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.
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