Induction, Nuclear Transcription Factor kappa B (NFkB)

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a generic term comprising a family of proteins that play a critical role in both innate and adaptive immunity, cell proliferation, apoptosis and inflammation.  Induction of NF-κB can triggered by a variety of factors, including interleukins, tumor necrosis factors, viral proteins, ultraviolet radiation, reactive oxygen species, nitric oxide and pro-inflammatory cytokines (Li and Verma, 2002), but the most studied mechanism is induction by toll-like receptor signaling – in particular TLR2 and TLR4 (Arancibia et al., 2007).

The mammalian NF-κB family of proteins consists of NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB and c-Rel, which are present as dimers in the cell cytosol, attached to inhibitors of κB (IκB) until activated (Gilmore, 2006). Induction of NF-κB occurs when an upstream signal activates IKB kinase (IKK) leading to phosphorylation and degradation of IκB. Degradation of IκB releases NF-κB subunit dimers and allows for subsequent phosphorylation and translocation to the cell nucleus. Inside the nucleus,  NF-κB dimers bind to DNA motifs and are critical transcriptional regulators in the innate immune response, mediating rapid upregulation of gene transcription (Li and Verma, 2002).

When NF-κB dimers containing p65 form complexes with CREB-binding protein (CBP) they can bind to DNA, inducing the transcription and rapid upregulation of a variety of proteins depending on the cell and tissue type. Additionally, NF-κB can work in concert with activator protein-1 (AP-1) to further stimulate gene transcription (Ye et al., 2014), and plays a regulatory role in T cell function (Gerondakis et al., 2014). In immune cells in the periphery, and glia within the CNS, p65/CBP binding to DNA is associated with a potent inflammatory response and results in upregulation of  pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and Interleukin 1beta (IL-1β) (Zhong et al., 2002; Giridharan and Srinivasan, 2018), and plays a complex role in the priming, but not necessarily the activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome (Kelley et al., 2019). Nuclear NF-κB also upregulates IκB repressor transcription in a negative-feedback loop which mediates deactivation and transportation of NF-κB dimers back to the cytosol (Giridharan and Srinivasan, 2018).

Induction of NF-κB is rapid and transient and is primarily detected using in vitro assays. Phosphorylated NF-κB (which is a necessary step in activation) can be detected by the application of phospho-specific antibodies to members of the NF-κB family of proteins such as p65 and p50, which form homo and heterodimers that enable translocation to the nucleus.  These proteins can then be detected using flow cytometry (Maguire et al., 2015).  Alternatively, phosphorylation can be measured using commercially available antibodies in western blots or immunohistochemistry/microscopy.  Numerous other methods exist for detecting NF-κB complexes bound to DNA, including quantitative enzyme-linked immunosorbent assay (ELISA), Electrophoretic mobility shift assay (EMSA) for detecting NF-κB binding to nucleic acid proteins,  chromatin immunoprecipitation combined with PCR (CHIP-seq), and live-cell imaging using GFP-tagged reporter cell-lines (for a full review of available techniques see (Ernst et al., 2018)).

NF-κB was first discovered 35 years ago in B cells (Sen and Baltimore, 1986) and was subsequently discovered to be family of related proteins. NF-κB protein subunit expression (homologs and orthologs) is conserved across most animal species including mammals, invertebrates, cnidarians and insects, (with the exception of yeasts and c. elegans), although some species-specific polymorphisms exist (Graef et al., 2001; Sullivan et al., 2009). By far, mammalian NF-κB is the most studied: Subunits p50, p52, and p65 are present in most cell types, whereas RelB and c-Rel are only found in a subset of immune cells (Gilmore and Gerondakis, 2011; Oeckinghaus and Ghosh, 2009).

The induction of NF-κB has been directly measured in a large variety of cell types, both in vivo and vitro, primarily using human and mouse tissue, for which there now exist commercial reporter lines for human HEK293, ME-180, HeLa, Jurkat and THP-1 cells, mouse RAW 264.7 cells, Chinese hamster ovary (CHO-K1) cells. More recently reporter mouse lines have been developed, with 3 different models available from JAX.ORG and a newly developed ROSA26 knock-in NF-κB reporter (KappaBle) line that allows visualization of NF-κB activity in any cells which stably express and activate NF-κB  (Tortola et al., 2022).

The study of NF-κB is broadly expressed in cells of both sexes, however sex differences have been found in basal NF-κB activation in mice (Villa et al., 2018) and down-stream gene expression impacting neuroprotection in human neurons (Ruiz-Perera et al., 2018) and murine bone mass (Zarei et al., 2019).  

NF-κB is present in differentiated cells at all life stages and is crucial during development, with loss of function leading to severe developmental and often fatal defects in mice and humans (Espín-Palazón and Traver, 2016). NF-κB regulates cell differentiation in adult stem cells, but is not present in mouse and human embryonic stem cells (Kaltschmidt et al., 2021). NF-κB has been implicated in the development and treatment of cancer, aging, autoimmune and degenerative disorders in humans with many studies leveraging mouse models (Miraghazadeh and Cook, 2018; Salminen and Kaarniranta, 2009; Sun et al., 2013; Xia et al., 2014; Zhang et al., 2021). Numerous knockout mouse models have been developed to study human disease-causing polymorphisms in NF-κB genes, with the biggest species differences being not in the NF-κB family of proteins but in the inhibitors of NF-κB, the IKK proteins (Zhang et al., 2017).

While 15 possible subunit dimer combinations have been identified, the most common dimer studied to date is the p65/p50 complex which has been well-documented to rapidly upregulate inflammatory cytokines  (Smale, 2012).  As of 2006, there were over 25,000 publications on the study of NF-kB (Gilmore, 2006), with many comprehensive and detailed reviews on the complex pathways, polymorphisms, and biological implications written prior to and since that time. Boston University Biology maintains a web site devoted to tracking the hundreds of genes identified as targets of NF-κB (https://www.bu.edu/nf-kb/gene-resources/target-genes/).  

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