Dynamic Characterization of the IKK:IkappaBalpha:NF-kappaB Negative Feedback Loop Using Real-Time Bioluminescence Imaging.

摘要

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→IκBα-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..