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.
- transcription factor
Abbreviations used: AcP, IL-1R accessory protein; BCA, bicinchoninic acid; EMSA, electrophoretic mobility shift assay; (E)GFP, (enhanced) green fluorescent protein; IL-1, interleukin-1; IL-1RI, type I IL-1 receptor; MKK, mitogen-activated protein kinase kinase; NF-κB, nuclear factor-κB; I-κB, inhibitor of NF-κB.
- The Biochemical Society, London ©2003