The airway epithelium responds to cytokines and viruses through changes in its genetic program, an important event in the initiation of pulmonary inflammation. Collaborating with Project Leaders in this PPG, we have shown that cytokine (TNF) stimulation and RSV replication activate the NF-kappaB transcription factor and that inhibition of its activity reduces inflammation in mice. Here we will investigate mechanisms underlying novel experimental findings that the NF-kappaB-dependent genes are activated in 3 distinct kinetic groups. We have also identified a DMSO-sensitive step important in control of NF-kappaB dependent genes, where target gene expression can be inhibited after NF-kappaB binds DNA, and have shown that NF-kappaB/Rel A forms a nuclear complex with the NF-kappaB inducing kinase (NIK), a MAP3K kinase that forms a DNA- associated complex with IKB Kinase-alpha (IKKalpha) and the p300 coactivator. We will pursue the hypothesis that the NF-kappaB transcription factor is a central regulator of airway inflammation in the stimulated epithelial cell. NF-kappaB activation of target promoters requires chromatin remodeling, is redox-sensitive, and requires formation of a macromolecular complex with NIK, IKKalpha and p300.
Our aims are to: 1. Identify mechanisms for distinct temporal genomic responses to NF-kappaB. Chromatin immunoprecipitation (ChIP) assays will identify the kinetics of NF-kappaB recruitment, coactivator p300/CBP binding, and histone modifications of transcriptional targets and investigate how DMSO inhibits NF-kappaB-dependent chromatin remodeling. 2. Map interacting domains of the NF-kappaB inducing kinase (NIK) -with NF-KB/Rel A and the p300 coactivator, determine their functional consequences and explore NIK's role in the epithelial NF-kappaB dependent gene network. Coimmunoprecipitation (co-IP) assays will identify the domains of NIK required for interaction with NF-KB/Rel A. The finding that NIK binds p300 will be validated, and we will determine if NIK modifies p300 transcriptional activity or its histone acetyl transferase activity. The effect of siRNA-mediated NIK """"""""knockdown"""""""" will be determined. 3. Determine the role of the NIK-IKKalpha, NIK-Rel A, and NIK- p300 complexes in NF-kappaB dependent gene expression. IKKalpha is a member of the IKK complex that shuttles into the nucleus where it initiates chromatin remodeling. We will measure NIK and IKKalpha recruitment to NF-kappaB-dependent genes by ChIP. We will inducibly express interacting domains (of NIK) to disrupt the NIK-IKKalpha, NIK-Rel A and NIK-p300 complexes and determine effects on NF-kappaB-dependent activation and chromatin remodeling. 4. Develop a protein interaction letwork map of NF-KB/Rel A, IKKalpha, and NIK in cytokine stimulated and viral infected epithelial cells. """"""""Antibody arrays"""""""" of nuclear and cytoplasmic proteins isolated from cytokine-stimulated and viral infected cells will be used to build a network map of NF-KB/Rel A, NIK and IKKalpha - associated proteins. These studies will enhance aims proposed in and require support. Upon completion, we will elucidate novel pathways that can be targeted for anti-inflammatory drugs to treat pulmonary inflammation.
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