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Development and application of stress-signal transduction system as an assessment ofbiomaterials

生物材料评估的应激信号转导系统的开发与应用

基本信息

批准号：
13557150
负责人：
TAKEDA Kohsuke
金额：
$ 8.51万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13557150/
关键词：
Stress Signal transduction MAP kinase Biomaterials

项目摘要

Among the three MAP kinase cascades, two of them that converge on JNKs and p38 MAP kinases are preferentially activated by cytotoxic stresses such as UV radiation, X-ray, heat shock and osmotic shock, and by proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1. One of the important biological responses mediated through these stress-activated MAP kinase pathways appears to be the decision of cell fate by regulating apoptosis. In this research project, we intended to develop a novel, rapid and sensitive assessment system for various biomaterials by assaying the activation status of the stress-activated MAP kinase pathways. We first established the sensitive assay system for INK and p38 activation, and clearly showed that apoptosis signal^egulating kinase 1 (ASK1), which is known to regulate the JNK and p38 MAP kinase pathways, is required for oxidative stress-nduced activation of JNK and p38. We next established the sensitive assay system for ASK1 activation. We found that phosphorylation of Thr845 in the kinase domain of ASK1 is required for the activation of ASK1, and raised a polyclonal antibody that specifically recognized the activation status of Thr 845 of ASK 1. This anti-phospho-ASKl antibody could detect the activation state of ASK1 with much higher sensitivity than the conventional "in vitro kinase assay". By the various biochemical analyzes using this antibody and the further investigation of ASKl-deficient mice, we have found that ASK1 is necessary for not only oxidative stress- but also ER stress-induced apoptosis, suggesting that the ASK1-MAP kinase pathways is a common signal mediator of various kind of stressors. Thus, accurate and sensitive detection of the activation status of the ASK1-MAP kinase pathways appears to be useful for the development and application of our assessment system for various biomaterials.

在三个MAP激酶级联中，两个聚集在JNKs和p38上的MAP激酶优先被紫外线、X射线、热休克和渗透休克等细胞毒性应激激活，以及肿瘤坏死因子和白介素1等促炎细胞因子激活。通过这些应激激活的MAPK通路介导的重要生物反应之一似乎是通过调节细胞凋亡来决定细胞命运。在本研究项目中，我们打算通过分析应激激活的MAPK通路的激活状态来开发一种新颖、快速和灵敏的各种生物材料的评估系统。我们首先建立了敏感的INK和p38激活检测系统，并清楚地表明，调控JNK和p38 MAP激酶通路的凋亡信号调节蛋白1(ASK1)是氧化应激诱导JNK和p38激活所必需的。接下来，我们建立了检测ASK1活性的灵敏检测体系。我们发现ASK1激活区Thr845的磷酸化是ASK1激活所必需的，并提出了一种特异性识别ASK1的Thr845激活状态的多克隆抗体。这种抗Ask1磷酸化抗体可以比传统的“体外激活法”更敏感地检测ASK1的激活状态。通过使用该抗体的各种生化分析和对Ask1基因缺陷小鼠的进一步研究，我们发现ASK1不仅是氧化应激所必需的，而且在内质网应激诱导的细胞凋亡中也是必需的，这表明ASK1-MAP激酶通路是各种应激源的共同信号介质。因此，准确和灵敏地检测ASK1-MAP激酶通路的激活状态对于我们开发和应用各种生物材料的评估系统是有用的。