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.