Regulation of nuclear translocation of nuclear factor-kappaB relA: evidence for complex dynamics at the single-cell level.
We have analysed activation of nuclear factor-kappaB (NF-kappaB) in response to interleukin-1 (IL-1) in human fibroblasts by tracking intracellular distribution and levels of endogenous relA, NF-kappaB1 and inhibitor of kappaB (I-kappaB) alpha using semi-quantitative confocal microscopy. Nuclear translocation of endogenous relA correlated with I-kappaBalpha degradation during stimulation with IL-1, whereas no effects were seen on levels or localization of NF-kappaB1. 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-kappaBalpha. 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-kappaB activity, as measured by EMSA. The analysis shows that stimulation by IL-1 results in transient perturbation of the NF-kappaB 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-kappaB 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.
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007375:Interleukin-1 / Q000378:metabolism*; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D017472:Receptors, Interleukin-1 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D051996:Transcription Factor RelA
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D007375:Interleukin-1; D016328:NF-kappa B; D017472:Receptors, Interleukin-1; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA