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Real-time imaging of ligand-induced IKK activation in intact cells and in living mice

Nature Methods volume 2, pages 607–614 (2005)Cite this article

Abstract

The transcription factor NF-κB is a key regulator of cellular activation, proliferation and apoptosis. Defects in the NF-κB pathway contribute to a broad array of malignant, neurodegenerative and chronic inflammatory diseases. IKK-dependent IκBα degradation by the 26S proteasome is a critical NF-κB 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 IκBα–firefly luciferase (IκBα-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 IκBα-FLuc reporter now permits continuous readout of IKK activation in vivo, facilitates development and validation of target-specific therapeutics, and complements conventional NF-κB transcriptional reporters for more complete temporal and regional investigations of the NF-κB signaling pathway in health and disease.

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Figure 1: Schematic representation of stimulus-induced degradation of IκBα-FLuc.
Figure 2: Validation of IκBα-FLuc functionality in vitro.
Figure 3: Characterization of steady-state turnover of the IκBα-FLuc reporter.
Figure 4: Time- and concentration-dependent effects of various NF-κB modulators on TNFα-induced IKK activation and NF-kB transactivation.
Figure 5: Imaging pharmacological modulation of LPS-induced IKK activation in living mice.
Figure 6: Real-time imaging of IKK inhibition in tumors of PS-1145-treated mice.

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Acknowledgements

The authors thank S. Gammon for insightful discussions and help with statistical analyses. Funded by National Institutes of Health grant P50 CA94056.

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Authors and Affiliations

  1. Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 S. Kingshighway Blvd., Box 8225, St. Louis, 63110, Missouri, USA

    Shimon Gross & David Piwnica-Worms

  2. Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 510 S. Kingshighway Blvd., Box 8225, St. Louis, 63110, Missouri, USA

    David Piwnica-Worms

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  1. Shimon Gross
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Correspondence to David Piwnica-Worms.

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Gross, S., Piwnica-Worms, D. Real-time imaging of ligand-induced IKK activation in intact cells and in living mice. Nat Methods 2, 607–614 (2005). https://doi.org/10.1038/nmeth779

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