NF-κB Signal Pathway
Background of NF-κB Signal Pathway
What is NF-κB Signal Pathway?
The most basic components of NF-κB signaling pathways include receptors, signal adapter proteins, IκB kinase (IKK) complex, IκB protein and NF-κB dimer. When cells are subjected to various intracellular and extracellular stimuli, IKK is activated, resulting in phosphorylation and ubiquitination of IκB protein, and then IκB protein is degraded and NF-κB dimer is released. The NF-κB dimer is further activated by various post-translational modifications and is transferred to the nucleus. In the nucleus, it binds to the target gene and promotes the transcription of the target gene. The NF-κB signaling pathway includes both canonical and non-canonical signaling pathways. In the canonical NF-κB signaling pathway, the degradation of IκB protein releases the NF-κB dimer. The non-canonical NF-κB signaling pathway is activated through the processing of p100 to p52.
Canonical NF-κB signaling
NF-κB/p65: p50 dimer activation through the canonical pathway, such as those transduced through TNFR1, involves signal responsive activation of IKK. In general, the catalytic activity of IKKβ is required for canonical signaling. In response to a variety of inflammatory stimuli, the IKK complex phosphorylates IκBs at specific N-terminal serine residues (serine 32 and serine 36 for IκBα). Subsequent β-TrCP-mediated ubiquitination of canonical IκBs promotes complete proteasomal degradation of the inhibitors to liberate bound NF-κB dimers. Interestingly, neither phosphorylation nor ubiquitination is sufficient to dissociate IκBs from the p65: p50 dimer and proteasomal degradation of IκBs is absolutely required for p65: p50 nuclear translocation. This mode of NF-κB activation in the canonical pathway is protein synthesis-independent. NF-κB transcriptional activity is thought to be further modulated through phosphorylation and other posttranscriptional modifications of p65, but a clear understanding that relates p65 modification with its function is yet to emerge.
Non-canonical NF-κB signaling
LTβR stimulation was shown to induce nuclear accumulation of RelB: p52 via the NIK/IKK1 pathway and the phosphorylation and processing of de novo synthesized, rather than preexisting p100 protein. Indeed, a first phase of p65 activity was proposed to induce p100 synthesis to amplify RelB: p52 dimer activation, but recent evidence indicates that p65-responsive expression of RelB is more important in this regard. Signaling through multiple other TNF receptor superfamily members, such as BAFFR, CD40R, BCMA, TAC1 or RANK, was also reported to activate the RelB: p52 dimer via the non-canonical pathway.
NF-κB Family
The NF-κB protein family consists of five members: p50, p52, p65 (RelA), c-Rel, and RelB. They are encoded by the NFKB1, NFKB2, RELA, REL, and RELB genes, respectively. Structurally, they all have an N-terminal Rel homology domain (RHD) responsible for their binding to DNA and dimerization. In addition, p65, c-Rel and RelB have a transcriptional activation region (TAD) that positively regulates gene expression. p50 and p52 do not have transcriptional activation regions, and their homodimers can inhibit transcription.
Figure 2. Schematic representation of NF-κB family of proteins.
IκB Family
The IκB protein family includes 7 members: IκBα, IκBβ, IκBζ, IκBε, Bcl-3, p100, and p105. IκB binds to the NF-κB dimer in the cytoplasm and plays an important role in signal responses. The IκB protein structure has an ankyrin repeat region, i.e., a plurality of closely linked hook repeats each containing 33 amino acids. The main role of IκB protein is to mask the nuclear localization signal of NF-κB, prevent its entry into the nucleus and its binding to DNA, and make NF-κB exist in the cytoplasm of cells in an inactive form. Therefore, for the study of NF-κB signaling pathway, the study of IκB protein is very important.
Figure 3. Schematic representation of IκB family of proteins.
IκB kinase (IKK)
The IKK complex comprises three subunits, IKKα (IKK1), IKKβ (IKK2), and the regulatory subunit IKKγ (NEMO). Among specific NF-κB signaling pathways, IKKα and IKKβ are selectively required. IKKα and IKKβ have high sequence homology and similar structures. At the N-terminus, there is a protein kinase region, a leucine zipper region (LZ) and a helix-loop-helix (HLH) near the middle region. NEMO includes a large section of coiled-coli and a leucine zipper region near the C-terminus.
Figure 4. Structures of the IKK complex. (A) Domain organizations. (B) Crystal structure of IKKβ dimer. (C) Crystal structure of IKKβ NBD in complex with the N-terminal kinase-binding domain (HLX1) of NEMO. (Napetschnig J, 2013)
Through a series of studies, IKK activation models have been established. In the resting state, IKKs in the IKK complex are inhibited by interacting with NEMO. In the presence of stimuli, NEMO binds to a RIP protein, exposes the IKK protein kinase domain, induces trans-autophosphorylation of the T-loop serine residue, or phosphorylates T-ring serine residues by IKK-K. Activated IKK phosphorylates downstream enzyme substrates (e.g., IκB), thereby activating the NF-κB signaling pathway. Activated IKK can also phosphorylate serine 740 of IKKβ and serine 68 of NEMO, allowing the separation of NEMO dimers from IKK, preventing the repeated activation of kinases. Cdc37/HSP-90 (chaperone) and PP2A/PP2Cβ (phosphorylase) can mediate the recombination of the IKK complex.
NF-κB Signal Pathway Related Diseases
The continuous activation of NF-κB signaling pathway is closely related to the occurrence and development of many diseases. In the inflammatory response, the activation of NF-κB promotes the expression of inflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1), which are involved in regulating immune cell activation and inflammatory processes. However, when the NF-κB signaling pathway is over-activated, it can lead to chronic inflammation and autoimmune diseases such as rheumatoid arthritis. In addition, the sustained activation of NF-κB in tumor cells helps cells escape apoptosis, promotes tumor growth and metastasis, and is associated with the occurrence of a variety of cancers. It can also help cancer cells resist the apoptosis-inducing effects of chemotherapy drugs by regulating the expression of anti-apoptotic genes. Therefore, abnormal activation of the NF-κB signaling pathway plays a key role in pathological states and is an important target for therapeutic strategies in many diseases.
Case Study
Case Study 1: Recombinant Rat GSK3B Protein (GSK3B-2728R)
Inflammation can influence the pluripotency and self-renewal of mesenchymal stem cells (MSCs), thereby altering their cartilage regeneration ability. Sprague-Dawley (SD) rat bone marrow mesenchymal stem cells (BMSCs) were isolated and found to be defective in differentiation potential in the interleukin-1β- (IL-1β-) induced inflammatory microenvironment. Glycogen synthase kinase-3β (GSK-3β) is an evolutionarily conserved serine/threonine kinase that plays a role in numerous cellular processes. The role of GSK-3β in inflammation may be related to the nuclear factor-κB (NF-κB) signaling pathway and the Wnt/β-catenin signaling pathway, whose mechanism remains unclear. In this study, the researchers found that GSK-3β can inhibit chondrogenesis of IL-1β-impaired BMSCs by disrupting metabolic balance and promoting cell apoptosis. By using the inhibitors LiCl and SN50, they demonstrated that GSK-3β regulates the chondrogenesis via the NF-κB and Wnt/β-catenin signaling pathways and possibly mediates the cross-reaction between NF-κB and β-catenin in the nucleus.
Fig1. GSK-3β regulates the NF-κB signaling pathway in IL-1β-induced inflammation. (Zhenggang Wang, 2022)
Case Study 2: Active Recombinant Human CD19 protein (CD19-3309HP)
Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors (LVs) are widely used to reliably generate genetically modified, clinical-grade T-cell products. However, the results of genetically modifying natural killer (NK) cells with VSV-G LVs have been variable. The authors explored whether inhibition of the IKK-related protein kinases TBK1 and IKKε, key signaling molecules of the endosomal TLR4 pathway, which is activated by VSV-G, would enable the reliable transduction of NK cells by VSV-G LVs. The authors activated NK cells from peripheral blood mononuclear cells using standard procedures and transduced them with VSV-G LVs encoding a marker gene (yellow fluorescent protein [YFP]) or functional genes (chimeric antigen receptors [CARs], co-stimulatory molecules) in the presence of three TBK1/IKKε inhibitors (MRT67307, BX-795, amlexanox). NK cell transduction was evaluated by flow cytometry and/or western blot and the functionality of expressed CARs was evaluated in vitro. Focusing on MRT67307, the authors successfully generated NK cells expressing CD19-CARs or HER2-CARs with an inducible co-stimulatory molecule. CAR NK cells exhibited increased cytolytic activity and ability to produce cytokines in comparison to untreated controls, confirming CAR functionality.
Fig2. Representative flow cytometry histogram plot depicting CD19-CAR expression with indicated MOIs. (Peter Chockley, 2021)
Case Study 3: Recombinant Human IL1B protein (IL1B-02H)
Most lung transplants are obtained from brain-dead donors. The physiopathology of brain death involves hemodynamics, the sympathetic nervous system, and inflammatory mechanisms. Administering methylprednisolone 60 min after inducing brain death in rats has been shown to modulate pulmonary inflammatory activity. This study aims to evaluate the effects of methylprednisolone on transplanted rat lungs from donors treated 60 min after brain death. All of the animals were observed and ventilated for 2 h prior to being submitted to lung transplantation. We evaluated the hemodynamic and blood gas parameters, histological score, lung tissue levels of thiobarbituric acid-reactive substances, level of superoxide dismutase, level of tumor necrosis factor-alpha, and level of interleukin-1 beta. After transplantation, a significant reduction in the levels of tumor necrosis factor-alpha and IL-1β was observed in the group that received methylprednisolone. There were no significant differences in tumor necrosis factor-alpha and superoxide dismutase levels between the control and methylprednisolone groups.
Fig3. Pulmonary interleukin-1 beta assay. (Luiz Felipe Lopes Araujo, 2014)
Related Products
NF-κB (nuclear factor κB) signaling pathway is an important nuclear transcription factor in the cell, it plays a key role in the body's inflammatory response, immune response, and can regulate apoptosis and stress response. Creative BioMart can provide a list of core protein products of the NF-κB pathway covering classical path, bypass path and atypical path. Please feel free to contact us if you’re interested.
