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Science 324 (5924): 242-246

Copyright © 2009 by the American Association for the Advancement of Science

Pulsatile Stimulation Determines Timing and Specificity of NF-{kappa}B-Dependent Transcription

Louise Ashall1*, Caroline A. Horton1*, David E. Nelson1*, Pawel Paszek1*, Claire V. Harper1, Kate Sillitoe1, Sheila Ryan1, David G. Spiller1, John F. Unitt2, David S. Broomhead3, Douglas B. Kell4, David A. Rand5, Violaine Sée1, and Michael R. H. White1{dagger}

1 Centre for Cell Imaging, School of Biological Sciences, Bioscience Research Building, Crown Street, Liverpool, L69 7ZB, UK.
2 Molecular Biology Department, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, LE11 5RH, UK.
3 School of Mathematics, The Alan Turing Building, The University of Manchester, Oxford Road, Manchester, M13 9PL, UK.
4 Manchester Centre for Integrative Systems Biology, School of Chemistry, and Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester, M1 7DN.
5 Warwick Systems Biology, Coventry House, University of Warwick, Coventry CV4 7AL, UK.

Figure 1 Fig. 1.. RelA oscillations at the single-cell level. (A and 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 differently colored lines. (B and D) The number of cells (separate experiments are 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 after TNF{alpha} stimulation. [View Larger Version of this Image (58K GIF file)]

Figure 2 Fig. 2.. Response of SK-N-AS cells to various TNF{alpha} pulse frequencies. (A) Time course of RelA-dsRedxp N:C ratio in transiently transfected cells pulsed three times with TNF{alpha} for 5 min at intervals of 60, 100, or 200 min (five 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 TNF{alpha}. Results were normalized to the amplitude of peak 1 (+SD). Asterisks indicate P values for a one-sample Wilcoxon test for peak amplitude equal to 1. (C) Western blot of Ser32 phospho-I{kappa}B{alpha} (p-I{kappa}B{alpha}), I{kappa}B{alpha}, Ser536 phospho-RelA (p-RelA), RelA, and cyclophilin A (cyclo A) amounts in cells stimulated with TNF{alpha} pulses 200 min apart. (D) Ratio of p-I{kappa}B{alpha}/total I{kappa}B{alpha} (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-{kappa}B signaling pathway showing IKK and the base module. (F and G) Computational analysis of existing (F) (21) and proposed (G) IKK structures. Heat maps [poor (red) to good (green)] represent the ability of the model to quantitatively fit the experimental data for a range of selected parameter values (table S5). A20 degradation rate (c4) was varied on a logarithmic scale two orders of magnitude above and below 0.0009 s–1. The best fit is highlighted and the corresponding simulated N:C ratio shown (F and G, bottom) for all TNF{alpha} stimulation conditions; c4 = 0.00143 s–1 in (F) and c4 = 0.0045 s–1 in (G). [View Larger Version of this Image (54K GIF file)]

Figure 3 Fig. 3.. Role of the I{kappa}B{epsilon} feedback loop. (A) Diagram of NF-{kappa}B signaling pathway, including three feedback mechansims. (B) Stochastic NF-{kappa}B–dependent regulation of I{kappa}B{alpha}, A20 (upper) and delayed I{kappa}B{epsilon} (lower) genes. (C) RelA and (D) RNA polymerase II DNA binding to the I{kappa}B{alpha} and I{kappa}B{epsilon} promoters after continuous TNF{alpha} stimulation by means of ChIP analysis. (E and F) Simulations of single-cell trajectories and the 100-cell average (black line) for (E) wild-type and (F) I{kappa}B{epsilon} knockdown conditions. (G and H) Time course of N:C ratio of RelA-dsRedxp in cells transiently transfected with RelA-dsRedxp and either (G) nonspecific or (H) I{kappa}B{epsilon} siRNA. The average population (nonspecific siRNA, n = 57 cells; I{kappa}B{epsilon} siRNA, n = 61 cells) response is shown by a black line. [View Larger Version of this Image (39K GIF file)]

Figure 4 Fig. 4.. Stimulation frequency determines differential gene expression. Cells were exposed to a single 5-min TNF{alpha} pulse or repeated pulses at 200-, 100-, or 60-min intervals or continuous treatment. (A) ChIP analysis of RelA-DNA binding at the I{kappa}B{alpha} promoter after a repeated TNF{alpha} pulse every 200 min. (B) Densitometric analysis of data with binding levels normalized to highest intensity. The dashed line represents the average peak 1 and peak 2 times, and the gray box shows±2 SD (from data in Fig. 2A). (C) Quantitative RT-PCR analysis of I{kappa}B{alpha}, I{kappa}B{epsilon}, MCP-1, and RANTES mRNA abundance in response to various TNF{alpha} stimulation frequencies. (D) Quantitative RT-PCR analysis of I{kappa}B{alpha}, I{kappa}B{epsilon}, MCP-1, and RANTES mRNA abundance in response to various frequencies of TNF{alpha} treatments. Amounts are expressed as percentages of continuous TNF{alpha} stimulation. [View Larger Version of this Image (39K GIF file)]

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