Abstract 4879: Real-time imaging of differential ligand-induced IKK activation and impact on the IκBα:NF-κB negative feedback loop within living cells and mice

Abstract

Abstract 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