Dynamic Characterization of the IKK:κBα:NFκB Negative Feedback Loop Using Real-Time Bioluminescence Imaging
Biology and Biomedical Sciences: Molecular Cell Biology
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
The transcription factor NF-κB is a pivotal regulator of mammalian cell function, modulating genes implicated in cellular stress responses, proliferation, differentiation, cell survival and apoptosis, as well as immune and inflammatory responses. Improper regulation of NF-κB signaling has been implicated in a myriad of human pathological disorders, including cardiovascular and neurodegenerative diseases, chronic inflammation, and various cancers. A key regulatory node within canonical NF-κB signaling is the IKK:NF-κB: IκBα negative feedback loop that plays a major role in regulating the strength and duration of NF-κB transcriptional activity. We have developed and characterized an unique bioluminescent reporter (κB5àIκBα-FLuc) that recapitulates this transcriptionally coupled negative feedback loop, and have extensively utilized this reporter to interrogate how diverse stimuli (i.e., ligand type, duration, concentration, sequential stimulation, etc.) impact the IKK:NF-κB: IκBα negative feedback loop in cellulo and in vivo. We found that the negative feedback loop exhibits differential and reproducible dynamic patterns in response to modulation of TNFα; concentration or pulse duration, and that responses to TNFα exhibited a remarkable degree of synchronicity at the level of single cells, cell populations, and in vivo. Furthermore, we discovered a TNFα-induced transient refractory period (lasting up to 120 min) during which cells were unable to fully degrade IκBα following a second TNFα challenge, and identified nuclear export of NF-κB: IκBα complexes as a rate-limiting step that may impact this refractory period. A high-throughput RNAi screen to identify new phosphatase and kinase regulators of TNFα-induced IKK:NF-κB: IκBα negative feedback loop dynamics revealed a vast array of different IκBα-FLuc dynamic profiles, highlighting the large number and diverse activities of kinases and phosphatases regulating the NF-κB pathway. Two of these hits, PTPRJ and DAPK3, have been validated and are the subjects of current investigations to understand the physiological and/or pathophysiological relevance in NF-κB, especially in the context of TNFα signaling during cancer and inflammation in the liver. In conclusion, our studies using dynamic, real-time bioluminescence imaging have demonstrated the utility of employing bioluminescent reporters alongside traditional biochemical assays, in silico modeling, and cell/molecular biology techniques to rigorously interrogate how diverse stimuli impact the IKK:NF-κB: IκBα negative feedback loop in single cells, cell populations, and at the organ- and tissue-level in vivo.
Moss, Britney, "Dynamic Characterization of the IKK:κBα:NFκB Negative Feedback Loop Using Real-Time Bioluminescence Imaging" (2011). All Theses and Dissertations (ETDs). 622.
Permanent URL: http://dx.doi.org/10.7936/K7ZW1HXK