In every cell of our body, there exists a complex and precise regulatory network responsible for sensing changes in the external environment and responding accordingly. At the heart of this network lies a special family of proteins that act as the "commanders" within the cell, controlling the expression of many important genes and playing a crucial role in regulating inflammation, immune response, cell growth, and apoptosis. This family is known as the NF-κB (nuclear factor-kappa B) transcription factor family.

Discovery and Research of the NF-κB Family

The discovery of NF-κB dates back to 1986 when researchers Ranjan Sen and David Baltimore at MIT identified this new nuclear factor while studying the regulatory mechanisms of immunoglobulin κ light chain genes in B lymphocytes. They found that NF-κB could specifically bind to certain DNA sequences in the κ light chain gene enhancer, thereby regulating its expression.

This discovery quickly attracted significant attention in the scientific community. Subsequent research revealed that NF-κB is not only present in B cells but also expressed in nearly all cell types. Moreover, many gene promoters or enhancers contain specific NF-κB binding sites, indicating that NF-κB may be involved in regulating the expression of numerous genes.

Over the years, scientists have come to understand that NF-κB plays a key role in cellular responses to various external stimuli. Bacterial and viral infections, inflammatory stimuli, UV radiation, and oxidative stress are just some of the factors that can activate NF-κB. These findings established NF-κB as a critical bridge linking external stimuli to gene regulation.

Figure 1. Members of the NF-κB, IκB, and IKK families. (Oeckinghaus A, et al., 2008)

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Members and Structural Characteristics of the NF-κB Family

In mammals, the NF-κB family comprises five members: p65 (RelA), RelB, c-Rel, p105/p50 (NF-κB1), and p100/p52 (NF-κB2). These proteins can dimerize in various combinations to exert their transcriptional regulatory functions. All NF-κB family members share a highly conserved Rel homology domain (RHD) of about 300 amino acids, which is responsible for several key functions:

1. Dimerization: The RHD enables NF-κB proteins to form homo- or heterodimers.

2. DNA Binding: The N-terminal part of the RHD specifically recognizes and binds to κB sites on DNA.

3. Interaction with IκB Inhibitors: The C-terminal part of the RHD is involved in binding with IκB proteins.

4. Nuclear Localization: The RHD contains a nuclear localization signal (NLS) that directs NF-κB into the nucleus.

Despite these shared features, the NF-κB family members exhibit significant structural and functional differences:

1. Transcriptional Activation: p65, RelB, and c-Rel contain transcriptional activation domains (TADs) that can directly activate gene transcription, while p50 and p52 lack TADs and require association with other family members or co-factors to activate gene expression.

2. Precursor Proteins: p50 and p52 are derived from larger precursor proteins, p105 and p100, respectively, which contain ankyrin repeats similar to IκB proteins that can inhibit NF-κB activity.

3. Special Functions: RelB plays a critical role in the non-canonical NF-κB pathway, primarily forming heterodimers with p52, while c-Rel is mainly involved in immune cell function.

Mechanisms of NF-κB Activation

In most resting cells, NF-κB exists in an inactive form bound to IκB inhibitory proteins in the cytoplasm. When the cell is stimulated, a signaling cascade is triggered, leading to the phosphorylation and degradation of IκB, allowing the NF-κB dimers to be released and translocated into the nucleus to activate target gene transcription. NF-κB activation occurs primarily through two pathways:

1. Canonical Pathway

The canonical pathway is the major route for NF-κB activation and can be triggered by various stimuli, including cytokines like TNFα and IL-1, pathogen-associated molecular patterns like LPS, antigen receptors like TCR and BCR, growth factors, and physical or chemical stresses such as UV radiation and oxidative stress. Central to this pathway is the IκB kinase (IKK) complex, particularly the IKKβ subunit. Upon activation, IKKβ phosphorylates IκBα or IκBβ, leading to their ubiquitination and degradation by the 26S proteasome. This process releases the NF-κB dimers (mainly p65/p50), exposing the nuclear localization signal and allowing them to rapidly translocate to the nucleus. In the nucleus, NF-κB dimers bind to κB sites on target gene promoters or enhancers, recruiting co-activators to initiate transcription. These target genes include cytokines, chemokines, adhesion molecules, and others involved in regulating immune response, inflammation, and cell survival.

Figure 2. The two NF‑κB signaling pathways. (Taniguchi K, et al., 2018)

2. Non-Canonical Pathway

The non-canonical pathway is primarily activated by specific members of the TNF receptor superfamily, such as the BAFF receptor, CD40, lymphotoxin-β receptor (LTβR), and receptor activator of NF-κB (RANK). Unlike the canonical pathway, the non-canonical pathway mainly relies on the IKKα subunit and does not require IKKβ or NEMO. The key event in this pathway is the activation of NF-κB-inducing kinase (NIK), which then activates IKKα. IKKα selectively phosphorylates p100, which undergoes partial proteolysis to generate p52, leading to the release and nuclear translocation of p52/RelB dimers. The activation of the non-canonical pathway is slower but can be sustained for longer periods. It plays an essential role in lymphoid organ development, B-cell maturation, and adaptive immune responses.

These two pathways are not entirely independent, with cross-regulation and interaction occurring between them. For instance, canonical pathway activation can induce the expression of p100 and RelB, setting the stage for non-canonical pathway activation.

Fine-tuning of NF-κB Regulation

Given the broad range of biological processes regulated by NF-κB and the potential consequences of its aberrant activation, cells have developed sophisticated multi-layered mechanisms to precisely control NF-κB activity:

1. IκB Protein Family

The IκB proteins are the primary inhibitors of NF-κB, including classic members such as IκBα, IκBβ, and IκBε, as well as the precursor proteins p100 and p105. They all contain multiple ankyrin repeats that can bind to the RHD of NF-κB. IκBα, the most studied member, forms a complex with NF-κB that shuttles between the cytoplasm and the nucleus, maintaining a dynamic balance. This is because IκBα only masks the nuclear localization signal of p65, leaving the p50 NLS exposed. Additionally, IκBα itself contains a nuclear export signal, allowing the complex to continuously move in and out of the nucleus.

The degradation of IκBα disrupts this balance, enabling a significant amount of NF-κB to enter the nucleus and bind DNA. Meanwhile, activated NF-κB promotes the transcription of the IκBα gene, with newly synthesized IκBα entering the nucleus to remove NF-κB from DNA and relocate it back to the cytoplasm. This forms a crucial negative feedback loop, ensuring a timely shutdown of NF-κB activity. Other IκB proteins have unique functions. For instance, IκBβ binds to NF-κB more stably and does not shuttle between the nucleus and cytoplasm, possibly contributing to the sustained expression of certain genes. IκBε may regulate oscillations in NF-κB activity.

2. IKK Complex

The IKK complex is the critical checkpoint in the NF-κB signaling pathway, comprising two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ (also known as NEMO). IKKβ plays a central role in the canonical pathway, efficiently phosphorylating IκBα and IκBβ. Mice lacking IKKβ die due to massive hepatocyte apoptosis, highlighting its crucial role in the TNF signaling pathway. IKKα is essential in the non-canonical pathway, mainly responsible for phosphorylating p100. Additionally, IKKα may regulate gene expression by modifying histones and p65.

IKKγ acts as a scaffold protein, essential for the assembly and activation of the complex. It interacts with various upstream signaling molecules, transmitting different stimuli to the IKK complex. The activation of the IKK complex involves complex mechanisms, including proximal kinase phosphorylation and ubiquitination-mediated interactions. Once activated, the IKK complex phosphorylates IκB proteins, triggering their degradation and thus activating NF-κB.

3. Post-translational Modifications of NF-κB Subunits

NF-κB subunits themselves are regulated by various post-translational modifications, including phosphorylation, acetylation, and ubiquitination. These modifications can influence NF-κB's DNA binding ability, transcriptional activity, and interactions with co-factors. For example, p65 can be phosphorylated at multiple sites by various kinases:

Ser276: Phosphorylated by PKA or MSK1/2, enhancing p65's interaction with co-activators like CBP/p300 and boosting transcriptional activity.

Ser536: Phosphorylated by IKKβ, TBK1, or PKCζ, promoting p65 nuclear translocation and transcriptional activation.

Ser468: Phosphorylated by IKKβ, GSK-3β, or Casein kinase II (CK2), leading to decreased p65 activity and recruitment of repressor complexes.

p65 acetylation occurs mainly at Lys310, mediated by CBP/p300. Acetylation enhances DNA binding and transactivation, and deacetylation by HDACs decreases p65 activity and promotes its nuclear export. p50 and p52 can also be post-translationally modified. For instance, p50 acetylation at Lys431 and Lys440 increases its DNA binding, while deacetylation by HDAC3 inhibits transcription. Additionally, p50 can interact with other transcription factors, such as BCL-3, to regulate gene expression.

The Role of NF-κB in Disease

Given the wide range of biological processes regulated by NF-κB, its dysregulation is closely associated with the pathogenesis of many diseases. These include chronic inflammatory diseases (e.g., rheumatoid arthritis, inflammatory bowel disease), autoimmune diseases, cancer, cardiovascular diseases, and neurodegenerative diseases.

Chronic Inflammatory and Autoimmune Diseases: NF-κB plays a central role in the regulation of inflammation and immune response. Dysregulation of NF-κB activation can lead to persistent inflammation and autoimmunity. For instance, rheumatoid arthritis is characterized by excessive NF-κB activation, leading to the production of pro-inflammatory cytokines, matrix metalloproteinases, and other mediators that cause joint damage. In inflammatory bowel disease, abnormal NF-κB activation contributes to the production of inflammatory mediators in the gut, promoting chronic inflammation and tissue damage.

Cancer: NF-κB is frequently constitutively active in many cancers, including lymphomas, breast cancer, prostate cancer, and colorectal cancer. In cancer, NF-κB promotes tumor cell proliferation, survival, angiogenesis, invasion, and metastasis. It also plays a crucial role in creating a tumor microenvironment that favors tumor progression. For instance, in colorectal cancer, aberrant activation of the canonical NF-κB pathway leads to the production of pro-inflammatory cytokines like TNFα, IL-6, and IL-1β, which promote tumor growth and resistance to apoptosis. Additionally, NF-κB activation in cancer cells can lead to chemoresistance by upregulating anti-apoptotic proteins like Bcl-2 and Bcl-xL.

Cardiovascular Diseases: NF-κB is involved in the pathogenesis of atherosclerosis, a major cause of cardiovascular diseases. In atherosclerosis, NF-κB is activated in endothelial cells, smooth muscle cells, and macrophages, leading to the production of pro-inflammatory cytokines, chemokines, and adhesion molecules. These mediators promote the recruitment of immune cells to the vessel wall, contributing to plaque formation and progression. NF-κB activation is also involved in other cardiovascular conditions, such as myocardial infarction and heart failure.

Neurodegenerative Diseases: NF-κB has been implicated in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). In these conditions, NF-κB is activated in neurons and glial cells, leading to the production of pro-inflammatory cytokines and reactive oxygen species that contribute to neuronal damage and death. For example, in Alzheimer's disease, NF-κB activation is associated with the production of amyloid-beta peptides and the formation of neurofibrillary tangles, both of which are hallmarks of the disease.

Future Directions and Challenges

The NF-κB signaling pathway is a multifaceted regulatory network within cells, involving both canonical and noncanonical pathways that control critical physiological processes such as inflammation, immune responses, cell proliferation, and survival. The canonical pathway typically responds rapidly to broad stimuli, activating NF-κB dimers through a multi-level regulatory process, including the IKK complex, and is self-limited by negative feedback mechanisms. In contrast, the noncanonical pathway is more specific, activated mainly through TNF superfamily receptors, relying on the collaborative actions of NIK and IKKα and the conversion of p100 to p52, to regulate the maturation and development of immune cells. Aberrant activation of NF-κB is closely linked to various diseases, including cancer, autoimmune disorders, and chronic inflammation, making it a crucial target for drug development. Understanding the regulatory mechanisms of the NF-κB signaling pathway is essential for developing effective therapeutic strategies and improving patient outcomes.