Abstract LB-419: Multi-scalar approaches to interrogate the IκBα:NF-κB negative feedback loop: Quantitative dynamics in single cells, cell populations, and live animals in vivo

Abstract

Abstract 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