induction of the osmolytes genes appears to be specific to osmotic stress. These two adaptive mechanisms allow kidney cells to survive and function in the hypertonic environment that exists on routine basis in kidney medulla. In mammalian cells, hypertonicity induces three mitogen-activated protein kinase pathways: ERK (extracellular regulated kinase), JNK (Jun N-terminal kinase), and p38. ERK activation by osmotic stress is a consistent finding in many cells, but it is not essential for transcriptional regulation of mRNA for transporter of organic osmolyte betaine. While the growth of yeast cells on NaCl-supplemented medium is dependent on HOG1 pathway, it is still unclear which pathway mediates the adaptation to osmotic stress in mammalian cells. Here, we show that inhibition of p38 kinase activity, using the specific inhibitor SB203580 (4-(fluorophenyl)-2-(4-methylsulfonyl-phenyl)-5-(4-pyridyl) imidazole), abolishes the hypertonicity-mediated induction of mRNAs for HSP70 and betaine transporter in Madin-Darby canine kidney cells. The inhibition is dose-dependent and correlates with the in situ activity of native p38 kinase, determined as MAPKAPK-2 activity in cell extracts. As reported previously, the activities of ERK-1 and -2 were not affected by SB203580, but surprisingly, inhibition of native p38 kinase activity correlates with up-regulation of native JNK-1 activity in osmotically stressed cells. p38 mRNA is induced by hypertonic stress and is attenuated with p38 kinase inhibition. We also find that thermal induction of HSP70 mRNA is not affected by p38 kinase inhibition. Such findings suggest that p38 kinase activity is essential for the induction of genes involved in the adaptation of mammalian cells to osmotic stress and that the increased activity of JNK-1 during p38 kinase inhibition is consistent with regulation of JNK-1 by p38 kinase in osmotically stressed cells. In addition, the transduction pathways mediating HSP70 mRNA induction by different stresses appear to be divergent; osmotic induction of HSP70 is p38 kinase-dependent, while thermal induction is not.
在肾细胞中，高渗状态可诱导热休克蛋白（HSP70、αB-晶状体蛋白）相关基因，以及直接参与保护性有机渗透溶质代谢和转运的酶及转运蛋白。虽然热休克蛋白可由包括渗透压应激在内的多种应激诱导产生，但渗透溶质相关基因的诱导似乎对渗透压应激具有特异性。这两种适应性机制使肾细胞能够在肾髓质中常规存在的高渗环境中存活并发挥功能。在哺乳动物细胞中，高渗状态可诱导三条丝裂原活化蛋白激酶信号通路：细胞外调节激酶（ERK）、Jun氨基末端激酶（JNK）和p38。渗透压应激激活ERK在许多细胞中是一个一致的发现，但它对于有机渗透溶质甜菜碱转运蛋白mRNA的转录调控并非必不可少。虽然酵母细胞在添加氯化钠的培养基上生长依赖于HOG1信号通路，但目前仍不清楚在哺乳动物细胞中是哪条信号通路介导对渗透压应激的适应。在此，我们表明，使用特异性抑制剂SB203580（4-（氟苯基）-2-（4-甲基磺酰基苯基）-5-（4-吡啶基）咪唑）抑制p38激酶活性，可消除高渗状态介导的Madin-Darby犬肾细胞中HSP70和甜菜碱转运蛋白mRNA的诱导。这种抑制呈剂量依赖性，并与细胞提取物中以MAPKAPK-2活性测定的天然p38激酶的原位活性相关。如先前报道，ERK-1和-2的活性不受SB203580影响，但令人惊讶的是，天然p38激酶活性的抑制与渗透压应激细胞中天然JNK-1活性的上调相关。高渗应激可诱导p38 mRNA表达，而p38激酶抑制可使其减弱。我们还发现，p38激酶抑制并不影响HSP70 mRNA的热诱导。这些发现表明，p38激酶活性对于诱导参与哺乳动物细胞适应渗透压应激的基因至关重要，并且在p38激酶抑制过程中JNK-1活性的增加与渗透压应激细胞中p38激酶对JNK-1的调控一致。此外，介导不同应激诱导HSP70 mRNA的信号转导途径似乎不同；HSP70的渗透压诱导依赖于p38激酶，而热诱导则不然。
