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. 2009 Apr 10;324(5924):242-6.
doi: 10.1126/science.1164860.

Pulsatile stimulation determines timing and specificity of NF-kappaB-dependent transcription

Louise Ashall  1 , Caroline A Horton, David E Nelson, Pawel Paszek, Claire V Harper, Kate Sillitoe, Sheila Ryan, David G Spiller, John F Unitt, David S Broomhead, Douglas B Kell, David A Rand, Violaine Sée, Michael R H White
Affiliations

Pulsatile stimulation determines timing and specificity of NF-kappaB-dependent transcription

Louise Ashall et al. Science. .

Abstract

The nuclear factor kappaB (NF-kappaB) transcription factor regulates cellular stress responses and the immune response to infection. NF-kappaB activation results in oscillations in nuclear NF-kappaB abundance. To define the function of these oscillations, we treated cells with repeated short pulses of tumor necrosis factor-alpha at various intervals to mimic pulsatile inflammatory signals. At all pulse intervals that were analyzed, we observed synchronous cycles of NF-kappaB nuclear translocation. Lower frequency stimulations gave repeated full-amplitude translocations, whereas higher frequency pulses gave reduced translocation, indicating a failure to reset. Deterministic and stochastic mathematical models predicted how negative feedback loops regulate both the resetting of the system and cellular heterogeneity. Altering the stimulation intervals gave different patterns of NF-kappaB-dependent gene expression, which supports the idea that oscillation frequency has a functional role.

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Figures

Fig. 1
Fig. 1. RelA oscillations at the single cell level
(A & C) Time-course of the ratio of nuclear to cytoplasmic localization (N:C) of RelA-dsRedxp in (A) SK-N-AS (stably transfected) and (C) MEF (transiently transfected) cells. Single cell dynamics are shown by different colored lines. (B & D) The number of cells (separate experiments represented by different colors) showing RelA-dsRedxp translocations in (B) stably transfected SK-N-AS (imaged for at least 350-min; 0 cells failed to respond) and (D) transiently transfected MEF cells (imaged for at least 250 min; 1 cell failed to respond). (E) Time-lapse confocal images of a typical RelA-dsRedxp-transfected MEF cell following TNFα stimulation.
Fig. 2
Fig. 2. Response of SK-N-AS cells to various TNFα pulse frequencies
(A) Time-course of RelA-dsRedxp N:C ratio in transiently transfected cells pulsed 3 times with TNFα for 5-min at intervals of 60-, 100- or 200-min (5 typical cells shown for each). RelA-dsRedxp N:C ratio was normalized to peak 1 intensity. (B) Amplitude of successive peaks of RelA-dsRedxp localization after pulses or continuous exposure of cells to TNFα. Results were normalized to the amplitude of peak 1 (+SD). Asterisks: p-values for 1-sample Wilcoxon test for mean peak amplitude=1, vs. ≠ alternative. (C) Western blot of Ser32 phospho-IκBα (p-IκBα), IκBα, Ser536 phospho-RelA (p-RelA), RelA, and cyclophilin A (cyclo A) amounts in cells stimulated with TNFα pulses 200-min apart. (D) Ratio of p-IκBα/total IκBα (relative to that recorded at t=5 min) in cells stimulated 60-, 100- and 200-min apart (+SD) (data based on (C), Fig. S4). p1 and p2 indicate time after pulse 1 or 2 for each stimulation protocol. (E) Two-feedback NF-κB signaling pathway showing IKK and Base Module. (F & G) Computational analysis of existing (F) (27) and proposed (G) IKK structures. Heat maps (red-poor to green-good) represent the ability of the model to quantitatively fit the experimental data for a range of selected parameter values (see Table S5). A20 degradation rate (c4) was varied on a logarithmic scale two orders of magnitude above/below 0.0009s−1. The best fit is highlighted, and corresponding simulated N:C ratio shown (F & G, bottom) for all TNFα stimulation conditions, (F) c4=0.00143s−1, or (G) c4=0.0045s−1
Fig. 3
Fig. 3. Role of the IκBε feedback loop
(A) Diagram of NF-κB signaling pathway including three feedback mechansims. (B) Stochastic NF-κB-dependent regulation of IκBα, A20 (upper) and delayed IκBε (lower) genes. (C) RelA and (D) RNA polymerase II DNA binding to the IκBα and IκBε promoters after continuous TNFα stimulation by ChIP analysis. (E & F) Simulations of single cell trajectories and the 100-cell average (black line) for (E) wild-type and (F) IκBε knock-down conditions. (G & H) Time-course of N:C ratio of RelA-dsRedxp in cells transiently transfected with RelA-dsRedxp and either (G) non-specific or (H) IκBε siRNA. The average population (non-specific siRNA n=57, IκBε siRNA n=61) response is shown by a black line.
Fig. 4
Fig. 4. Stimulation frequency determines differential gene expression
Cells were exposed to a single 5 min TNFα pulse or repeated pulses at 200-, 100-, or 60-min intervals or continuous treatment. (A) ChIP analysis of RelA-DNA binding at the IκBα promoter after a repeated TNFα pulse every 200 min. (B) Densitometric analysis of data with binding levels normalized to highest intensity. Dashed line represents the average peak 1 and peak 2 times with the grey box showing ± 2 SD (from data in Fig. 2A). (C) qRT-PCR analysis of IκBα, IκBε, MCP-1 and RANTES mRNA abundance in response to various TNFα stimulation frequencies. (D) qRT-PCR analysis of IκBα, IκBε, MCP-1 and RANTES mRNA abundance in response to various frequencies of TNFα treatments. Amounts are expressed as percentages of continuous TNFα stimulation.
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