Quantitative assessment of NFκB transcription factor activity

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Abstract

The Nuclear Factor Kappa B (NFκB) pathway is an important signalling pathway in the immune system. Single gene defects in the NFκB pathway are described in a number of immunodeficiency diseases. These conditions provide a unique opportunity to investigate the mechanisms of NFκB function and how genetic mutations that disrupt this function lead to human disease. Here we describe a robust method for quantifying small differences in the functional activity of the NFκB pathway.

Peripheral blood mononuclear cells from healthy donors were stimulated over several days, with a combination of anti-IgM antibody and multimeric CD40 ligand. Nuclear proteins were thereafter extracted and tested for the ability of activated transcription factors, to bind known NFκB DNA binding motifs.

Repeatability experiments showed that the DNA binding Activity can be quantified with an average inter and intra assay coefficient of variation of less than 10% (RelB and p52) and less than 15% (p50 and RelA). In healthy individuals there is a significant increase in the DNA binding activity of NFκB transcription factors in response to stimulation, although the magnitude of this response varies across individuals. The kinetics of the DNA binding activity also differs between the canonical and non-canonical transcription factors. P50 and RelA DNA binding activity responds within hours of stimulation, whilst RelB and p52 response was delayed to more than a day after stimulation.

Activation of NFκB signalling in response to B cell specific stimulation, can be precisely measured to distinguish individuals with differences in the functional activity of this pathway. This test may prove to be an important biomarker for investigating the functional impact of genetic variants on NFκB signalling.

Introduction

Nuclear factor kappa B (NFκB) represents a protein complex of inducible transcription factors that form homo and hetero dimers, which bind DNA differentially at kappa B enhancer sites (Hoffmann et al., 2003). NFκB transcription factors, RelA, RelB, c-Rel, NFKB1/p50 and NFKB2/p52 are structurally similar. They contain an N-terminal Rel homology domain, essential for dimerization and binding to cognate DNA elements. NFκB transcription factors are regulated through canonical and non-canonical pathways (Hayden and Ghosh, 2008). Both activation pathways control NFκB activity through the degradation of ‘inhibitor of kappa B’ (IκB) proteins. In the steady state, RelA, RelB and c-Rel are sequestered in the cytoplasm where their DNA binding domain is associated with ankyrin repeat regions of IκB proteins, which render them transcriptionally inactive. Processing and cleavage of similar ankyrin repeat regions in NFκB precursor proteins, NFKB1/p105 and NFKB2/p100, results in release of p50 and p52 proteins (Hoffmann et al., 2006).

Canonical and non-canonical pathways differ in their response to stimulation, due to the variation in receptor and adaptor molecules, critical kinases, IκB proteins and the transcription factors activated. The canonical pathway responds rapidly to a wide and diverse range of stimuli, including cytokines, growth factors, mitogens, microbial components and stress factors. Stimulation through this pathway typically leads to a cascade of signalling events that culminate in the ubiquitination and proteasomal degradation of inhibitory proteins. This releases canonical NFκB heterodimers, predominantly RelA/p50 and p50/c-Rel dimers, which translocate to the nucleus for transcription of response genes (Shih et al., 2011; Beinke and Ley, 2004).

Activation of the non-canonical pathway however, is slow, persistent, and tightly regulated through specific receptors (Almaden et al., 2016). Activation through this pathway leads to the recruitment of adaptor molecules, which stabilise and allow accumulation of the NFκB inducing kinase (NIK) and its downstream kinase, IκB Kinase α (IKKα). IKKα induces proteasomal processing of the NFκB precursor protein, p100 to p52. Typically, p100 is associated with RelB, so proteasomal possessing induces the formation of p52/RelB dimers, which translocate to the nucleus where they can activate an array of target genes (Sun, 2010; Solan et al., 2002).

The NFκB pathway regulates the expression of a broad range of genes involved in immune responses and homeostasis (Pahl, 1999). This includes acute phase proteins, adhesion molecules, cytokines and chemokines, receptors of both innate and adaptive immunity, enzymes, anti-apoptotic factors, cell cycle regulators and other transcription factors (Werner et al., 2005; Liu et al., 2017a, Liu et al., 2017b). This ability to regulate the expression of multiple genes makes abnormal NFκB function and/or control, an important contributor to a wide spectrum of human diseases (Fraser, 2006).

An important adjunct to studies of the mechanisms by which genetic defects of NFκB cause human disease, are functional tests of the pathway. Several methods are widely used to assess NFκB function. Western blot is commonly used to detect phosphorylated NFκB proteins in nuclear extracts as well as IκB degradation (Maguire et al., 2011). The electrophoretic mobility shift assay (EMSA) measures the DNA binding capacity of NFκB proteins through the use of radiolabelled oligonucleotides containing the consensus NFκB binding site. These are resolved by polyacrylamide gel electrophoresis and autoradiography (Hellman and M, 2007). Luciferase and β-galactosidase reporter gene promoter assays measure the ability of NFκB proteins to derive a reporter gene expression through NFκB sensitive promoters (Badr et al., 2009; Matsuda et al., 2007). Cell lines that stably express NFκB subunits, fused to green florescent protein (GFP) are used to quantify nuclear translocation of NFκB proteins to the nucleus (Bartfeld et al., 2010).

These tests suffer certain deficiencies. Western blot does not measure the DNA binding activity of NFκB proteins. Phosphorylation of RelA, the most studied NFκB subunit, is merely thought to be correlated with activity. Whilst IκB degradation does not provide information about whether NFκB is nuclear or able to bind DNA. Moreover, nuclear abundance measurements of NFκB proteins do not provide information about the NFκB dimers. The luciferase/β-galactosidase assays require cells to be transfected, transduced or genetically modified with a reporter and does not provide information about the specific dimer. Whereas cell lines with fluorescent proteins do not provide direct quantitation of downstream NFκB activity. The EMSA provides the most direct measurement of DNA binding activity present in the cell or nucleus and provides information about the specific dimer involved. However, the EMSA usually requires highly radioactive reagents.

Transcription factor DNA binding assays, based on the enzyme linked immunosorbent assay (ELISA) technique, are more sensitive and scalable than gel shift assays. Although they do not provide information on specific dimers binding, they provide quantitative results on the DNA binding activity of NFκB transcription factors within hours. The DNA binding complex of NFκB proteins recognises a consensus nucleotide sequence, -GGGRNWYYCC- (N is any base; R is a purine; W is an adenine or thymine; and Y is a pyrimidine), termed κB, found in the enhancer or promoter regions of target genes (Hoffmann and D., 2006). ELISA-based methods use the κB consensus oligonucleotide sequence, immobilised on plastic plates, as a substrate for binding activated transcription factors. Primary antibodies detect specific epitopes on active bound NFκB subunits, which themselves are detected by secondary conjugated antibodies. Reactions are developed via chromogenic or chemiluminescent substrates to produce semi-quantitative readouts.

The TransAM transcription factor assay (Active Motif, Belgium), was the first of these ELISA-based methods that measure NFκB activity to be developed. It uses HRP-conjugated antibodies and a chromogenic substrate to produce a sensitive colorimetric readout that can easily be quantified by spectrophotometry. Here we describe how this was further optimised and validated to produce a functional assay that accurately and reliably quantifies small differences in the DNA binding activity of the NFκB transcription factors in response to stimulation.

Section snippets

Setting up a standard curve

Transforming the TransAM NFκB Activation Assay from a semi-quantitative method to a robust quantitative assay required stable standard calibrators with known transcription factor binding activity. The DNA binding activity in this method is calibrated against the reference response of recombinant human NFκB transcription factors (Active Motif, Belgium; SignalChem, Canada).

The lowest limit of quantitation (LLOQ) was determined as the calibration standard that produced values 10 standard

Discussion

We have optimised the TransAM NFκB activation assay (TAMNA) to be a robust method for accurately quantifying the DNA binding activity of NFκB transcription factors. The TAMNA assay can be used to accurately quantify NFκB activity across samples, individuals or experiments. This ensures the method is not only useful for paired functional analysis in NFκB research but when clinically validated, would makes it applicable as a functional diagnostic tool to evaluate the response of the pathway,

Sample collection

Whole blood samples were taken from the healthy population of volunteers at King's College Hospital with informed consent, according to the study protocol. This protocol was approved by a National Research Ethics Committee (16/LO/2130) and the Health Research Authority. This study was also approved by the King's College hospital NHS Trust Research and Innovations division. Large pools of leukocytes were procured in cones from the NHS Blood and Transplant Service according to their approved

Declaration of Competing Interest

None.

Acknowledgements

This project is supported by King's College Hospital, Viapath LLP and King's College Hospital Charitable Trust. The authors are grateful to Steve Ley for useful suggestions and staff colleagues for generously donating blood and being very supportive.

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