Novel Role of Pre-B-Cell Colony Enhancing Factor in ALI

前 B 细胞集落增强因子在 ALI 中的新作用

• 批准号: 7095414
• 负责人: Shui Qing Ye
• 金额: $ 34.48万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7095414
• 关键词: adult respiratory distress syndrome; biomarker; cytokine; gel mobility shift assay; gene induction /repression; genetic promoter element; genetic susceptibility; genetic transcription; genetically modified animals; inflammation; laboratory mouse; lung injury; molecular pathology; pulmonary edema; respiratory epithelium; single nucleotide polymorphism; vascular endothelium; vascular endothelium permeability

DESCRIPTION (provided by applicant): Pre-B-cell colony enhancing factor (PBEF) is a unique, little-studied cytokine, whose relevance to the lung pathophysiology is unknown. Recent work by the PI has provided the first evidence of PBEF expression in lung tissues and overexpression in acute lung injury (ALI) as well as the association of PBEF genetic variants with susceptibility to ALI. The haplotype weighted analysis of single nucleotide polymorphism (SNP) T-1001G and C-1543T in the PBEF gene promoter revealed a susceptible haplotype GC with a 7.7-fold higher risk of ALI. Reporter gene assays indicated that theses variants affected transcription rate. Our recent data suggest that PBEF affects pulmonary endothelial cell permeability in vitro and increased pulmonary edema in C57 BL/6 mice in vivo. In addition, stealth PBEF siRNA significantly inhibited TNF-alpha mediated increase of IL-8 secretion, a known edematogenic factor in ALI, in pulmonary cells. All these results support PBEF as a potential novel candidate gene and biomarker in ALI. To further detail underlying molecular mechanisms of PBEF in the pathogenesis of ALI, we hypothesize that patients with the genetically- determined susceptible haplotype in the PBEF promoter region are more susceptible to the upregulation by mechanical forces and inflammatory stimuli, two most inciting risk factors to ALI; increased PBEF expression contribute to the increased susceptibility to ALI by stimulating expression of other inflammatory cytokines such as IL-8, which leads to the pulmonary artery endothelial and epithelial cell barrier dysfunction and increased pulmonary permeability, resulting in the pathogenesis of ALI. The goal of this application is to define the physiologic and molecular significance of the identified PBEF polymorphisms. Furthermore, we will determine the pathophysiological role of PBEF in inflammatory lung injury with a specific focus on a role of PBEF in increased lung vascular leak/permeability, a pathophysiological feature of ALI, both in tissue culture system in vitro and in Pbef gene knock out C57 BL/6 mice in vivo. Specific Aim 1 will functionally characterize PBEF gene promoter SNPs utilizing the reporter gene and electrophoretic mobility shift assays. Specific Aim 2 will examine the role of PBEF gene expression in endothelial cell barrier regulation and contractility in vitro by evaluating endothelial cell transendothelial electric resistance, stress fiber formation, and phosphorylation of myosin light chains. Specific Aim 3 will define the signal transduction pathways of PBEF-mediated endothelial barrier regulation by examining PBEF- regulated genes and interacting partners. Specific Aim 4 will examine the in vivo role of PBEF in a murine model of ALI using Pbef knock out and over-expressing animals. Together, these novel and highly translational studies using the genomic and genetic approaches will provide new insight into molecular mechanisms of PBEF function in ALI, which may lead to the development of novel diagnostic modalities and therapeutic strategies in ALI.

描述（由申请人提供）：前B细胞菌落增强因子（PBEF）是一种独特的，较少的细胞因子，其与肺病理生理学的相关性尚不清楚。 PI的最新工作提供了PBEF在肺组织中表达和急性肺损伤（ALI）的过表达的第一个证据，以及PBEF遗传变异与ALI敏感性的关联。 PBEF基因启动子中单核苷酸多态性（SNP）T-1001G和C-1543T的单倍型加权分析显示，易感的单倍型GC，ALI风险高7.7倍。记者基因测定表明，这些变体影响了转录率。我们最近的数据表明，PBEF在体外影响肺内皮细胞通透性，并在体内C57 BL/6小鼠中增加肺水肿。此外，隐形PBEF siRNA显着抑制了TNF-α介导的IL-8分泌的增加，IL-8分泌是ALI的已知肿瘤因子，肺细胞中的已知肿瘤因子。所有这些结果都支持PBEF作为ALI中潜在的新型候选基因和生物标志物。为了进一步详细介绍PBEF在ALI发病机理中的分子机制，我们假设在PBEF启动子区域中具有遗传确定的易感单倍型的患者更容易受到机械力和炎症刺激的上调，这是两个最刺激的风险因素对ALI的刺激性。 PBEF表达的增加通过刺激其他炎症性细胞因子（例如IL-8）的表达增加了对ALI的敏感性，这会导致肺动脉内皮和上皮细胞屏障功能障碍和肺部通透性增加，从而导致ALI病原体。该应用的目的是定义已鉴定的PBEF多态性的生理和分子意义。此外，我们将确定PBEF在炎症性肺损伤中的病理生理作用，特别关注PBEF在增加的肺血管泄漏/渗透性中的作用，这是ALI的病理生理特征，在体外和PBEF基因在VIVO中的C57 BL/6小鼠中，ALI在组织培养系统中的病理生理特征。特定的目标1在功能上将表征PBEF基因启动子SNP，利用报告基因和电泳移动性转移测定法。特定的目标2将通过评估内皮细胞跨内皮电阻，应力纤维形成以及肌球蛋白光链的磷酸化来研究PBEF基因表达在体外内皮细胞屏障调节和收缩性中的作用。特定的目标3将通过检查PBEF调节的基因和相互作用的伴侣来定义PBEF介导的内皮屏障调节的信号转导途径。特定的目标4将使用PBEF敲除和表达过度表达的动物来检查PBEF在ALI的鼠模型中的体内作用。共同使用基因组和遗传学方法的这些新颖的高度转化研究将为ALI中PBEF功能的分子机制提供新的见解，这可能导致ALI中新型诊断方式和治疗策略的发展。