Identification of a ligand-induced transient refractory period in nuclear factor-κB signaling

In response to a variety of extracellular ligands, nuclear factor-κB (NF-κB) signaling regulates inflammation, cell proliferation, and apoptosis. It is likely that cells are not continuously exposed to stimulating ligands in vivo but rather experience transient pulses. To study the temporal regulation of NF-κB and its major regulator, inhibitor of NF-κBα (IκBα), in real time, we utilized a novel transcriptionally coupled IκBα-firefly luciferase fusion reporter and characterized the dynamics and responsiveness of IκBα processing upon a short 30-s pulse of tumor necrosis factor α (TNFα) or a continuous challenge of TNFα following a 30-s preconditioning pulse. Strikingly, a 30-s pulse of TNFα robustly activated inhibitor of NF-κB kinase (IKK), leading to IκBα degradation, NF-κB nuclear translocation, and strong transcriptional up-regulation of IκBα. Furthermore, we identified a transient refractory period (lasting up to 120 min) following preconditioning, during which the cells were not able to fully degrade IκBα upon a second TNFα challenge. Kinase assays of IKK activity revealed that regulation of IKK activity correlated in part with this transient refractory period. In contrast, experiments involving sequential exposure to TNFα and interleukin-1β indicated that receptor dynamics could not explain this phenomenon. Utilizing a well accepted computational model of NF-κB dynamics, we further identified an additional layer of regulation, downstream of IKK, that may govern the temporal capacity of cells to respond to a second proinflammatory insult. Overall, the data suggested that nuclear export of NF- κB·IκBα complexes represented another rate-limiting step that may impact this refractory period, thereby providing an additional regulatory mechanism.