DIFFERENTIAL CHEMOKINE GENE EXPRESSION IN THE LUNG

我们之前曾报道从肺中克隆了一个基因IkappaBR 上皮细胞。最近我们已经证明了过度表达 肺上皮细胞IkappaBR导致RANTES上调，但 不是白介素8(IL-8)基因的表达，尽管两者都 基因受核因子-kappaB的调控。这种选择性的上调 与独特的RANTES-kappaB结合增加的结合相关 已知的p50同源二聚体的活性和结合减少 作为某些kappaB位点的抑制物。这些加在一起， 观察结果提示我们假设：1.独特的核因子-kappaB家族 上皮细胞中存在的蛋白质可以选择性地上调 趋化因子基因在肺部炎症中的表达。2.有能力 IkappaBR对隔离抑制性p50同源二聚体起重要作用 在这个过程中。为了解决这一假设，我们将：目标#I。 IkappaBR介导的细胞特异性及其机制的研究 Rantes基因上调。(A)不同的刺激是否会激活 细胞因子和病毒等RANTES基因的表达也会增强 将研究IkappaBR基因的表达。(B)核糖核酸酶保护 检测、DNA足迹检测、酶联免疫吸附检测和 将使用电泳迁移率变化分析来研究这种影响 IkappaBR在RANTES上的过度表达及IL-8基因表达的研究 不同的细胞类型。(C)IkappaBalpha的显性否定形式 (IkappaBalphaM)在IkappaBR过表达肺中诱导表达 决定上皮细胞对经典p50/p65的需求 异源二聚体在这些细胞中的RANTES基因表达。(D)影响 核因子-kappaB(p50/p65)活化特异性抑制物对RANTES基因的影响 这一独特的复合体的表达和形成将被研究。目标# 确定构成独特复合体的蛋白质的特性。一个 将使用分子克隆方法，即酵母双杂交系统 鉴定独特的RANTES-kappaB结合复合体。目标#三. 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 在使用可诱导转基因系统的小鼠中。(A)原位杂交 将使用技术来确定IkappaBR基因的表达 在人类哮喘中表达上调。(B)多西环素诱导剂 将使用我们实验室最新建立的转基因系统 在体内过表达IkappaBR或IkappaBalphaM。(C) IkappaBR或IkappaBalphaM过表达对RANTES基因表达的影响 在呼吸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.