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

• 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
• 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

(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.

（申请人的摘要）周围血液嗜酸性粒细胞的机制 （PBEO）区分为基于气道的效应细胞负责 哮喘的病理生理学知之甚少。为此的候选人 过程是粒细胞巨噬细胞集落刺激因子（GM-CSF），这是一种有效的 由嗜酸性粒细胞产生的细胞因子。 GM-CSF通常在BAL流体中升高 有症状的哮喘患者。在体外分泌激活的pbeo 可以检测到的可检测到的GM-CSF和表达GM-CSF mRNA。 Pbeos也表达 细胞表面GM-CSF受体，这表明GM-CSF功能是关键 体外和体内自分泌生长和生存因子。尽管有 GM-CSF的功能意义可能很少，对 分子机制控制其生产和释放 嗜酸性粒细胞。最近，我们表明GM-CSF mRNA稳定性为 用肿瘤坏死因子α（TNF）处理的PBEO显着增强 和纤连蛋白或来自过敏原的BAL衍生的嗜酸性粒细胞挑战 志愿者。使用酵母3混合筛选，我们确定了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 特定于体外结合到富含AU的3'UTR不稳定性决定因素 GM-CSF mRNA。当转染到外周血嗜酸性粒细胞中时，YB-1增强了 体外存活率3-5倍，抗GM-CSF完全阻止 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 激活嗜酸性粒细胞中GM-CSF mRNA的转录后调节。因此， 该项目的目的是1）。表征YB-1如何增加GM-CSF pbeos中的mRNA，2）。 纤连蛋白使YB-1能够与GM-CSF mRNA相互作用并调节3）。 确定yb-1是该系统中的唯一效应子还是与 调节GM-CSF mRNA的其他蛋白质成分，4）。确定哪个 yb-1的域是/csf转录后基因所必需的 规定。在汇总中，这些研究将阐明分子机制 激活的嗜酸性粒细胞中的基本GM-CSF mRNA调节，因此， 为预防和治疗提供其他新颖的治疗靶标 哮喘。