The transcription factor nuclear factor-κB (NF-κB) regulates immunity, inflammation, and survival and is implicated in a variety of human diseases, including many types of cancer. A key regulatory node of this pathway is sequestration of NF-κB in the cytoplasm, through binding to members of the inhibitor of NF-κB family (IκB's); subsequent stimulus-induced activation of IκB Kinase (IKK), which phosphorylate IκB's and targets them for proteasomal destruction, frees NF-κB's to enter the nucleus and begin activating transcription of target genes. To directly monitor regulation of IKK activation in intact organisms, we designed and utilized an IκBα-firefly luciferase fusion reporter which, like endogenous IκBα, is degraded upon IKK activation, resulting in a rapid decrease in bioluminescence. This reporter has been used to image pharmacological inhibition of IKK activity in a mouse zenograft model (Figure 1). A second generation transcriptionally-coupled reporter (pκB5-IκBα-FLuc) was recently used (Figure 2) for real-time in cellulo studies of ligand-induced IKK activation and subsequent NF-κB-dependent upregulation of IκBα (which represents a critical negative feedback loop governing NF-κB activity).
Figure 1
Nature Methods 2005.
Figure 2
J Biol Chem 2008.
References:
Gross S, Piwnica-Worms D. Real-time imaging of ligand-induced IKK activation in intact cells and in living mice. Nature Methods 2005; 2(8): 607-14.
Moss BL, Gross S, Gammon ST, Vinjamoori A, Piwnica-Worms D. Identification of a ligand-induced transient refractory period in nuclear factor-kappaB signaling. J Biol Chem 2008; 283(13): 8687-98.
