A TNF-induced gene expression program under oscillatory NF-kappaB control

Abstract

Background: The cytokine tumor necrosis factor (TNF) initiates tissue inflammation, a process mediated by the NF-kappaB transcription factor. In response to TNF, latent cytoplasmic NF-kappaB is activated, enters the nucleus, and induces expression of inflammatory and anti-apoptotic gene expression programs. Recently it has been shown that NF-kappaB displays two distinct activation modes, monophasic and oscillatory, depending on stimulus duration. Characterization of temporal expression patterns for the NF-kappaB network and determination of those genes under monophasic- or oscillatory control has not been experimentally addressed.

Results: To identify the kinetics of NF-kappaB-dependent gene expression and determine whether these two types of NF-kappaB translocation modes control distinct gene programs, a detailed kinetic analysis of a validated microarray data set was performed on 74 unique NF-kappaB-dependent genes in response to TNF. Hierarchical clustering identified distinct expression profiles termed the "Early", "Middle", "Late" response groups, peaking 1, 3, and 6 h after stimulation, respectively. These expression patterns were validated by Quantitative Real Time PCR (Q-RT-PCR) and NF-kappaB binding was demonstrated by chromatin immunoprecipitation (ChIP) assays. Each response group was mapped to its molecular function; this analysis indicated that the Early group encodes cytokines or negative regulators of the IKK-NF-kappaB pathway, and the Late group encodes cell surface receptors, adhesion molecules and signal adapters. That similar coordinated sequential cascades of gene expression were also seen in response to stimulation by the cytokine IL-1, and expression patterns observed in MRC-5 fibroblasts indicated that the epithelial NF-kappaB program is relatively stimulus- and cell type-independent. Bioinformatic analysis of the Early and Late gene promoters indicates that although both groups contain similar patterns of NF-kappaB-binding sites, only the Early gene promoters contain NF-kappaB-binding sites located in phylogenetically conserved domains. Stimulation protocols designed to produce either monophasic or oscillatory NF-kappaB activation modes showed that the oscillatory mode is required only for expression of the Late genes.

Conclusion: This analysis provides important insights into the TNF-regulated genetic response program in epithelial cells, where NF-kappaB controls sequential expression patterns of functionally distinct genes that depend on its oscillatory activation mode.

Figure 1

Schematic diagram of microarray data analysis. HeLa^{TetO-FLAG-IκBα Mut} cells were plated in parallel into cultures in the absence or presence of Dox (2 μg/ml). After four days, cells were stimulated without (0 h) or with rhTNFα (25 ng/ml) at 6 h, 3 h, and 1 h prior to simultaneous harvest for RNA extraction. Experiments were conducted four independent times. Data sets were scaled for comparison. NF-κB dependent genes were identified using 2-way ANOVA where Dox treatment and TNF treatment were considered independent variables. Those changed by Dox treatment at a p-values [Pr(F)< 0.01] were then filtered for 3-fold change at any point during the experiment (signal intensity with NF-κB vs signal intensity without NF-κB).

Figure 2

Temporal Cascades of NF-κB Regulated Genes. (a) The Signal Intensity values from 74 probe sets identified as being NF-κB dependent were Z-score normalized and subjected to hierarchical clustering. Red corresponds Z > +2.5, green indicates Z < 0, and black indicates Z > 0.5. Expression groups are indicated at right by vertical line. (b) Distinct Expression Profiles. The normalized SI measurements for each of the genes in Clusters I–IV are presented as a percentage of the maximum value for any point across the stimulation.

Figure 3

Validation of expression profiles and NF-κB dependence. (a) Early gene profiles. HeLa^{tTA/FLAG-IκBα Mut} cells were plated in parallel in the absence or presence of Dox (2 μg/ml) and stimulated with rhTNFα. Changes in mRNA abundance (normalized by 18S) determined by Q-RT- PCR from total RNA. For each of the indicated mRNA transcripts, values are expressed as fold change relative to unstimulated cells and plotted on a logarithmic scale. +/-Dox, data obtained from cells cultured with or without Dox. (b) Late gene profiles. Experiment and data analysis are as in Figure 3a. (c) ChIP for NF-κB subunit binding to Early Gene promoters. ChIP was performed on control or TNFα-stimulated (30 min, 20 ng/ml) HeLa cells using the antibodies indicated at left. Shown is an ethidium-bromide stained agarose gel of the PCR products performed under linear amplification conditions. The target gene is indicated at the bottom. NC, negative control reaction (no template is added to the PCR reaction); PC, positive control reaction (25 ng of genomic DNA is used as template in PCR). (d) ChIP for NF-κB subunit binding to Late Gene promoters. ChIP was performed on HeLa cells stimulated as in Figure 3c.

Figure 4

Ingenuity Pathway Analysis of biological pathways controlled by Early and Late genes. (a) Early gene pathway. Shown is a graphical representation of the highest scoring pathway controlled by the genes in Cluster III. Shown are labeled nodes representing individual protein functions and their relationship represented by edges. Nodes are colored by changes in expression, with red indicating > 10 fold change; pink > 2-fold and < = 10-fold change; no color indicating < = 2-fold change or data is not present. Squares indicate cytokines, circles indicate chemokines, and ovals indicate transcription factor. For the edges, an arrow indicates "acts on". Horizontal lines indicate the most likely subcellular location for the protein encoded by each node. See Legend to Table II for the index of relevant abbreviations. (b) Late gene pathway. Graphical representation of the highest scoring pathway controlled by the genes in Cluster III. See Fig. 2A for explanation of figure and symbols.

Figure 5

(a) IL-1 induces sequential cascades of NF-κB dependent gene expression. HeLa^{tTA/FLAG-IκBα Mut} cells were plated in parallel in the absence or presence of Dox (2 μg/ml) and stimulated with IL-1α. Changes in mRNA abundance (normalized by 18S) was then determined by Q-RT- PCR from total RNA. Shown is a Z-score representation, where red corresponds to Z > +2.5, green indicates Z < 0, and black indicates Z > 0.5. The common name of each gene is indicated at right. (b) TNF sequential cascades of NF-κB dependent gene expression in MRC-5 fibroblasts. Human MRC-5 fibroblasts were stimulated for the times indicated at top with TNFα (20 ng/ml) and RNA extracted. Shown is a northern blot hybridization of 20 μg RNA using probes specific to IL-8 (top) and Naf-1 (bottom). Asterix indicates apparent plateau of gene expression.

Figure 6

(a) Hierarchical clustering of high-affinity NF-κB DNA-binding sites. The probability over 100 bp intervals for finding a high-affinity NF-κB-binding site was used for hierarchical clustering (data from Table I) of the early and late NF-κB dependent gene promoters. Data is shown as a heat map, where green = 0, red = 1. The common name of each gene is shown at right. Note that there is no separation of early and late gene promoters based on the pattern or location of the NF-κB-binding sites. (b) Co-occurrence of high-affinity NF-κB- and AP-1 DNA-binding sites. Superimposed on the NF-κB binding site analysis is the presence and location of high-affinity AP-1 DNA-binding sites. The location of each AP-1 DNA-binding site is indicated in black.

Figure 7

Phylogenetic analysis of NF-κB dependent promoters. (a) Early gene promoters. Promoters spanning from -1000 bp to the first nontranslated exon were aligned between human and mouse genes. Shown are the VISA identity curves [49]. For each curve, the percent sequence conservation is plotted over a sliding 20 base pair window (from 0–100% identity). Shaded regions indicated significant regions of sequence conservation. The location of NF-κB-binding sites within these conserved domains are displayed at top (location indicated by I). The presence of AP-1 sites is indicated by green asterix (*). (b) Late gene promoters. For each late gene promoter indicated, analysis as in 7a.

Figure 8

Late gene expression requires the NF-κB oscillatory mode. (a) Experimental Strategy. Schematic diagram of the tonic and pulse stimulation paradigm. Parallel plates of cells were stimulated with TNF continuously ("tonic" treatment), without removing the agonist. Pulse stimulated cells were exposed to TNF to activate the NF-κB pathway (activation is maximal within 15 min of stimulation), whereupon the agonist is removed from the medium. At identical times after application of the stimulus, cells are harvested for gel shift (Figure 8b) or Q-RT-PCR (Figures 8c, d). (b) NF-κB-binding in tonic- vs pulse-stimulated cells. Nuclear extracts from tonic- or pulse stimulated HeLa cells were prepared and NF-κB-binding measured. Shown is an autoradiogram of the bound NF-κB complexes by EMSA. The specific NF-κB/Rel A and NF-κB1 complexes previously identified by supershift analyses are indicated at left (see Ref [21] for further details). (c) IκB proteolysis and resynthesis in tonic- vs pulse-stimulated cells. Cytoplasmic extracts from tonic- or pulse stimulated HeLa cells were prepared and abundance of IκB determined by Western blot. IκB is rapidly proteolyzed, with both treatments, however, the steady state levels are reduced 3 and 6 h in tonic treated cells compared to those pulse-treated. (d) Early gene expression profiles. HeLa cells were treated as in Figure 8a, total RNA extracted and mRNA abundance (normalized by 18S) determined by Q-RT- PCR. For each of the indicated mRNA transcripts, values are expressed as fold change relative to unstimulated cells and plotted on a logarithmic scale. (e) Late gene expression profiles. Samples obtained as in Figure 8d. The mRNA transcript measured is indicated for each plot.