Regulation Of Cytosolic Phospholipase A2 In The Lung

肺中胞质磷脂酶 A2 的调节

• 批准号: 7167643
• 负责人: HARRY S NICK
• 金额: $ 35.96万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7167643
• 关键词: DNA footprinting; chromatin; cytokine; eicosanoid metabolism; fibroblasts; gel electrophoresis; gel mobility shift assay; genetic mapping; genetic regulatory element; glucocorticoids; inflammation; lung; phospholipase A2; protein structure function; respiratory epithelium; tissue /cell culture; transcription factor

DESCRIPTION (Applicant's Abstract): Of the known biologically active mediators generated by cells, both normally and during the inflammatory response, few have received more attention than those generated in the arachidonic acid cascade. Cytokines are established regulators of the arachidonic acid cascade in lung cells. The levels of various arachidonic metabolites distinguish between the normal and pathogenic states of the human lung. The arachidonyl-selective, cytosolic phospholipase A2 (cPLA2) is ubiquitously present in human lung and is most likely the rate-limiting step in eicosanoid generation. We therefore propose to study the molecular regulation of this pivotal gene in human lung fibroblasts and epithelial cells in response to glucocorticoids and pro-inflammatory cytokines. We will detect basal and stimulus-specific DNase I hypersensitive (HS) sites located in distant regions of the cPLA2 locus by utilizing a novel strategy involving field inversion gel electrophoresis (FIGE) to analyze altered regions of chromatin structure. A regulatory element trapping protocol will be employed to functionally identify regulatory sequences and complement our chromatin analysis. Deletion analysis will be used to precisely delineate cis-acting regulatory elements functionally by transient transfection in control, stimulated and repressed pulmonary cells. To identify, at single nucleotide resolution, the location of transcriptionally relevant protein-DNA contacts in living cells, we will employ in vivo footprinting using ligation mediated PCR (LMPCR) in conjunction with site-directed mutagenesis, EMSA and Transcription Factor Decoy (TFD) analysis. The knowledge gained by studying the molecular mechanisms controlling arachidonate levels will provide the basis for an understanding of the pathophysiology of acute lung injury that occurs in pulmonary diseases, such as asthma, acute respiratory distress syndrome, pulmonary fibrosis, and cystic fibrosis.

描述（申请人的摘要）：已知的生物活性介质 由细胞产生，无论是正常情况还是炎症反应期间，很少 比花生四烯酸中产生的物质受到更多关注 级联。细胞因子是花生四烯酸级联的既定调节剂 在肺细胞中。各种花生四烯酸代谢物的水平区分 人肺的正常状态和致病状态之间。这 花生四烯酰选择性胞质磷脂酶 A2 (cPLA2) 无处不在 存在于人类肺部，很可能是类二十烷酸的限速步骤 一代。因此，我们建议研究这种现象的分子调控 人肺成纤维细胞和上皮细胞响应的关键基因 糖皮质激素和促炎细胞因子。我们将检测基础和 位于远处区域的刺激特异性 DNase I 超敏感 (HS) 位点 利用涉及场反转凝胶的新策略对 cPLA2 基因座进行分析 电泳（FIGE）来分析染色质结构的改变区域。一个 调控元件捕获协议将用于功能识别 调控序列并补充我们的染色质分析。删除分析 将用于在功能上精确描述顺式作用调控元件 通过瞬时转染对照、刺激和抑制的肺细胞。 以单核苷酸分辨率鉴定转录位置 活细胞中相关的蛋白质-DNA接触，我们将在体内使用 使用连接介导的 PCR (LMPCR) 结合 定点诱变、EMSA 和转录因子诱饵 (TFD) 分析。 通过研究控制分子机制获得的知识 花生四烯酸水平将为理解 肺部疾病中发生的急性肺损伤的病理生理学，例如 哮喘、急性呼吸窘迫综合征、肺纤维化和囊肿 纤维化。