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J. Biol. Chem., Vol. 283, Issue 13, 8687-8698, March 28, 2008

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Identification of a Ligand-induced Transient Refractory Period in Nuclear Factor-B Signaling^*

Britney L. Moss¹², Shimon Gross¹, Seth T. Gammon, Anant Vinjamoori, and David Piwnica-Worms³

From the Molecular Imaging Center, Mallinckrodt Institute of Radiology, and the Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

In response to a variety of extracellular ligands, nuclear factor-B (NF-B) signaling regulates inflammation, cell proliferation, and apoptosis. It is likely that cells are not continuously exposed to stimulating ligands in vivo but rather experience transient pulses. To study the temporal regulation of NF-B and its major regulator, inhibitor of NF-B (IB), in real time, we utilized a novel transcriptionally coupled IB-firefly luciferase fusion reporter and characterized the dynamics and responsiveness of IB processing upon a short 30-s pulse of tumor necrosis factor (TNF) or a continuous challenge of TNF following a 30-s preconditioning pulse. Strikingly, a 30-s pulse of TNF robustly activated inhibitor of NF-B kinase (IKK), leading to IB degradation, NF-B nuclear translocation, and strong transcriptional up-regulation of IB. Furthermore, we identified a transient refractory period (lasting up to 120 min) following preconditioning, during which the cells were not able to fully degrade IB upon a second TNF challenge. Kinase assays of IKK activity revealed that regulation of IKK activity correlated in part with this transient refractory period. In contrast, experiments involving sequential exposure to TNF and interleukin-1β indicated that receptor dynamics could not explain this phenomenon. Utilizing a well accepted computational model of NF-B dynamics, we further identified an additional layer of regulation, downstream of IKK, that may govern the temporal capacity of cells to respond to a second proinflammatory insult. Overall, the data suggested that nuclear export of NF-B·IB complexes represented another rate-limiting step that may impact this refractory period, thereby providing an additional regulatory mechanism.

Received for publication, August 16, 2007 , and in revised form, January 8, 2008.

^* This work was supported in part by National Institutes of Health Molecular Imaging Center Grant P50 CA94056. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Supported in part by a National Science Foundation graduate research fellowship grant.

³ To whom correspondence should be addressed: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 S. Kingshighway Blvd., Box 8225, St. Louis, MO 63110. Tel.: 314-362-9359; Fax: 314-362-0152; E-mail: piwnica-wormsd{at}mir.wustl.edu.

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