Dynamic Characterization of the IKK:κBα:NFκB Negative Feedback Loop Using Real-Time Bioluminescence Imaging

<p>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 (κB₅_àIκBα-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 <em>in cellulo</em> and <em>in vivo</em>. 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, <em>in silico</em> 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 <em>in vivo.</em></p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biology and Biomedical Sciences: Molecular Cell Biology

Year dc:date.available

2011

Author dc:creator

• Moss, Britney

Contributors dc:contributor

• David Piwnica-Worms

• Cellular biology
• Systematic biology
• bioluminescence
• imaging
• liver
• luciferase
• nfkb
• TNFa
• Biology

Language dc:language

English (en)

DOI

https://doi.org/10.7936/K7ZW1HXK

OAI identifier oai:identifier

oai:openscholarship.wustl.edu:etd-1621