Molecular mechanisms that regulate eosinophil cytokine production

调节嗜酸性粒细胞细胞因子产生的分子机制

基本信息

批准号：
6565043
负责人：
James S Malter
金额：
$ 19.62万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-01-05 至 2006-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6565043
关键词：
RNA binding protein asthma cell differentiation colony stimulating factor eosinophil fibronectins gene expression genetic promoter element genetic transcription human tissue immunogenetics leukocyte activation /transformation messenger RNA posttranscriptional RNA processing tissue /cell culture transcription factor transfection tumor necrosis factor alpha

项目摘要

(Applicant's Abstract) The mechanisms by which peripheral blood eosinophils (PBEos) differentiate into airway based, effector cells responsible for the pathophysiology of asthma are poorly understood. A candidate mediator for this process is granulocyte macrophage colony stimulating factor (GM-CSF), a potent cytokine produced by eosinophils. GM-CSF is commonly elevated in the BAL fluid of symptomatic asthmatics. PBEos which are activated in vitro secrete immunologically detectable GM-CSF and express GM-CSF mRNA. PBEos also express cell surface GM-CSF receptors, suggesting GM-CSF functions as a critical autocrine growth and survival factor both in vitro and in vivo. Despite the likely functional significance of GM-CSF, very little is known about the molecular mechanism(s) which controls its production and release by eosinophils. Recently we have shown that GM-CSF mRNA stability was significantly enhanced in PBEos treated with tumor necrosis factor alpha (TNF) and fibronectin or in BAL derived eosinophils from allergen challenged volunteers. Using a yeast 3 hybrid screen, we identified YB-1, a known nucleic acid binding protein as a GM-CSF mRNA binding protein. Recombinant YB-1 specifically bound in vitro to the AU-rich, 3' UTR instability determinants of GM-CSF mRNA. When transfected into peripheral blood eosinophils, YB-1 enhanced in vitro survival by 3-5 fold, which was completely blocked by anti-GM-CSF antibodies. Finally, in preliminary studies, transfected YB-1 stabilized GM-CSF mRNA in PBEos. Therefore, we hypothesize that YB-1 mediates the post-transcriptional regulation of GM-CSF mRNA in activated eosinophils. Thus, the aims of this project are to 1). Characterize how YB-1 increases GM-CSF mRNA in PBEos, 2).Characterize the signaling cascades induced by TNFalpha, and fibronectin which enable YB-1 to interact with and regulate GM-CSF mRNA, 3). Determine if YB-1 is the sole effector in this system or interacts with additional protein components to regulate GM-CSF mRNA, 4). Determine which domain(s) of YB-1 is/are required for GM-CSF post-transcriptional gene regulation. In aggregate these studies will clarify the molecular mechanisms underlying GM-CSF mRNA regulation in activated eosinophils, and as such, provide additional, novel therapeutic targets for the prevention and treatment of asthma.

（申请人摘要）外周血嗜酸性粒细胞的机制 (PBEos) 分化为基于气道的效应细胞，负责人们对哮喘的病理生理学知之甚少。一位候选调解员过程是粒细胞巨噬细胞集落刺激因子（GM-CSF），一种有效的嗜酸性粒细胞产生的细胞因子。 BAL 液中的 GM-CSF 通常升高有症状的哮喘患者。体外激活的 PBEos 分泌免疫学可检测 GM-CSF 并表达 GM-CSF mRNA。 PBEos 还表达细胞表面 GM-CSF 受体，表明 GM-CSF 发挥着关键作用体外和体内自分泌生长和存活因子。尽管 GM-CSF 可能的功能意义，但人们对其知之甚少控制其产生和释放的分子机制嗜酸性粒细胞。最近我们发现 GM-CSF mRNA 稳定性经肿瘤坏死因子 α (TNF) 治疗的 PBEos 显着增强和纤连蛋白或来自过敏原挑战的 BAL 衍生的嗜酸性粒细胞志愿者。使用酵母 3 杂交筛选，我们鉴定了 YB-1，一种已知的核酸酸结合蛋白作为GM-CSF mRNA结合蛋白。重组YB-1 在体外与富含 AU 的 3'UTR 不稳定决定因素特异性结合 GM-CSF mRNA。当转染至外周血嗜酸性粒细胞时，YB-1 增强体外存活率提高 3-5 倍，但可被抗 GM-CSF 完全阻断抗体。最后，在初步研究中，转染的YB-1稳定 PBEos 中的 GM-CSF mRNA。因此，我们假设 YB-1 介导活化的嗜酸性粒细胞中 GM-CSF mRNA 的转录后调节。因此，该项目的目标是 1)。描述 YB-1 如何增加 GM-CSF PBEos 中的 mRNA，2). 表征 TNFα 诱导的信号级联，以及纤连蛋白使 YB-1 能够与 GM-CSF mRNA 相互作用并对其进行调节，3)。确定 YB-1 是否是该系统中的唯一效应器或与调节 GM-CSF mRNA 的其他蛋白质成分，4)。确定哪个 GM-CSF 转录后基因需要 YB-1 的结构域规定。总的来说，这些研究将阐明分子机制激活的嗜酸性粒细胞中 GM-CSF mRNA 的潜在调节，因此，为预防和治疗提供额外的、新颖的治疗靶点哮喘。