Osmoregulated Proteins in the Inner Medulla

内髓质中的渗透调节蛋白

• 批准号: 7270150
• 负责人: Tomas Berl
• 金额: $ 7.08万
• 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 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7270150
• 关键词: JUN kinase; RNA interference; confocal scanning microscopy; electron microscopy; gene expression; genetic regulation; genetic transcription; genetically modified animals; kidney function; laboratory mouse; mass spectrometry; microarray technology; mitogen activated protein kinase; northern blottings; phosphatidylinositol 3 kinase; phosphorylation; physiologic stressor; polymerase chain reaction; protein kinase; protein structure function; proteomics; renal medulla; sodium chloride; tissue /cell culture; two dimensional gel electrophoresis; western blottings

DESCRIPTION (provided by applicant): The survival of cells in the mammalian inner medulla requires an adaptive response to the hypertonic environment. This adaptation to changes in tonicity initially involves the activation of protein kinases and ultimately the upregulation of proteins that allow for cellular accumulation of inert osmolytes. It is now evident that a more complex array of both genes and proteins is involved in the adaptive response. The present proposal intends to examine the repertoire of genes and proteins that are both increased and decreased in cells adapted to live in hypertonic conditions (600 and 900 mOsm/kg) and in cells acutely (24- 48 hours) exposed to increased tonicity. In this setting the role of various signaling pathways in the genetic regulation will be assessed by employing inhibitors of such pathways as well as transfected cells with both gain and loss of function mutations. The in-vitro results will also be extended to the study in-vivo in rodents in various states of water balance. Both genomic and proteomic approaches are contemplated to unveil as full a repertoire as the presently available techniques allow. In its second aim the proposal will examine the mechanism whereby the subunit of Na-K-ATPase is upregulated by hypertonicity and how the protein provides a survival advantage in these conditions. More specifically, the transcriptional regulation of the gene will be studied. We hypothesize that the effect of JNK kinase to enhance gamma subunit synthesis is regulated at the transcriptional level. The hypothesis will be studied by employing inhibitors of JNK kinase, gain and loss of function transfectents of the kinase and knockout mice deficient in JNK1 and JNK2. The translational regulation will focus on the role of PI3 kinase in the process. The role of the subunit in osmoprotection will be studied by comparing the effects of hypertonicity in wild type cells to that of cells rendered incapable of synthesizing the protein by a silencing RNA technique. This approach could be extended to the study of relevant genes unveiled in the genomics-proteomic studies. Effects on alteration in cell Na concentration, uptake of an inert osmolyte (inositol) and finally cell survival itself will be assessed. These experiments should define genes and proteins involved in osmoregulation and particularly unveil the role of the subunit as a vital component in the adaption to the hypertonic environment of the mammalian inner medulla.

描述（由申请人提供）：哺乳动物内髓质细胞的生存需要对高渗环境的适应性反应。这种对张力变化的适应最初涉及蛋白激酶的激活，并最终涉及蛋白质的上调，从而允许惰性渗透剂的细胞积累。现在很明显，适应性反应涉及更复杂的基因和蛋白质阵列。本提案旨在检查在适应高渗条件（600 和 900 mOsm/kg）的细胞中以及在急性（24-48 小时）暴露于高渗环境的细胞中增加和减少的基因和蛋白质的全部成分。在这种情况下，将通过使用这些途径的抑制剂以及具有获得和丧失功能突变的转染细胞来评估各种信号传导途径在遗传调控中的作用。体外结果也将扩展到不同水平衡状态的啮齿动物的体内研究。基因组学和蛋白质组学方法都有望揭示目前可用技术允许的全部内容。该提案的第二个目标将研究高渗性上调 Na-K-ATP 酶亚基的机制，以及该蛋白质如何在这些条件下提供生存优势。更具体地说，将研究该基因的转录调控。我们假设 JNK 激酶增强 γ 亚基合成的作用是在转录水平上受到调节的。该假设将通过使用 JNK 激酶抑制剂、激酶功能转染子的获得和丧失以及 JNK1 和 JNK2 缺陷的敲除小鼠来研究。翻译调控将集中于PI3激酶在此过程中的作用。将通过比较野生型细胞中高渗性的影响与通过沉默 RNA 技术使其无法合成蛋白质的细胞的影响来研究该亚基在渗透保护中的作用。这种方法可以是 扩展到基因组学-蛋白质组学研究中揭示的相关基因的研究。将评估对细胞Na浓度变化、惰性渗透剂（肌醇）的吸收以及最终细胞存活本身的影响。这些实验应该定义参与渗透调节的基因和蛋白质，特别是揭示该亚基作为适应哺乳动物内髓质高渗环境的重要组成部分的作用。