Airway inflammatory diseases continue to inflict significant morbidity in the US. NF-KB/RelA is a cytosolic transcription factor that mediates the inflammatory response to cytokines, viruses, oxidative stressors, and allergens. Our studies in the past project period have demonstrated that the NF-KB/RelA activation pathway is bipartite, with one arm releasing RelA from cytoplasmic inhibition, and a separate arm inducing reactive oxygen species (ROS)-dependent, site-specific RelA Ser[276] phosphorylation, required for activating inflammation. Our preliminary studies using TNF-a as stimulus have shown that RelA Ser[276] phosphorylation is a switch required for RelA association with a complex containing the cyclin-dependent kinase (CDK)-9 and bromodomain-4 (Brd4) proteins. CDK9 phosphorylates the C-terminus of RNA Polymerase II, promoting entry into transcriptional elongation mode that produces full-length mRNA transcripts. During this next grant period P1 seeks to understand the mechanism(s) by which RSV modulates NF-KB signaling in the epithelial cell. Our central hypothesis is that NF-KB /RelA Ser[276] phosphorylation couples to an acetyl (Ac) RelA- Brd4-CDK9 pathway that mediates transcriptional elongation as a mechanism for inflammatory gene expression, leading to airway inflammation.
Aim 1 will test the hypothesis that RelA Ser[310] phosphorylation is required for RelA Lys[310] acetylation and Brd4-CDK9 recruitment to promote inflammatory gene expression in response to RSV and 8-oxoG in cultured airway cells.
Aim 2 will test the hypothesis that CDK9 mediates RNA Pol II C-terminal domain (CTD) phosphorylation, clearance and transcriptional elongation in NF-KB-dependent gene expression in cultured airway cells.
Aim 3 will test the hypothesis that NF-KB-induced CDK9 recruitment mediates transcriptional elongation in RSV-induced mucosal inflammation in a mouse model in vivo. This project is highly synergistic with Projects 2-4, informing hypothesis testing and shared experimental work, and relies upon the support of Cores A and B. PI is innovative because it will for the first time establish the mechanism for the RelA phosphorylation/acetylation-transcriptional elongation pathway in RSV and 8-oxoGinduced inflammatory gene expression, and establish this pathway as a central mediator of airway inflammation. These discoveries will lay the foundation for new approaches to selective NF-KB inhibition, for example using CDK9 inhibitors.
Asthma is a breathing disorder affecting about 10% of Americans, and is frequently worsened by cold viruses. This project seeks to understand how the lung tissues respond to signals produced by cold viruses that worsen the disease. This knowledge should lead to novel approaches to asthma treatment, based on blocking the signals elucidated in this project.
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