Identification of a Ligand-induced Transient Refractory Period in Nuclear Factor-κB Signaling

Abstract

In response to a variety of extracellular ligands, nuclear factor-B (NF-B) 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-B and its major regulator, inhibitor of NF-B␣ (IB␣), in real time, we utilized a novel transcriptionally coupled IB␣-firefly luciferase fusion reporter and characterized the dynamics and responsiveness of IB␣ processing upon a short 30-s pulse of tumor necrosis factor ␣ (TNF␣) or a continuous challenge of TNF␣ following a 30-s preconditioning pulse. Strikingly, a 30-s pulse of TNF␣ robustly activated inhibitor of NF-B kinase (IKK), leading to IB␣ degradation, NF-B nuclear translocation, and strong transcriptional up-regulation of IB␣. Furthermore, we identified a transient refractory period (lasting up to 120 min) following preconditioning, during which the cells were not able to fully degrade IB␣ upon a second TNF␣ 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 TNF␣ and interleukin-1␤ indicated that receptor dynamics could not explain this phenomenon. Utilizing a well accepted computational model of NF-B 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-B⅐IB␣ complexes represented another rate-limiting step that may impact this refractory period, thereby providing an additional regulatory mechanism.Adequate resolution of an inflammatory reaction is as equally important as initiation. Persistent or fulminant responses can cause detrimental consequences both locally and systemically (1), and resolution of inflammation is important for both termination of an acute response as well as for prevention of destructive chronic responses. It is therefore not surprising that mechanisms aimed at rapid and specific initiation of proinflammatory reactions have co-evolved with mechanisms that provide timely termination of such processes. From a systems biology perspective, such "switchability" can be achieved by intracellular feedback loops that permit ligand-induced desensitization and resensitization of proinflammatory signaling cascades (2).In this regard, recent studies have shown that nuclear factor-B (NF-B) 4 signaling plays a critical role in both initiation and resolution of inflammation (2, 3). The transcription factor NF-B is a key regulator of innate and adaptive immune responses as well as a mediator of cell survival and proliferation (4). Improper regulation of NF-B contributes to induction and progression of a wide range of human disorders, including a variety of pathological inflammatory conditions, neurodegenerative diseases as well as many types of cancer (5, 6). In resting cells...

Interrogation of Inhibitor of Nuclear Factor κB α/Nuclear Factor κB (IκBα/NF-κB) Negative Feedback Loop Dynamics

“…2A) rapidly decreased to a transient minimum (due to TNF␣-induced degradation of ⌱B␣) and then strongly rebounded above initial levels (due to NF-B-induced resynthesis of ⌱B␣). This rebound was previously shown to be consistent with de novo transcription and translation of ⌱B␣ (12) and with the previously reported ligand-induced stabilization of newly synthesized ⌱B␣ (31,32). A TNF␣ pulse as short as 5 s in duration was capable of inducing substantial ⌱B␣ degradation (35 Ϯ 9%, mean Ϯ S.E.…”

“…We hypothesized that many of these complex patterns were a consequence of the continuous presence of TNF␣ driving subsequent rounds of IKK activation and ⌱B␣ degradation during the resynthesis phase. This hypothesis was supported by our previous finding that HepG2 cells given a 30-s pulse of TNF␣ regain the capacity to fully re-initiate a second TNF␣-induced ⌱B␣ degradation only after a 60 -120-min refractory period, the approximate time frame during which maximal ⌱B␣ resynthesis and rollover occur (12).…”

“…Therefore, we utilized our previously validated B 5 3⌱B␣-FLuc reporter expressed at sub-endogenous levels in HepG2 (human hepatocellular carcinoma) cells to systematically evaluate the impact of short duration TNF␣ pulses on negative feedback loop dynamics within populations of cells in culture (Fig. 1, A and B) (12,26).…”

“…We then placed the fusion reporter under the control of an NF-B-responsive promoter (B 5 3 ⌱B␣-FLuc) and showed that it recapitulated the endogenous ⌱B␣ negative feedback loop (Fig. 1A) (12,13,27). The B 5 3⌱B␣-FLuc reporter offers the distinct advantage of monitoring protein activity within different subcellular compartments as opposed to simply measuring changes in total protein content or localization.…”

“…stimulus concentration, stimulus duration (pulse versus continuous), and pulse interval), which may be particularly relevant during cellular responses to inflammatory cytokines, such as TNF␣. Cytokines are likely perceived as transient pulses or waves occurring over a wide range of concentrations (12)(13)(14)(15)(16)(17). Thus, the NF-B pathway must rapidly decode different types of signal inputs and integrate intracellular information to control individual cell fate decisions (proliferation, apoptosis, differentiation, etc.)…”