NF-kB Signalling

Question 1

What are the five members of the NF-kB family?

Answer 1

In mammals, the family consists of Rel (c-Rel), RelA (p65), RelB, p50/p105 (NF-κB1), and p52/p100 (NF-κB2). Each polypeptide has a Rel-homology domain (RHD), which mediates both DNA binding and dimerization.

Question 2

What form does active NF-kB exist in? Give some examples.

Answer 2

Each NF-kB family member has a common domain: the Rel homology domain (RHD). This is important for the oligomerisation of NF-κB, which exists as a dimer.

The 5 members can form homo or heterodimers, the most common is p50:p65, and the different dimers induce different gene expressions. Possible combinations include:

• p50:p50
• p65:p65
• RelB:RelB
• p50:p65
• p50:RelB
• p52:RelB

Question 3

What is the negative regulator of NF-kB? How is this achieved?

Answer 3

NF-κB dimer is present in the cytosol as there is a physical linkage between the dimer and IκBα (negative regulator), which stops NF-κB entering the nucleus. The activation of NF-κB (by stress, hormones etc.) induces the phosphorylation of IκBα, which then inhibits the binding of NF-κB, allowing it to enter the nucleus.

Question 4

What are the five members of the IκB family?

Answer 4

The IκB protein family has five members, all of which contain an ankyrin domain (composed of a highly conserved alpha helix dimer backbone).

• IkBα
• IkBβ
• IkBε
• IkBγ
• Bcl3

These ankyrin domains bind to NF-kB. Mutations in this site prevent the inhibition of the NF-κB dimer.

Question 5

What kinases are responsibe for activating NF-kB?

Answer 5

Inhibitor-kappaB kinase (IKK) family members: the three members include IKK alpha, beta and gamma.

Question 6

What happens to NF-kB when there is a canonical (TNF-alpha, IL-1) stimulus?

Answer 6

The canonical pathway is induced by tumour necrosis factor-α (TNFα), interleukin-1 (IL-1) and many other stimuli, and is dependent on activation of IKKβ. This activation results in the phosphorylation (P) of IκBα at Ser32 and Ser36, leading to its ubiquitylation (Ub) and subsequent degradation by the 26S proteasome. Release of the NF-κB complex allows it to relocate to
the nucleus:

• Ubiquitous NF-κB dimers are activated rapidly by stimuli that induce the sequential phosphorylation and proteolysis of IκBs – a process that depends on the IκB kinase (IKK) complex and the ubiquitin/proteasome pathway. Upon removal of the inhibitors, NF-κB dimers enter nuclei to induce expression of coordinate sets of target genes that regulate innate and adaptive immunity, inflammation, cell growth and cell survival.

Question 7

What are the two pathways for NF-kB signalling? What proteins are involved?

Answer 7

1. Classical
2. Alternative

Question 8

Define cytokine.

Answer 8

Cytokines are a broad and loose category of small proteins that are important to cell signalling. Their release has an effect on the cells around them.

Question 9

Define chemokine.

Answer 9

Chemokines are a family of small cytokines, or signalling proteins secreted by cells. Their name is derived from their ability to induce directed chemotaxis in nearby responsive cells; chemotactic cytokines.

Question 10

What is NF-kB? What are the consequences of its misregulation?

Answer 10

NF-κB is an inducible transcription factor that controls the transcription of DNA, cytokine production and cell survival. It is involved in cellular responses to stimuli such as stress, cytokines, free radicals, heavy metals, UV, bacterial and viral antigens. It plays a key role in regulating the immune response to infection (critical to immunoglobulins). It exists as a dimer, and can contain different subunits.

Incorrect regulation has been linked to autoimmune diseases, septic shock, and cancer.

Question 11

Outline transcription factor structure.

Answer 11

Transcription factors have three domains: the DNA-binding domain (DBD), the optional signal sensing domain (SSD), and the trans-activating domain (TAD).

• The DBD consists of amino acids that recognise specific DNA bases near the start of transcription.
• The TAD contains binding sites for other proteins such as transcription co-regulators (frequently referred to as activation functions).
• The SSD senses external signals and, in response, transmits these signals to the rest of the transcription complex, resulting in up- or down-regulation of gene expression.

The DBD and SSD may be present on separate proteins that then associate in the transcription complex to regulate gene expression.

Question 12

What is the NF-kB DNA-binding domain (DBD)?

Answer 12

Question 13

What are the functions of NF-kB?

Answer 13

• Survival
• Proliferation
• Angiogenesis
• Invasion
• Metastasis

Question 14

What biological roles are related to NF-kB’s pro-survival function?

Answer 14

NF-κB has a number of biological roles related to its prosurvival function:

• Cellular responses to the triggering of TNF-Rs, TRAIL-Rs, and Fas.
• B lymphopoiesis.
• Bone morphogenesis.
• B- and T-cell costimulation (CD40, CD28, etc.)
• Liver development.

Question 15

How does NF-kB promote cell survival?

Answer 15

NF-κB promotes cell survival. The ability of this is based on that fact that it can block programmed cell death (PCD).

Question 16

What is the molecular mechanism by which NF-κB blocks cell death?

Answer 16

TNF-R1 signalling.

TNFα is an important cytokine in the immune system, and is a protein of the plasma membrane. When activated it induces caspases and MAPKs that lead to programmed cell death. TNFα binding induces cell death. Pro-survival genes block JNKs (c-jun n-terminal kinases) in the pathway. NF-κB inhibits TNFα induced apoptosis.

Question 17

What is the TNF family?

Answer 17

The tumor necrosis factor (TNF) superfamily refers to a superfamily of cytokines that can cause cell death (apoptosis).

TNF-α is a pleiotropic cytokine that plays a key role in inflammation, immunity, apoptosis and differentiation, and is arguably the most potent inducer of NF- κB.

Question 18

What is a MAPK?

Answer 18

A mitogen-activated protein kinase (MAPK or MAP kinase) is a type of protein kinase that is specific to the amino acids serine, threonine, and tyrosine (i.e., a serine/threonine-specific protein kinase). MAPKs are involved in directing cellular responses to a diverse array of stimuli, such as mitogens, osmotic stress, heat shock and pro-inflammatory cytokines. They regulate cell functions including proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis.

Question 19

What is a kinase?

Answer 19

Kinases are enzymes that phosphorylate proteins from ATP to a target protein. After phosphorylation the protein (substrate) is modified in function. This involves removing a phosphate group from ATP and covalently attaching it to an amino acid that has a free hydroxyl group. Most kinases act on both serine and threonine, others on tyrosine. This is an important regulatory mechanism.

Phosphatases do the opposite, removing a phosphate group from its substrate.

Question 20

How is NF-kB linked to cancer?

Answer 20

NF-kB is involved in the promotion of metastasis, invasion, proliferation, blocks programmed cell death (PCD) and differentiation.

• Genes encoding NF-κB-family members such as p52/p100, Rel, RelA and the IκB-like protein Bcl-3 are frequently rearranged or amplified in human lymphomas and leukemias, and inactivating mutations of IκBα occur in Hodgkin’s lymphoma (HL).
• Control of apoptosis by NF-κB is crucial to promotion of oncogenesis. In the early stages of tumorigenesis, NF-κB suppresses transformation-associated apoptosis induced by oncoproteins such as mutated H-Ras and Bcr-Abl. NF-kB is also needed for survival of a growing list of latestage tumors, including HL, diffuse large B-cell lymphoma (DLBCL), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML) and breast cancer.
• Elevated NF-κB activity has also been associated with tumor resistance to anticancer therapy, as well as to TNF-α-induced apoptosis, which might help these cells evade immune surveillance.
• Blockers of NF-κB such as proteasome inhibitors (PS-341) are now being used successfully to treat patients with MM. Glucorticoids, which also block NF-κB, are part of the therapeutic regimen for HL.

Question 21

What are some potential targets of anti-cancer therapy involving NF-kB?

Answer 21

NF-kB blockers for cancer treatment have been developed, but these have varied side effects. Therefore, it would be better to block more distinct pathways of NF-kB that do not compromise immune activity. This is done through identifying downstream pro-survival genes, however these are unknown.

Question 22

What are some novel genes downstream of TNF-alpha that can be used as potential anti-cancer therapy targets? How were these genes identified?

Answer 22

Genes that are important for survival downstream of TNF-alpha include the three novel genes:

• FHC (Blocks ROS)
• Gadd45beta (Blocks MKK7)
• Twist-1 (Blocks downstream of JNK)

‘Death trap’ screen: RelA knockout cells means that there is no NF-kB → no activation of TNF-alpha → cells able to die → no pro-survival pathway. When RelA knockouts are transfected with WT genes, some cells die and some survive. If they survive they have incorporated protective genes, which are used to identify genes using cDNA microarrays (probe hybridisation).

Question 23

Gadd45beta is a target gene downstream of TNF-alpha, outline its function.

Answer 23

Gadd45beta is a nuclear protein and is part of the Gadd45 family. Gadd45beta inhibits the JNK cascade. Ectopic/over expression in the cell is able to block cell death induced by TNF-alpha.

Gadd45beta physically interacts with MKK7 at two specific regions on MKK7 and blocks its function. One region contains K149 (lycine), which is important for the binding of ATP, nn doing so Gadd45beta inactivates MKK7 by blocking the catalytic pocket of MKK7.

This was discovered using a kinase assay, to determine the activity of kinases:

• Small peptides were synthesised (p1 and p7, 20 aa in length). P1 mimics K149 region, p7 mimics the second region. Combining them together showed that p1 blocks the inhibitory role of gadd45beta and p1 disrupted the interaction. Cell death pathway still active. Cells treated with p1 compared to other peptides died more. P1 treatment now in clinical trials.

MKK7 activation is the crucial point between cell survival and cell death.

Question 24

FHC is a target gene downstream of NF-kB, outline it’s function.

Answer 24

Ferritin is a multimeric protein important for storage of iron in our body. It is composed of two types of polypeptide: light and heavy. These polypeptides form a spherical shell around a pocket of iron. FHC is one of these polypeptides, so it has an important function for storing iron in a non-toxic form.

Iron is present in two forms: ferrous and ferric. The ferrous form can react with free radicals and form reactive oxygen species (ROS), ferritin stores iron in the ferric form. ROS can damage cells: lipid peroxidation, protein interactions, DNA damage. Ferritin blocks the production of ROS, promoting cell survival.

Question 25

In addition to marshalling _______ and ____________ responses, transcription factors of the NF-κB family control cell survival. This control is crucial to a wide range of biological processes, including B and T lymphopoiesis, adaptive immunity, ___________ and cancer _______________. During an inflammatory response, NF-κB activation antagonizes apoptosis induced by _____ ______________ (___)-_, a protective activity that involves suppression of the Jun N-terminal kinase (___) cascade. This suppression can involve upregulation of the Gadd45- family member _______/______, which associates with the JNK kinase MKK7/JNKK2 and blocks its catalytic activity. Upregulation of XIAP, A20 and blockers of reactive oxygen species (ROS) appear to be important additional means by which NF-κB blunts JNK signaling. These recent findings might open up entirely new avenues for therapeutic intervention in chronic _____________ diseases and certain ________; indeed, the Gadd45β-MKK7 interaction might be a key target for such intervention.

Answer 25

In addition to marshalling immune and inflammatory responses, transcription factors of the NF-κB family control cell survival. This control is crucial to a wide range of biological processes, including B and T lymphopoiesis, adaptive immunity, oncogenesis and cancer chemoresistance. During an inflammatory response, NF-κB activation antagonizes apoptosis induced by tumor necrosis factor (TNF)-α, a protective activity that involves suppression of the Jun N-terminal kinase (JNK) cascade. This suppression can involve upregulation of the Gadd45- family member Gadd45β/Myd118, which associates with the JNK kinase MKK7/JNKK2 and blocks its catalytic activity. Upregulation of XIAP, A20 and blockers of reactive oxygen species (ROS) appear to be important additional means by which NF-κB blunts JNK signaling. These recent findings might open up entirely new avenues for therapeutic intervention in chronic inflammatory diseases and certain cancers; indeed, the Gadd45β-MKK7 interaction might be a key target for such intervention.

Question 26

What is the importance of NF-kB in programmed cell death?

Answer 26

Normally, stimulation with the cytokine (TNF-alpha) has no apoptotic effect unless NF-κB activation or protein synthesis is blocked.

Programmed cell death (PCD) is crucial to tissue homeostasis, organ development and the elimination of defective or ‘dangerous’ cells, such as cancerous and virus-infected cells. Underscoring the importance of this process, numerous diseases arise from defects in the pathways controlling PCD. For instance, defective and excessive cell death respectively contribute to cancer and neurodegenerative disorders such as Alzheimer’s disease. Ultimately, the balance between life and death might depend on the ability of the cell to sustain activation of transcription factors of the NF-κB family.

Question 27

What is the problem with targeting NF-kB for treatment of illnesses?

Answer 27

Inhibitors of NF-κB are becoming drugs of choice in the treatment of an increasing number of illnesses. However, as NF-κB has numerous functions, particularly in immunity, there is a need for new compounds that selectively target the pro-survival activity of NF-κB and thereby minimize deleterious effects on the immune system. Remarkably, this goal now appears realistic, because it is becoming increasingly clear that – although integrated – the functions of NF-κB in immunity and programmed cell death are executed through distinct subsets of target genes.

Question 28

What does the JNK MAP kinase cascade promote?

Answer 28

Apoptosis

Question 29

Constitutive NF-kB activity is needed for the survival of late-stage tumours such as:

Answer 29

• Hodgkin lymphoma (HL)
• Multiple myeloma (MM)
• Chronic myelogenous leukaemia (CML)
• Breast cancer

Question 30

What is seen in RelA -/- cells and mice?

Answer 30

• Cells: In RelA -/- knockout cells cultures, much fewer cells survive after exposure to TNFα compared to the WT. NF-κB is the crucial survival factor downstream of TNFα. The cell death that was observed in RelA -/- knockouts was associated with activation of a JNK. When there is no RelA, there is no NF-kB. The cells die when TNFα is present as the cells cannot inactivate the JNK pathway. In RelA -/- knockouts there is a sustained activation of JNK, whereas it is not in the WT.
• Mice: Deletion of RelA in mice causes embryonic lethality at mid-gestation owing to massive liver apoptosis.

Question 31

Outline the molecular interactions resulting from TNF-alpha binding TNFR1. What are the two outcomes?

Answer 31

Upon ligand engagement, TNFR1 recruits to its cytoplasmic tail the death domain (DD)-containing proteins TRADD and RIP1, which then form one of two complexes.

1. Complex I – which also contains TRAF2 – binds to the TNFR1 tail, inducing NF-κB activation and, thereby, cell survival.
2. When ubiquitylated, the TRADD-RIP1 complex instead localizes to the cytosol, where it associates with FADD, yielding complex II, which then recruits and activates procaspase-8 and procaspase-10 to induce cell death.

Question 32

In what way is suppression of apoptosis by NF-kB a transcriptional event?

Answer 32

Although other modes of action might exist, the suppression of apoptosis by NF-κB is, by and large, a transcriptional event. NF-κB-regulated genes that are capable of blocking programmed cell death have been identified. Interestingly, the NF-κB-activated pro-survival program appears to be specifically tailored for each tissue and biological context. The bases for this plasticity are not fully understood, but the program seems to be dictated by the particular milieu of NF- κB dimers and transcription factors present in each tissue, and the specific network of interactions and modifications induced by the apoptotic stimulus.

Question 33

How does NF-kB act on the JNK pathway? What genes are involved?

Answer 33

NF-κB inhibits the JNK cascade by inducing target genes.

• Gadd45β is an effector of the suppressive activity of NF-κB, inhibiting the JNK pathway. The JNK kinase MKK7/JNKK2 is a target of Gadd45β and, indirectly, of NF-κB. Gadd45β associates tightly with MKK7 and inhibits its catalytic activity by contacting crucial residues in the catalytic pocket, including the ATP-binding residue Lys149. MKK7 is a selective activator of JNK, and its ablation in fibroblasts completely abolishes JNK induction by TNF-α. The Gadd45β-MKK7 interaction represents an important molecular link between the NF-κB and JNK pathways

Question 34

How is NF-kB linked to human disease?

Answer 34

TNF-α is a potent activator of NF-κB, which in turn is a potent inducer of TNF-α. This positive feedback is key to chronic inflammatory conditions such as rheumatoid arthritis and inflammatory bowel disease. Indeed, the standard therapy for these conditions includes NF-κB blockers such as aspirin and glucocorticoids, and neutralizing anti-TNF-α antibodies represent an effective new tool.

JNK and NF-κB have seemingly opposing effects in tumor cells. Whereas NF-κB activation is required to suppress transformation-associated apoptosis, activators of the JNK cascade (e.g. MKK4, JNK3 and BRCA1) are tumor suppressors. Several oncogene products, including oncogenic Ras and Her-2/Neu, are potent inducers of JNK, and thus cancerous cells might need constitutively active NF-κB to suppress JNK-mediated apoptosis induced by these products.