HUMAN MAST CELL CHYMASE AND CATHEPSIN G EXPRESSION IN AIRWAY INFLAMMATION

这里提出的研究将比较校长的表达 正常和炎症的人肥大细胞的糜蛋白酶样蛋白酶 并将表征遗传和核元素 负责它们的调节表达。 这些酶，糜酶和 组织蛋白酶G是MC/TC分泌颗粒的主要成分 肥大细胞亚群。 它们具有强效的促分泌素、肽酶和 基质降解活性并释放到细胞外环境中 在肥大细胞被抗原结合的IgE激活后，神经肽和 其他刺激。 肥大细胞中含有糜蛋白酶和 组织蛋白酶G在不同组织中具有显著的生理差异 肺和气道的微环境，以及分布模式 以及假设在肺和气道中变化的产生水平 疾病该项目将探索酶的调节， 基因的水平。 糜酶的水平、定位和细胞来源， 组织蛋白酶G mRNA表达将在正常和炎症气道中进行探索 组织，使用定量聚合酶链反应和原位 杂交方法14号染色体上的顺式作用元件位于 两种酶的紧密连锁基因将通过转染来确定 用含有蛋白酶的质粒对凝乳酶转录细胞系进行了 与报告基因相连的调控元件。特定DNA序列 参与与肥大细胞特异性核调节蛋白结合的蛋白质将 通过DNA足迹、凝胶迁移率变化和甲基化来定义 干扰测定。最后，参与肥大细胞的调节蛋白 将分离糜蛋白酶和组织蛋白酶G表达本身， 表征了这些研究探讨了有关 生物学上重要的肥大细胞糜蛋白酶样蛋白的表达 酶，并可能提供深入了解的现象， 表型变异 在人肺肥大细胞中。

The studies proposed here will compare the expression of the principal chymotrypsin-like proteases of human mast cells in normal and inflamed airway tissues and will characterize genetic and nuclear elements responsible for their regulated expression. These enzymes, chymase and cathepsin G, are major components of secretory granules in the MC/TC subset of mast cells. They have potent secretagogue, peptidase and matrix-degrading activity and are released into the extracellular milieu following activation of mast cells by antigen-bound IgE, neuropeptides and other stimuli. The proportion of mast cells that contains chymase and cathepsin G exhibits striking physiological variation in different tissue microenvironments of the lung and airways, with patterns of distribution and levels of production that are hypothesized to vary in lung and airway diseases. This project will explore the regulation of the enzymes at the level of the gene. The level, location and cellular source of chymase and cathepsin G mRNA expression will be explored in normal and inflamed airway tissues, using the quantitative polymerase chain reaction and in situ hybridization. The cis-acting elements on chromosome 14 flanking the closely linked genes for the two enzymes will be defined by transfection of chymase-transcribing cell lines with plasmids containing protease regulatory elements linked to reporter genes. Specific DNA sequences involved in binding to mast cell-specific nuclear regulatory proteins will be defined by DNA footprinting, gel mobility shift, and methylation interference assays. Finally, regulatory proteins involved in mast cell chymase and cathepsin G expression themselves will be isolated and characterized. These studies address basic questions regarding the expression of the biologically important mast cell chymotrypsin-like enzymes and may provide insights into the poorly understood phenomenon of phenotype variation in mast cells of human lung.