Molecular mechanisms that regulate eosinophil cytokine production

(申请人摘要)外周血嗜酸性粒细胞的作用机制 (PBEos)分化为基于呼吸道的效应细胞，负责 哮喘的病理生理学还知之甚少。这件事的候补调解人 过程是粒细胞巨噬细胞集落刺激因子(GM-CSF)的一种强效 嗜酸性粒细胞产生的细胞因子。BAL液中GM-CSF普遍升高 有症状的哮喘患者。在体外激活的PBEos分泌 免疫检测GM-CSF，并表达GM-CSF mRNA。PBEos还表示 细胞表面GM-CSF受体，提示GM-CSF是一种关键的 体外和体内的自分泌生长和存活因子。尽管 GM-CSF的可能功能意义，人们对其知之甚少 控制其产生和释放的分子机制(S) 嗜酸性粒细胞。最近我们发现GM-CSF mRNA的稳定性是 肿瘤坏死因子α(TNF)治疗后外周血Eos显著增强 和纤维连接蛋白或BAL来源的嗜酸性粒细胞中的过敏原 志愿者。通过酵母3杂交筛选，我们鉴定了YB-1，一个已知的核 酸结合蛋白作为GM-CSF的mRNA结合蛋白。重组YB-1 在体外与富含AU的3‘非编码区不稳定性决定因素特异性结合 GM-CSF基因的表达。将YB-1基因导入外周血嗜酸粒细胞后，YB-1基因的表达增强 体外存活3-5倍，这可被抗GM-CSF完全阻断 抗体。最后，在初步研究中，转基因YB-1稳定了下来 PBEos中GM-CSF基因的表达。因此，我们假设YB-1介导了 活化的嗜酸性粒细胞中GM-CSF mRNA的转录后调控。因此， 这个项目的目标是1)。YB-1如何提高GM-CSF的特性 PBEos中的信使核糖核酸，2).表征由肿瘤坏死因子α诱导的信号级联，以及 纤维连接蛋白使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器还是与 调节GM-CSF mRNA的额外蛋白质组分，4)。确定是哪一个 GM-CSF转录后基因需要YB-1的结构域(S) 监管。总的来说，这些研究将阐明分子机制。 在激活的嗜酸性粒细胞中潜在的GM-CSF mRNA调控，因此， 为预防和治疗提供额外的、新的治疗靶点 哮喘的症状。

(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.