We have analysed activation of nuclear factor-κB (NF-κB) in response to interleukin-1 (IL-1) in human fibroblasts by tracking intracellular distribution and levels of endogenous relA, NF-κB1 and inhibitor of κB (I-κB) α using semi-quantitative confocal microscopy. Nuclear translocation of endogenous relA correlated with I-κBα degradation during stimulation with IL-1, whereas no effects were seen on levels or localization of NF-κB1. During pathway activation, relA was transported up a concentration gradient, resulting in a 3—4-fold increase in nuclear levels, but without any significant decrease in cytoplasmic concentration. IL-1 stimulation caused translocation of only 20% of the relA, but resulted in degradation of up to 70% of the cytoplasmic I-κBα. RelA nuclear translocation in fibroblasts correlated with DNA-binding activity measured by electrophoretic mobility shift assay (EMSA), both with respect to kinetics and IL-1 concentration-dependence. Clonal populations of cells demonstrated a marked degree of heterogeneity in the response to IL-1. The single-cell assay revealed the presence of responder and non-responder subpopulations, with an enhanced proportion of responder cells, and prolonged responses at higher concentrations of IL-1. Comparing different cell types demonstrated that whereas HepG2 cells, as fibroblasts, showed good correlation between nuclear translocation of relA and activation of DNA binding by relA-containing dimers, EL4 thymoma cells showed no effect on relA localization, even during induction of significant levels NF-κB activity, as measured by EMSA. The analysis shows that stimulation by IL-1 results in transient perturbation of the NF-κB system, which cycles between the resting and active states with net redistribution of a minor proportion of its DNA-binding component. In addition, it demonstrates significant cell-to-cell variations, as well as cell-type-specific differences in net relA nuclear transport in response to stimuli. The data are consistent with NF-κB constituting a dynamic and versatile system, regulated to a significant degree by binary events involving bidirectional trafficking between the cytoplasmic and nuclear compartments during pathway activation.
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January 2003
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Research Article|
January 15 2003
Regulation of nuclear translocation of nuclear factor-kappaB relA: evidence for complex dynamics at the single-cell level
Kenneth SCHOOLEY;
Kenneth SCHOOLEY
∗Department of Biochemistry, Immunex Research and Development Corporation, 51 University Street, Seattle, Washington 98101, U.S.A.
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Ping ZHU;
Ping ZHU
†Department of Pathology, University of Washington, Seattle, Washington 98101, U.S.A.,
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Steven K. DOWER;
Steven K. DOWER
‡Division of Genomic Medicine, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield S10 2JF, U.K.
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Eva E. QWARNSTRÖM
Eva E. QWARNSTRÖM
1
†Department of Pathology, University of Washington, Seattle, Washington 98101, U.S.A.,
1To whom correspondence should be addressed (e-mail [email protected]).
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Publisher: Portland Press Ltd
Received:
September 05 2002
Revision Received:
September 27 2002
Accepted:
October 01 2002
Accepted Manuscript online:
October 01 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 369 (2): 331–339.
Article history
Received:
September 05 2002
Revision Received:
September 27 2002
Accepted:
October 01 2002
Accepted Manuscript online:
October 01 2002
Citation
Kenneth SCHOOLEY, Ping ZHU, Steven K. DOWER, Eva E. QWARNSTRÖM; Regulation of nuclear translocation of nuclear factor-kappaB relA: evidence for complex dynamics at the single-cell level. Biochem J 15 January 2003; 369 (2): 331–339. doi: https://doi.org/10.1042/bj20020253
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