DIFFERENTIAL CHEMOKINE GENE EXPRESSION IN THE LUNG

我们以前报道了从人肺组织中克隆了一个基因IkappaBR， 上皮细胞 最近我们发现， 肺上皮细胞中的IkappaBR导致RANTES的上调， 而不是白细胞介素-8（IL-8）基因表达，尽管事实上， 基因由NF-κ B调节。 这种选择性上调 与独特的RANTES-kappaB结合增加相关 活性和减少的p50同二聚体的结合，这是已知的， 作为某些kappaB位点的阻遏物发挥作用。 综上所述各项 观察促使我们假设：1。独特的NF-κ B家族 蛋白质存在于上皮细胞中， 趋化因子基因在肺部炎症中的表达。 2.的能力 IkappaBR在螯合抑制性p50同源二聚体方面发挥重要作用 在这个过程中。 为了解决这个假设，我们将：目标# I。 表征IkappaBR介导的细胞特异性和机制 RANTES基因上调。 (a)是否不同的刺激激活了 RANTES基因表达，如细胞因子和病毒也增加 将研究IkappaBR基因表达。 (b)rna酶保护 测定、DNA足迹测定、酶联免疫吸附测定和 将使用电泳迁移率变动分析来研究 IkappaBR过表达对RANTES和IL-8基因表达的影响 不同的细胞类型 (c)IkappaB α的一种显性阴性形式 （IkappaBalphaM）在IkappaBR过表达肺中以诱导方式表达 上皮细胞，以确定对经典p50/p65的需求 在这些细胞中RANTES基因表达的异源二聚体。 (d)效果 RANTES基因NF-κ B（p50/p65）激活的特异性抑制剂 将研究独特复合物的表达和形成。 目标编号 二.表征构成独特复合物的蛋白质。 一 将使用分子克隆方法，酵母双杂交系统 以表征独特的RANTES-kappaB结合复合物。目标三。 研究IkappaBR在人哮喘中的表达及其作用 IkappaBR或IkappaBalphaM过表达对RANTES基因表达的影响 在小鼠中使用可诱导的转基因系统。 (a)原位杂交 技术将用于确定IkappaBR基因表达是否 在人类哮喘中表达上调。 (b)强力霉素诱导的 本实验室最近建立的转基因系统 以在体内过表达IkappaBR或IkappaBalphaM。 (c)的影响 RANTES基因表达上的IkappaBR或IkappaBalphaM过表达将 在气道炎症的抗原和病毒模型中进行研究。

We previously reported the cloning of a gene IkappaBR from lung epithelial cells. Recently we have shown that overexpression of IkappaBR in lung epithelial cells results in upregulation of RANTES but not interleukin-8 (IL-8) gene expression despite the fact that both genes are regulated by NF-kappaB. This selective upregulation correlated with increased binding of a unique RANTES-kappaB binding activity and decreased binding of p50 homodimers which are known to function as repressors of certain kappaB sites. Taken together, these observations prompted us to hypothesize that: 1. Unique NF-kappaB family proteins exist in epithelial cells which can selectively upregulate chemokine gene expression in lung inflammation. 2. The ability of IkappaBR to sequester inhibitory p50 homodimers plays an important role in this process. To address this hypothesis we will: Aim # I. Characterize the cell-specificity and mechanisms of IkappaBR-mediated RANTES gene upregulation. (a) Whether different stimuli that activate RANTES gene expression such as cytokines and viruses also augment IkappaBR gene expression will be investigated. (b) RNase protection assays, DNA footprinting assays, enzyme-linked immunosorbent assays and electrophoretic mobility shift assays will be used to study the effect of IkappaBR overexpression on RANTES and IL-8 gene expression in different cell types. (c) A dominant negative form of IkappaBalpha (IkappaBalphaM) in an inducible fashion in IkappaBR-overexpressing lung epithelial cells to determine the requirement for the classical p50/p65 heterodimer in RANTES gene expression in these cells. (d) The effect of specific inhibitors of NF-kappaB (p50/p65) activation on RANTES gene expression and formation of the unique complex will be studied. Aim # II. Characterize the proteins constituting the unique complex. A molecular cloning approach, the yeast two-hybrid system, will be used to characterize the unique RANTES-kappaB binding complex. Aim # III. Investigate the expression of IkappaBR in human asthma and the effect of overexpression of IkappaBR or IkappaBalphaM on RANTES gene expression in mice using an inducible transgenic system. (a) In situ hybridization techniques will be used to determine whether IkappaBR gene expression is upregulated in human asthma. (b) The doxycycline-inducible transgenic system recently established in our laboratory will be used to overexpress IkappaBR or IkappaBalphaM in vivo. (c) The effect of IkappaBR or IkappaBalphaM overexpression on RANTES gene expression will be investigated in antigen and viral models of airway inflammation.