Investigating the role of intra- and extra-cellular modulators of the transcription factor NF-kappaB

Abstract

The nuclear factor-kappa B (NF-kappaB) family of transcription factors play a pivotal role in the inflammatory and immune responses, as well as cell growth and survival. Since its discovery 25 years ago, NF-kappaB has emerged as one of the most intensely studied eukaryotic transcription factors, due to its pleiotropic functions and its inducible pattern of expression that is subject to multi-level regulation. Many modulatory events affect NF-kappaB function and thus to have a better understanding of the NF-kappaB pathway and its gene regulation will require additional studies of NF-kappaB's modulators. The goal of this thesis was to investigate the role of intra- and extra-cellular modulators of NF-kappaB. To study how intracellular modulators affect NF-kappaB function, various genetic and biochemical approaches were used to identify potentially new NF-kappaB's interacting proteins. Two AGC family kinases, STK38 and STK38L, were chosen for further study. These two proteins were found to not only interact with NF-kappaB's p65 subunit but also act as its kinases in vitro. shRNA mediated knockdown combined with gene expression analysis revealed that STK38 and STK38L via their kinase function regulated p65 activity on some, but not all NF-kappaB-responsive inflammatory genes in response to TNFa stimulation. To study the role of extracellular modulators on the apoptotic behaviors of cells, a panel of human immortalized and cancer liver cell lines were pre-treated with pro-inflammatory cytokines TNFa and IL-1a followed by TRAIL, a potent inducer of cell death. This context-dependent approach revealed that TNFa and IL-1a had diverse effects on cell death behaviors and inhibiting the NF-kappaB pathway was enough to block pro-inflammatory cytokines-mediated pro-death or pro-survival outcomes. Chromatin immunoprecipitation followed by high throughput sequencing, realtime PCR and Western blotting were used to identify NF-kappaB's target genes involved in the cell death pathway. From these data, new regulatory mechanisms that affect cell death behaviors were proposed. Dysregulation of NF-kappaB activation has been implicated in various pathologies including inflammatory diseases and cancer. The results described here represent progress toward understanding the NF-kB pathway and its gene regulation program. The more we understand this pathway, the better we are at targeting it for disease treatments.