Identification of Novel Regulators of Nuclear Factor Kappa B Repression by the Glucocorticoid Receptor
- Author(s): Murphy, Samantha Harriet
- Advisor(s): Verma, Inder M
- et al.
Nuclear factor kappa B (NF-kappaB) is a family of transcription factors that has an essential regulatory function in inflammation, the immune response, cell proliferation, and apoptosis. Constitutive activation of the NF-kappaB pathway is often associated with cancer and chronic inflammatory diseases such as multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, and asthma. Glucocorticoids, which bind to the glucocorticoid receptor (GR), are among the strongest anti-inflammatory agents and one of the most common forms of treatment to suppress inflammation. GR is a transcription factor that plays an important role in a variety of cellular processes including reproduction, development, differentiation, and metabolism, and it is fundamental to the counteraction of immune and inflammatory responses by acting as a key repressor of NF-kappaB. Proposed mechanisms of NF-kappaB repression by GR are complex and varied, and a universal mechanism of repression has yet to be elucidated. By discovering novel genes that play a role in GR repression of NF-kappaB, potential therapeutic targets and their molecular mechanisms can be identified to specifically improve the use of glucocorticoids in clinical applications. We performed high throughput screens using siRNA oligonucleotide library collections to identify novel genes that affect glucocorticoid repression of NF-kappaB activity after activation of both the NF-kappaB and GR pathways. The work presented in this thesis provides evidence that p53 is involved in glucocorticoid receptor repression of NF-kappaB. We initially establish p53 as a gene of interest by high throughput screening, and validate this finding using luciferase assays in physiologically relevant cell lines and by qPCR. We also confirm its biological significance in vivo in a mouse model of LPS shock. Additionally, we demonstrated that p53 does not play a role in upstream NF-kappaB or GR signaling cascades, that p53 loss impairs glucocorticoid repression of NF-kappaB target gene transcription, and that p53 loss impairs GR target gene transcription. We conclude that p53 is an important gene involved in GR repression of NF-kappaB, a finding that may explain why glucocorticoid treatment is often an ineffective therapy in the repression of inflammation associated with the tumor microenvironment and why NF-kappaB is often up-regulated in many human cancers. Additionally, we have identified 24 other novel genes that play a role in NF-kappaB repression by GR, and have created a survey outlining which of these genes affect aspects of certain NF-kappaB signaling components. Ultimately, we believe that agonist development targeting one of these genes may have the potential to lead to better anti-inflammatory therapies.