Abstract
Simple SummaryIn stress and disease situations, cells must rapidly and in a coordinated manner change their gene expression patterns to respond adequately. The NF-κB system comprises five transcription factors that are released from the cytosol to enter the nucleus in response to a wide range of extracellular stimuli via a complex cytosolic signaling system. In the nucleus, activated NF-κB dimers bind to specific chromatin loci across the entire genome and induce the expression of a broad repertoire of genes that regulate immune and inflammatory responses. Consistent with its biological importance, the extent of NF-κB activity is regulated and controlled at multiple levels. The aim of this review is to comprehensively present and discuss the currently available conceptual and methodological approaches to monitor the molecular activation status of the NF-κB system, including multi-level single cell analysis.The NF-κB signaling system plays an important regulatory role in the control of many biological processes. The activities of NF-κB signaling networks and the expression of their target genes are frequently elevated in pathophysiological situations including inflammation, infection, and cancer. In these conditions, the outcome of NF-κB activity can vary according to (i) differential activation states, (ii) the pattern of genomic recruitment of the NF-κB subunits, and (iii) cellular heterogeneity. Additionally, the cytosolic NF-κB activation steps leading to the liberation of DNA-binding dimers need to be distinguished from the less understood nuclear pathways that are ultimately responsible for NF-κB target gene specificity. This raises the need to more precisely determine the NF-κB activation status not only for the purpose of basic research, but also in (future) clinical applications. Here we review a compendium of different methods that have been developed to assess the NF-κB activation status in vitro and in vivo. We also discuss recent advances that allow the assessment of several NF-κB features simultaneously at the single cell level.
Highlights
The NF-κB pathway has been discovered 36 years ago and its broad role in diversephysiological responses has sparked a tremendous and ongoing interest to determine its activation status in cells from healthy or diseased individuals [1,2]
Cells with constitutively active NF-κB such as B cells are characterized by elevated turnover of endogenous IκBα and IκBß, a feature that can be measured by determining their quantity at various time points after inhibition of de novo protein synthesis [66]
The inducible nuclear translocation of NF-κB subunits can be readily measured by a number of techniques including subcellular fractionation, where DNA-binding subunits are detected in the soluble nuclear fraction and to a minor extent in the insoluble chromatin fraction (Figure 3D)
Summary
The NF-κB pathway has been discovered 36 years ago and its broad role in diverse (patho)physiological responses has sparked a tremendous and ongoing interest to determine its activation status in cells from healthy or diseased individuals [1,2]. The NF-κB system plays an important role in multiple processes that require the induction of appropriate gene responses in order to cope with all types of stressful conditions, but some tissues or cell types use this pathway for constitutive functions [3]. In this review, based on many years of experimental work in the Schmitz and Kracht laboratories, we attempt to give a comprehensive overview on methods employed to study the NF-κB pathway and discuss some of their intrinsic and conceptual limitations. We use published and some unpublished preliminary data to illustrate the outcome of typical experiments and briefly discuss the advantages or disadvantages of individual assays used in the field to monitor NF-κB activity
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