Real-time imaging of ligand-induced IKK activation in intact cells and in living mice.
The transcription factor NF-kappaB is a key regulator of cellular activation, proliferation and apoptosis. Defects in the NF-kappaB pathway contribute to a broad array of malignant, neurodegenerative and chronic inflammatory diseases. IKK-dependent IkappaB alpha degradation by the 26S proteasome is a critical NF-kappaB regulatory control point, which is emerging as an important target for drug development. To directly monitor regulation of IKK activation in intact organisms, we engineered an IkappaB alpha-firefly luciferase (IkappaB alpha-FLuc) fusion reporter. In cultured cells and living animals, the reporter provided a continuous, noninvasive readout of the kinetics of ligand-induced IKK activation and the pharmacodynamics of selective inhibitors of both IKK and the 26S proteasome. This IkappaB alpha-FLuc reporter now permits continuous readout of IKK activation in vivo, facilitates development and validation of target-specific therapeutics, and complements conventional NF-kappaB transcriptional reporters for more complete temporal and regional investigations of the NF-kappaB signaling pathway in health and disease.
Gross
Shimon
S
Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 S. Kingshighway Blvd., Box 8225, St. Louis, Missouri 63110, USA.
Piwnica-Worms
David
D
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003199:Computer Systems; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D049409:Luciferases, Firefly; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014018:Tissue Distribution; D035602:Transcriptional Elongation Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D002352:Carrier Proteins; C114720:Elp1 protein, human; D020672:I-kappa B Proteins; C491968:Ikbkap protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D011993:Recombinant Fusion Proteins; D035602:Transcriptional Elongation Factors; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D049409:Luciferases, Firefly