REGULATION OF THE ICAM-1 GENE IN THE PULMONARY AIRWAY

肺气道中 ICAM-1 基因的调节

• 批准号: 2210780
• 负责人: Dwight C Look
• 金额: $ 8.1万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2210780
• 关键词: DNA binding protein; cell adhesion molecules; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; human tissue; inflammation; interferon gamma; messenger RNA; plasmids; respiratory epithelium; tissue /cell culture; transfection; vascular endothelium

The central hypothesis of the research proposal is that airway epithelial and vascular endothelial cells modulate inflammation through the coordinated expression of cell surface adhesion receptors. Preliminary studies demonstrate that intercellular adhesion molecule-1 (ICAM-1) is selectively expressed on airway epithelial cells after interferon-gamma (IFN-gamma) stimulation and that ICAM-1 interaction with corresponding beta2-integrins on neutrophils and T-lymphocytes is a critical mechanism for epithelial-leukocyte adhesion. The epithelial cell profile for cytokine responsiveness is distinct from the one for vascular endothelial cells which express functional ICAM-1 in response to other cytokines but not after IFN-gamma treatment. The complimentary profile of cytokine responsiveness is proposed to allow for efficient localization of leukocytes under different inflammatory conditions. The level of ICAM-1 expression appears to be regulated at the level of mRNA transcription in both airway epithelial and vascular endothelial cells, but the mechanism for the differences in gene activation between cell types is uncertain We propose to study the transcriptional control of ICAM-1 gene in airway epithelial cells with initial emphasis on the mechanism(s) that control IFN-gamma selectivity. The principal cell system to be employed for this work is primary cultures of human airway epithelial cells. A model cell system will also be developed using transformed bronchial epithelial cells which also express functional ICAM-1 and exhibit selective responsiveness to IFN-gamma. Distinct features of the epithelial ICAM-I gene will be defined by comparison to (i) ICAM-I gene regulation in endothelial cells and (ii) HLA-A gene regulation in epithelial and endothelial cells. Specific aims are to: 1) Identify cis-acting regulatory elements in the ICAM-1 gene promoter that mediate activation by IFN-gamma in airway epithelial cells. This aim will be accomplished using a transient DNA transfection system with cultured airway epithelial cells and plasmid constructs composed of the 5'-flanking region of the ICAM-1 gene linked to a reporter gene. 2) Identify and characterize the specific trans-acting proteins which bind to IFN-gamma response elements in the ICAM-1 gene. This aim will be accomplished using nuclear protein extracts from IFN-gamma stimulated cells in DNA-protein binding assays such as the electrophoretic mobility shift assay.

研究建议的中心假设是气道上皮 和血管内皮细胞通过 细胞表面粘附受体的协调表达。 初步的 研究表明，细胞间粘附分子1（ICAM-1）是 干扰素伽马后在气道上皮细胞上有选择地表达 （IFN-GAMMA）刺激以及ICAM-1与相应的相互作用 中性粒细胞和T-淋巴细胞上的β2-积聚是一种关键机制 用于上皮白细胞粘附。上皮细胞剖面 细胞因子反应与血管内皮的反应性不同 对其他细胞因子响应的功能性ICAM-1的细胞，但 不接受IFN-gamma治疗。细胞因子的免费特征 提出了响应能力，以便有效地定位 白细胞在不同的炎症条件下。 ICAM-1的水平 表达似乎是在mRNA转录水平的调节 气道上皮和血管内皮细胞都 对于细胞类型之间基因激活的差异是不确定的 建议研究气道中ICAM-1基因的转录控制 上皮细胞最初强调控制的机制 IFN-gamma选择性。为此使用的主要单元系统 工作是人类气道上皮细胞的主要培养物。模型单元格 系统还将使用转化的支气管上皮细胞开发 这也表达功能性ICAM-1并具有选择性响应性 到ifn-gamma。上皮ICAM-I基因的不同特征将是 通过与（i）ICAM-I基因调节的内皮细胞相比定义 （ii）上皮细胞和内皮细胞中的HLA-A基因调节。 具体目的是： 1）确定ICAM-1基因启动子中的顺式作用调节元件 这种介导IFN-gamma在气道上皮细胞中的激活。这个目标 将使用具有瞬态DNA转染系统来完成 培养的气道上皮细胞和质粒构建体由 ICAM-1基因的5'-频型区域与记者基因有关。 2）识别并表征结合的特定跨作用蛋白 在ICAM-1基因中的IFN-GAMMA响应元件中。这个目标将是 使用刺激的IFN-GAMMA的核蛋白提取物完成 DNA-蛋白结合测定的细胞，例如电泳迁移率 换档测定。