Background: Understanding the biological significance of feedback loops requires interrogation at multiple scales. Results: A nuclear factor B (NF-B) negative feedback reporter revealed stimulus-specific dynamics in cells and animals in vivo. Conclusion: Circulating tumor necrosis factor ␣ (TNF␣) doses are perceived by the liver as pulses. Significance: Bioluminescent imaging of live single cells and cell populations revealed reproducible behaviors that informed interpretation of in vivo data.

The IKK (IκB Kinase) complex is a key regulator of the NF-κB signaling cascade and is a pharmacological target for cancer, autoimmune, and inflammatory therapies. IKK is a direct regulator of the IκBα:NF-κB negative feedback loop, a critical regulatory node within the NF-κB pathway. We have developed a systems-level reporter that utilizes bioluminescence imaging for real-time read-out of the dynamics of this negative feedback loop, enabling quantitative characterization of cell signaling in populations of live cells in vitro and in vivo. This novel transcriptionally-coupled IκBα-firefly luciferase fusion reporter (κB5→IκBα-FLuc), expressed in HepG2 cells and murine livers, monitors both IKK-induced degradation of IκBα and the subsequent NF-κB-driven re-synthesis of IκBα. Of particular interest was systematically evaluating the response of this negative feedback loop to subtle and/or drastic changes in ligand type, concentration, and exposure duration.To probe the effect of ligand type and concentration, HepG2 cells expressing κB5→IκBα-FLuc were stimulated with TNFα (0.57 – 570 pM) or IL-1β (0.06 – 60 pM) and the luciferase photon flux was followed for six hours. Increasing the concentration of either ligand increased the degree of degradation (EC50 = 6.7 pM for TNFα and 1.4 pM for IL-1β) and decreased the time required to achieve maximum degradation (from 53 to 29 min and 60 to 30 min, respectively). While higher concentrations of either ligand resulted in faster re-synthesis kinetics, IL-1β elicited a biphasic increase in IκBα re-synthesis amplitude and TNFα elicited increasing levels of IκBα re-synthesis up to a threshold (57 pM) beyond which higher amounts of TNFα actually elicited lower levels of re-synthesis (i.e. a “rollover” back down to 74 ± 3% of maximum levels). Concomitant modulation of TNFα concentration and duration revealed a prominent re-synthesis rollover when TNFα is given as a long pulse (> 15 min) that is less prominent or non-existent for short (30 sec to 10 min) pulses. Re-synthesis rollover was also observed for endogenous IκBα by Western blot analysis (although this technique reveals smaller changes in the IκBα levels) and within a computational model of the NF-κB pathway. Furthermore, in vivo somatic gene transfer of the κB5→IκBα-FLuc reporter into murine livers demonstrated TNFα dose-dependent increases in IκBα degradation and to a lesser extent re-synthesis, both with significantly faster kinetics than seen in vitro.In summation, a κB5→IκBα-FLuc reporter has revealed significant dynamic differences in the response of the IκBα:NF-κB negative feedback loop within populations of cells in vitro and in vivo to a variety of TNFα and IL-1β stimulation regimens, and represents a novel tool to qualitatively and quantitatively analyze regulation of negative feedback loops within complex biological signaling cascades. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4879. doi:10.1158/1538-7445.AM2011-4879

Cells have evolved complex molecular networks to sense environmental signals, transmit this information through the cell, and elicit appropriate biological responses. In particular, negative feedback loops represent a widely-utilized network motif capable of eliciting transient responses. To truly understand the biological significance of negative feedback processes, it is critical to study them at multiple scales: in single cells, in cell populations, and in animals. The IκBα:NF-κB negative feedback loop, a pivotal regulatory node of innate immunity and inflammation active in both immune cells and non-immune tissues, represents a model system for the use of multi-scalar reporter systems. To this end, we have utilized the κB5→IκBα-FLuc bioluminescent reporter to study dynamics of this transcriptionally-coupled negative feedback loop in response to diverse modes of stimulation which may be particularly relevant during cellular responses to inflammatory cytokines, such as TNFα. The κB5→IκBα-FLuc reporter enabled rigorous evaluation of the stimulus-specific dynamics of βκγα degradation and the downstream consequences of NF-κΔ nuclear translocation (i.e., NF-κΔ transcriptional activity) in single cells, cell populations and live animals in vivo. In response to modulation of TNFα concentration and pulse duration, complex, differential patterns in βκγα degradation and re-synthesis were discovered in both cell populations and single cells. Furthermore, IκBα dynamics observed in live animals in vivo upon modulation of TNFα dose strongly resembled those observed in single cells and cell populations upon modulating TNFα pulse duration, suggesting that increased doses of circulating TNFα were perceived by hepatocytes in vivo as pulses of increasing duration. Thus, a single bioluminescent reporter strategy enabled correlative quantitation of dynamic NF-κα:βκβα negative feedback loop responses in live single cells, cell populations, and tissues in vivo with a variety of rapid, low-cost, high-throughput approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-419. doi:1538-7445.AM2012-LB-419