NF-κB Signaling Pathway

Introduction

Nuclear factor-κB (NF-κB) is a class of transcription factors that are ubiquitous in various cell types in living organisms. In 1986, Sen and Baltimoer first detected a nuclear protein factor that specifically bound to the immunoglobulin κ light chain gene enhancer κB sequence (GCGACTTTC) from mature B lymphocyte nuclear extract, called NF-κB. It is widely present in eukaryotic cells and can specifically bind to specific sites of various cellular gene promoters or enhancer sequences to promote transcription and expression. It is also closely related to important pathophysiological processes such as inflammatory response, immune response and cell proliferation, transformation and apoptosis.

Figure 1 The NF-κB signaling pathway

Classical activation pathway

Two NF-κB signaling pathways have been identified including classical and non-canonical activation pathways.

The classical signaling pathway is mainly triggered by the combination of the ligands and T cell receptor (TCR), B cell receptor (BCR) or toll-like receptor (TLR) - interleukin-1 receptor (IL-1R) family members. And it terminates in the transcription of target genes encoding chemokines, cytokines, adhesion molecules, inflammatory responses, and cell survival.

When the cells are in a resting state, NF-κB usually binds to the inhibitory proteins IκBs (IκBα, IκBβ, IκBε) and exists in the cytoplasm in an inactive form and the combination of the two masks the nuclear localization signal of NF-κB. When the cells are stimulated, the inducer of NF-κB activates the IKK in the cytoplasm through the cell membrane to phosphorylate the serine at positions 32 and 36 of IκBα. Phosphorylated IκB is rapidly ubiquitinated by ubiquitin-binding enzymes and is rapidly degraded by the 26s proteasome. The free NF-κB in the cytoplasm is transported into the nucleus and binds to the NF-κB binding site in the promoter region of the DNA molecule target gene, thereby initiating transcription of the target gene to generate corresponding mRNA and protein.

Non-classical activation pathway

The classical pathway is characterized by the induction of rapid, transient transcriptional responses, while the non-canonical pathway is delayed and induces sustained transcriptional responses. Lymphotoxin B receptor, B lymphocyte activating factor, NF-κB activation receptor, and cell differentiation antigen 40 can phosphorylate NF-κB kinase α(IκKα) by NF-κB inducing enzyme (NIK). Activated IκKα phosphorylates p100 at Ser 866 /870 and processes it into mature p52. The P52/RelB heterotrimer translocates into the nucleus and is characterized by a specific gene. Activation of NF-κB stimulates the production of TNFα and IL-1, and the increase of these inflammatory factors promotes the activation of NF-κB. Thus, activation of NF-κB leads to increased expression of TNFα and IL-1, and these substances act as agonists to further act on NF-κB, forming a positive feedback regulation of NF-κB activation. Activation of this pathway regulates the development of lymphoid organs and the adaptive immune system.

Upstream signaling of NF-κB

In many NF-κB signaling pathways, many signal intermediates are common, especially upstream signals of the IKK complex. Different signal paths can utilize some common signal element activation and suppression paths.

TRAFs: TNF receptor-related factors

TRAFs family, the TNF receptor-associated factor, is a large class of intracellular adaptor proteins that bind directly or indirectly to a variety of TNF and IL-1/Toll-like receptor family members. It affects the survival, proliferation, differentiation, and death of cells and participates in many biological processes. In almost all NF-κB signaling pathways, TRAFs are key signaling intermediates. The TRAF protein family has a total of seven members, namely TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, and TRAF7.

By TRADD, TRAF2 binds to the receptor TNFR1 of TNF-α, signaling down and activating IKK. In this process, its RING finger region is necessary as an E3 ligase. In the TNFR1 signaling pathway, with a single knockout of TRAF2 or TRAF5, activation of the NF-κB signaling pathway will still occur. However, double knockout of TRAF2 and TRAF5 results in defects in the activation of the IKK complex in the NF-κb signaling pathway. Therefore, in the TNFR1 signaling pathway, a synergistic effect of TRAF2 and TRAF5 is required.

RIPs: Receptor acting protein

RIPs are key adaptor proteins in the classical NF-κB signaling pathway. RIPs can act directly upstream of the signaling pathway through the protein-binding region, or activate the IKK complex by binding to NEMO. And RIPs are involved in most TRAF-dependent signaling pathways. RIPs have a death domain that can be linked to the death structure of other adaptor proteins and receptors. They can not only recruit IKK complexes but more importantly activate IKK complexes.

TAK1/NIK

TAK1/NIK appears as an IKK kinase in the NF-κB signaling pathway. Among them, TAK1 is involved in the classical signal path. In the non-canonical signaling pathway, NIK has the effect of inducing IKKα activation and P100 phosphorylation. TAK1 is generally involved in the signaling pathway for the activation of IKK by RIP proteins. In different classical signaling pathways, the TAK1 gene is knocked out, and the activation of the NF-κB signaling pathway shows various degrees of defects.

Downstream signaling of NF-κB

The inhibitory effect of NF-κB in the mitochondrial pathway can be achieved by modulating the transcription of inhibitor of apoptosis proteins (IAP) family members X-linked IAP (XIAP), c-IAP-1, and c-IAP-2. XIAP is a key factor in the inhibition of apoptosis by NF-κB, which specifically binds to and inhibits the activation of the apoptotic effectors caspase 3, 7 and 9, thereby blocking the release of Cytc in cells and inhibiting apoptosis. In addition, the anti-apoptotic effect of NF-κB can also be achieved by up-regulating the expression of anti-apoptotic genes. Bcl-2 family proteins control the permeability of mitochondrial membranes and play a key role in the mitochondrial apoptotic pathway.

JNK is a member of the MAPK family and regulates different stress responses inside and outside the cell. Quantitative studies have shown that NF-κB activation can inhibit JNK-mediated apoptosis. Nakano et al. found that the level of intracellular reactive oxygen species can be down-regulated by NF-κB activation, thereby inhibiting the JNK cascade response signal and inhibiting apoptosis. Studies have shown that NF-κB plays an anti-apoptotic role by inhibiting JNK signaling.

NF-κB and tumor

Studies have shown that inflammatory breast cancer phenotype usually shows a high activity of NF-κB expression, and is often in advanced stage at diagnosis, mostly estrogen receptor negative and human epidermal growth factor receptor II (HER2) positive. Studies have shown that activation of NF-κB in breast cancer occurs downstream of the EGFR (ERBB1 /HER1) signaling pathway. Overexpression of the HER-2/ neu gene leads to activation of the PI3K /Akt signaling pathway and NF-κB (p50/p65). Studies in hepatocellular carcinoma have shown that alcohol consumption causes accumulation of intracellular ROS, leading to activation of NF-κB and up-regulation of VEGF and MCP-1. VEGF and MCP-1 promote tumor angiogenesis and may mediate the progression/metastasis of alcohol-stimulated liver cancer.

Figure 2 The role of the NF-kB signaling pathway in the regulation of ethanol-enhanced the progression of HCC

HTLV-1-infected cells have strong activity through the NF-κB pathway compared to normal T cells. In contrast, by obtaining miR-31-dependent NIK expression, T cell leukemia (ATL) cells maintain strong NF-κb activity without being affected by Tax. Activation of the non-canonical pathway eventually joins the classical pathway, leading to transactivation of many target genes, which play an important role in cell survival, cytokine production and invasiveness. HTLV-1-infected cells have strong activity through the NF-κB pathway compared to normal T cells.

Figure 3 NF-κB signaling pathway in ATL

NF-κB and rheumatoid arthritis

Spurlock et al. studied the expression of RNA-p21 (lincRNA-p21), NF-κB activity, and response to MTX in RA. It is found that the basal expression of lincRNA-p21 is decreased in RA patients, and the basal expression of NF-κB activity marker phosphorylation p65 (RelA) is elevated. Wang et al. detected the expression level of NF-κB negative regulator A20 and mucosa-associated lymphoid tissue lymphoma translocation gene (MALT)-1, MALT-V1, A20 by RA-PCR. It was found that compared with healthy controls, A20 expression was lower in RA patients, NF-κB was overexpressed, and MALT1 and MALT1-V expression were decreased. NF-κB has made some progress in the field of RA, but there are still many problems to be addressed in the targeted therapy of NF-κB.

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