Gene Specific Regulation of NFkB by Nuclear IkBa in Inflammation and Cancer

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DESCRIPTION (provided by applicant): Our long-term goal is to understand the mechanisms regulating transcription of NF?B-dependent anti-apoptotic and pro-inflammatory genes. Since NF?B activity and expression of NF?B-regulated pro-inflammatory and anti-apoptotic genes are increased in many inflammatory diseases, as well as in many types of human cancer and leukemia, inhibition of NF?B-dependent transcription thus represents an important therapeutic target. However, one of the main concerns regarding the NF?B inhibitors is their specificity, since many steps leading to NF?B activation are important for other cellular functions as well. Our previous studies have shown that an induction of nuclear translocation and accumulation of the NF?B inhibitor, I?B?, inhibits the in vitro NF?B DNA binding activity and induces apoptosis in human leukocytes and cancer cells. Importantly, our recent data indicate that the in vivo inhibition of NF?B-dependent transcription by nuclear I?B? is gene specific: while the transcription of pro-inflammatory cytokines TNF?, IL-12 and IL-6 in human leukocytes is inhibited by nuclear I?B?, transcription of IL-8 is not. However, at present, the mechanisms that regulate the in vivo nuclear interaction of I?B? with NF?B proteins and NF?B- dependent promoters are unknown. The central hypothesis of this proposal is that the regulation of NF?B-dependent transcription of pro-inflammatory and anti-apoptotic genes by nuclear I?B? is gene specific, and could thus provide a basis for novel anti-inflammatory and anti-cancer therapies aimed at the specific inhibition of NF?B activity by nuclear I?B?. The specific aims focus on analyzing the mechanisms that regulate the in vivo binding of I?B? to NF?B dimers and NF?B promoters, thus regulating NF?B-dependent transcription. In Aim 1, we will measure by chromatin immunoprecipitation the recruitment of NF?B and I?B? proteins to NF?B-regulated promoters of pro-inflammatory and anti-apoptotic genes in human U937 macrophages and leukemia Hut-78 cells. We will determine whether the ability of nuclear I?B? to inhibit NF?B-dependent transcription depends on the subunit composition of NF?B dimers. In Aim 2, we will identify the mechanisms that regulate the nuclear I?B?-NF?B interaction in U937 macrophages and Hut-78 leukemia cells, and we will test the hypothesis that the responsible mechanisms involve post-translational modification(s) of p65 NF?B, and/or gene specific recruitment of the proteasome. Identification of the mechanisms that regulate the gene specific transcription of NF?B-dependent genes by nuclear I?B? might provide a new strategy that would use the nuclear I?B? as a specific regulator of NF?B-dependent transcription. This approach could be applicable in both anti-inflammatory and anti-cancer therapies. In addition, this project will enhance the research environment at St. John's University by providing undergraduate students with numerous opportunities to learn the fundamentals of biomedical research. PUBLIC HEALTH RELEVANCE: This proposal focuses on the mechanisms that regulate ability of the nuclear inhibitory protein I?B? to associate with NF?B-regulated promoters and inhibit transcription of NF?B-dependent inflammatory and pro-survival genes. Since NF?B activity is increased in many human diseases including inflammatory disorders, cancer, and leukemia, identification of the mechanisms by which the nuclear I?B? inhibits NF?B-dependent transcription will lead to the development of more specific anti-inflammatory and anti-cancer therapies.