A major goal of this SCOR proposal is to determine how inflammatory cells infiltrate the pulmonary airway in asthma. In that context, we have proposed that epithelial cells lining the airway provide critical biochemical signals for immune-cell influx and activation. One major influence on epithelial-immune cell interaction is the level of epithelial expression of intercellular adhesion molecule-1 (ICAM-1). In turn, the level of ICAM-1 depends on a specific interferon-gamma (IFN-gamma)-driven signal transduction pathway that consists of the IFN-gamma-receptor, receptor-associated Jak1 and Jak2 kinases, the Stat1 transcription factor, and a specific Stat 1/DNA interaction in the ICAM-1 gene promoter region. This pathway may allow for selective localization and activation of immune cells during IFN-gamma-dependent host defense, and in some cases, may mediate virus-induced airway inflammation and hyperreactivity. The current proposal is based on three characteristics of IFN-gamma-driven, Stat1-dependent gene regulation in airway epithelial cells: (i) it appears to depend on two critical DNA/protein interactions--one between an inverted- repeat motif and activated Stat1 and another between a GC-box motif and the transcription factor Sp1; (ii) there is persistent and/or exaggerated activation of Stat1 and concomitant increases in expression of ICAM-1 and Stat1 itself; and (iii) Stat1 overexpression and consequent Stat-1-dependent gene activation appear to be features of subjects with moderately severe asthma. These features of IFN-gamma-driven activation of the ICAM-1 gene are shared with the gene for interferon regulator factor-1 (IRF- 1), so that IRF-1 may act in concert with ICAM-1 to mediate airway immunity and inflammation. Accordingly, our specific aims are to: I. Define the role of Stat1/Sp1 interactions in Stat 1-dependent gene activation.
This aim will take advantage of an airway epithelial cell model (primary-culture human tracheobronchial epithelial cells) for IFN-gamma-driven interaction between Stat1 and Sp1 and their respective DNA binding sites in the ICAM-1 and IRF-1 gene promoter regions. Structure-function studies of Stat1 and Sp1 will be accomplished by analyzing DNA/protein and protein/protein interactions in this model. II. Define the basis for prolonged and/or exaggerated Stat1- dependent gene activation in airway epithelial cells.
This aim will take advantage of the same cell model and its capacity for concomitant IFN-gamma-driven expression of Stat 1, ICAM-1, and IRF- 1. Regulation of Stat1 activity and its relationship to upstream and downstream events in the IFN-gamma-dependent signal transduction pathway will be examined under unstimulated and IFN- gamma-stimulated conditions. III. Define the characteristics of Stat1-dependent gene regulation in airway epithelial cells in nonasthmatic and asthmatic subjects.
This aim take advantage of airway epithelial biopsies and epithelial cell brushings from normal and asthmatic subjects. Stat1 behavior will be correlated with disease status (atopic, nonatopic, stable, flare, antigen-challenge) and with the status of IFN-gamma, IFN-gamma-receptor, Jak1, Jak2, Sp1, IRF-1, and ICAM-1.
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