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: 6630928
• 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 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6630928
• 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。