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(申请人提供)：前B细胞集落增强因子(PBEF)是一种独特的，很少被研究的细胞因子，其与肺部病理生理学的相关性尚不清楚。PI最近的工作首次证明了PBEF在肺组织中的表达和在急性肺损伤(ALI)中的过度表达，以及PBEF基因变异与ALI易感性的关系。PBEF基因启动子单核苷酸多态(SNP)T-1001G和C-1543T的单倍型加权分析显示，易感单倍型GC患ALI的风险增加7.7倍。报告基因分析表明，这些变异体影响转录速率。我们最近的数据表明，PBEF在体外影响肺内皮细胞的通透性，并在体内增加C57BL/6小鼠的肺水肿。此外，隐形PBEF siRNA显著抑制了TNF-α介导的肺细胞IL-8分泌增加，IL-8是ALI中已知的水肿性因子。这些结果支持PBEF作为ALI潜在的候选基因和生物标志物。为了进一步阐明PBEF在ALI发病机制中的潜在分子机制，我们假设PBEF启动子区域具有遗传易感单倍型的患者更容易受到机械力和炎症刺激的上调，这两个因素是ALI的最危险因素；PBEF表达增加通过刺激IL-8等其他炎性细胞因子的表达而增加ALI的易感性，从而导致肺内皮细胞和上皮细胞屏障功能障碍和肺通透性增加，从而导致ALI的发病。这项应用的目标是定义已识别的PBEF多态的生理和分子意义。此外，我们将确定PBEF在炎性肺损伤中的病理生理作用，特别是在体外组织培养系统和体内Pbef基因敲除的C57BL/6小鼠中，PBEF在增加肺血管渗漏/通透性方面的作用。特定目的1将利用报告基因和电泳迁移率改变分析对PBEF基因启动子SNPs进行功能表征。具体目的2将通过评估内皮细胞跨内皮细胞的电阻、应力纤维的形成和肌球蛋白轻链的磷酸化来研究PBEF基因表达在体外内皮细胞屏障调节和收缩中的作用。具体目标3将通过检测PBEF调节的基因和相互作用的伙伴来确定PBEF介导的内皮屏障调节的信号转导途径。具体目标4将利用PBEF基因敲除和过度表达的动物来检验PBEF在ALI小鼠模型中的体内作用。综上所述，这些采用基因组和遗传学方法进行的新的高度翻译的研究将为ALI中PBEF功能的分子机制提供新的见解，这可能导致开发新的ALI诊断模式和治疗策略。