An initial biological stimulus can induce intracellular signal oscillation, increasing the complexity and accuracy in the control of downstream targets [1]. Numerous studies have been focusing on the oscillation of second messengers, and the fact that different spiking patterns can trigger distinct activations of downstream pathways [2,3]. More recently, the oscillation in the localisation of transcription factors has been recognised as a new way regulating the specificity of gene expression, especially in response to signals arriving at irregular intervals.

Transcription factor NF-κB, regulates various gene expressions that are critical for stress response, cell growth, survival and apoptosis [4]. The binding of IκB prevents NF-κB translocating to nuclear and reduces its affinity to DNA. In response to tumour necrosis factor-α (TNF-α), IκB kinase (IKK) associates with NF-κB and IκB complex, promoting the degradation of IκB and the nuclear translocation of NF-κB. The activation of NF-κB facilitates expression of three IκB isoforms, IκBα, IκBβ, and IκBε, therefore forming a delayed negative feedback on NF-κB activity (Fig. 1).

Figure 1: The IκB-NF-κB signaling pathways. Figure taken from [5].

Figure 2: Experimental and computational analysis of NF-κB nuclear activity in genetically reduced systems. (A) Analysis of NF-κB nuclear level by EMSA of nuclear extracts of embryonic fibroblasts that contained only one IκB isoform, after persistently stimulation by TNF-α. Arrows indicate specific nuclear NF-κB binding activity; asterisks indicate nonspecific DNA binding complexes. (B) The quantification of NF-κB nuclear activity illustrated in (A). (C) The nuclear level of NF-κB obtained from stimulating the simplified computational models where only the indicated IκB isoform is expressed. Figure taken from [5].

Hoffmann et al. [5], showed that this delayed negative feedback can induce oscillation in NF-κB nuclear localisation, by using electrophoretic mobility shift assay (EMSA), western blotting and computational modelling.

By persistently stimulating the embryonic fibroblasts stem cells that only express one IκB isoform, with TNFα, they first showed that different IκB isoforms promote differed dynamic behaviours of NF-κB translocation. The isoform, IκBα, providing rapid negative feed back, causes oscillatory NF-kB nuclear activity; whereas IκBβ and IκBε stabilise its nuclear level (Fig. 2 A,B).

The authors then set up a two-compartment model, including cytoplasm and nucleus BIOMD0000000140. The association of IκB and NF-κB, the binding and IKK to the complex, the degradation of IκB, the translocation of IκB and NF-κB, and transcription and translation of IκB isoforms were all explicitly modelled. By setting the initial concentrations and transcription rates of two IκB isoforms to zero, three simplified models, each of which contains only one IκB isoform, could be derived (e.g. BIOMD0000000139).

Then the rates of transcription and translation of each IκB isoform was determined by fitting simulation results of one model to the corresponding experimental data introduced above (Fig. 2 C).

Simulation results from both computational model and the experiments indicate that NF-κB responses to sustained stimulation in two phases. In first phase, the concentration of NF-κB in nucleus reaches maximum within 30 minutes, then rapidly decays. The second phase appears after 2 hours, the nuclear NF-κB level increases, then slowly reduced to about half of the maximum and stabilises at this level. This damped oscillation is mostly regulated by the expression of IκBα, which provides strong negative feedback on NF-κB activity (Fig. 3 A,B).

Figure 3: Experimental and computational analysis of NF-κB nuclear activity in the complete system. (A) Analysis of NF-κB nuclear level by EMSA of nuclear extracts of embryonic fibroblasts that contained three IκB isoform, after persistently stimulation by TNF-α (top). Analysis of cytoplasmic expression level of IκB isoforms by western blotting (bottom). (B) Simulation results obtained from computational model for wild-type cells. Figure taken from [5]

Figure 4: Computational analysis of NF- κB nuclear activity by a transient stimulus. Analysis of NF- κB nuclear activity in response to stimuli of various durations. Figure taken from [5]

Figure 5: Computational analysis of NF-κB nuclear activity by stimulation of varying durations. Plot of the duration of above-threshold (20nM) NF- κB nuclear concentration, as a function of the duration of the stimulus. Figure taken from [5]

Authors further discovered that when a stimulus is shorter than 1 hour, the duration of NF-κB in the nuclear, is insensitive to the duration of the stimulus, although the maximum level of translocation does change (Fig. 4). However, when the stimulation is longer than 1 hour, the duration of NF-κB in nuclear is prolonged as well (Fig. 5). These simulation results indicate when the extracellular signal arrives at irregular intervals, NF-κB can activate different groups of genes. Some, for example the chemokine IP-10, can be regulated by transient NF-kB activation; while others, for example the chemokine gene RANTES, require at least 2 hours of stimulation (Fig. 6 A).

As stated previously, IκBα is responsible for negatively regulating NF-κB nuclear translocation. This prediction is verified by the fact that when stimulating cells lacking IκBα, a transient stimulation can induce sustained NF-κB nuclear activity, and trigger RANTES gene transcription (Fig. 6 B).

Therefore, different patterns of extracellular signal can be decoded into activation of distinct gene sets, by the oscillation of NF-κB nuclear activity. Furthermore, the dynamic behaviour of NF-κB activation is regulated by the expression level of different IκB isoforms, especially by IκBα. Thus, varying synthesis level of IκB isoforms may contribute to specificity in gene regulation. A mechanism can be used by cells to differentially response to environmental stimuli.

Figure 6: Experimental analysis of NF-κB nuclear activity and its qualitative effects on gene regulation. (A) NF-κB nuclear activity in response to persistent and transient stimuli in wild-type cells, by analysis of EMSAs. (B) NF-κB nuclear activity in response to persistent and transient stimuli in genetically modified cells without IκBα, by analysis of EMSAs. The transcription level of chemokine genes RANTES, IP-10 and housekeeping gene, GAPDH were monitored by ribonuclease protection assays. Figure taken from [5]