What is NFkB?

What is NFƘB?

technical briefing

Nuclear factor kappa-light-chain-enhancer of activated B cells, or simply nuclear factor kappa B (NFkB), is a small family of large regulatory proteins that is fundamental to a cell’s response to a wide variety of stimuli. [1,2]

The importance of NFkB and the centrality of its role in DNA transcription is well illustrated by the list of cellular functions and responses regulated by NFkB: [1,3-6]

· inflammation

· innate and adaptive immune responses

· cell survival (anti-apoptosis)

· cell replication

· cell differentiation/maturation

· timely apoptosis (programmed cell death)

· pain that is independent of inflammation

The positive roles of NFkB in neural plasticity, learning, and memory [4,7-9] are particularly interesting because they show us a more global, and perhaps even holographic, view of these regulatory proteins:

The NFkB family facilitates the reactive and adaptive responses required for the organism to survive and to thrive.

The fact that NFkB proteins are found in a wide variety of “simpler” organisms, such as sea anemones, coral, sponges, and insects, [1,7] further supports their core role in survival and adaptation.

NFkB family

As might be expected for such a range of functions, most of which are fundamental or essential to the cell’s and the organism’s survival, the NFkB proteins as individuals and as a group are quite complex. There are at least five distinct proteins in the mammalian NFkB family: [1,7]

· NFkB1, or p105 and its active subunit, p50

· NFkB2, or p100 and its active subunit, p52

· RelA, or p65

· RelB

· c-Rel, or simply REL

The individuality and complexity of each protein, coupled with their ability to form homodimers and heterodimers with each other (e.g., p50-RelA), [1,4,10] has in a way created a language or code that allows for the impressive repertoire of cellular responses orchestrated by NFkB.

This small family of regulatory proteins controls the expression of several hundred different genes, including cytokines, chemokines, and their modulators; immunoreceptors; proteins involved in antigen presentation; cell adhesion molecules; acute phase proteins; stress response genes; cell surface receptors; regulators of apoptosis; growth factors, ligands, and their modulators; early response genes; transcription factors and regulators; and enzymes. [3]

So, not only does this “language” enable the necessary housekeeping duties of cell/tissue maintenance, protection, and repair, it may also provide the sophistication or subtlety required for a truly adaptive response.

Constitutive and inducible NFkB

Also as might be expected for its range of regulatory functions, NFkB is both constitutively and inducibly expressed. In normal cells, it is active in small (basal) amounts until the cell is stimulated, at which point its expression is rapidly, and in some instances markedly, upregulated. [11-15]

The list of NFkB inducers is extensive, as could be expected for such a central regulator: microbes (bacteria, fungi, viruses) and their products, eukaryotic parasites, inflammatory cytokines, a wide variety of physiological mediators, physical stress, oxidative stress, environmental hazards (ultraviolet light, cigarette smoke, etc.), various drugs, modified proteins, overexpressed proteins, receptor ligands, apoptotic mediators, mitogens, growth factors, and a number of hormones. [2]

We see the dynamic of constitutive and inducible expression with various other biochemical families, such as the cyclo-oxygenases (COX) and the prostaglandins — not coincidentally, products of NFkB activation. [3] Evidently, the constitutively expressed forms or activities are important for normal “housekeeping” functions, which are essential for optimal tissue maintenance and repair; and the inducible forms or responsiveness are important for the cell’s and the organism’s ability to respond to potentially harmful stimuli.

Persistent upregulation of constitutively expressed (basal) NFkB is a feature of many tumours [6,14,16,17] and a plethora of other chronic inflammatory or degenerative diseases. [17] The precise mechanisms remain to be fully elucidated, and may in fact prove to be cell- and circumstance-specific, [7,16] but one thing is clear: restoration of normal basal NFkB activity is an important therapeutic goal.

But just as with COX inhibition, potent suppression of all NFkB expression is not a wise strategy, given the essential role that NFkB plays in normal physiology. [4,7,18] Rather, the combined use of low doses of inhibitors that target multiple steps in the NFkB signalling pathway is advised in order to effectively downregulate NFkB overexpression while avoiding adverse effects. [18]

Autoregulation of NFkB

Because NFkB is central to the inflammatory process, immunological responses, and cell replication, differentiation, and apoptosis, it is essential that this potent family of regulators is itself well regulated. The two aspects of autoregulation that are most relevant here are these:

1. Expression of NFkB is a multi-site and multi-step process, so inhibition may occur at any one of a number of sites/steps. [18,19]

(i) It begins at the cell membrane, with binding at any one of a number of receptors that trigger the activation of NFkB. Inhibitors of receptor activation block the process at this step.

(ii) It continues in the cytosol, where NFkB is stored in an inactive form, tethered to its main inhibitor, IkB (inhibitor of NFkB, actually a family of several distinct inhibitor proteins). This inhibitor blocks the nuclear localisation signals of NFkB, which keeps the NFkB bound in the cytosol. During NFkB activation, IkB is itself inhibited (phosphorylated and then proteosomally degraded) by activation of the IKK (IkB kinase) complex, which frees and thus activates NFkB.

(iii) The free NFkB then moves (translocates) into the nucleus, where it binds to the DNA in the region of the specific response elements (promoter or enhancer regions) on the target genes. Transcription of those genes ensues.

(iv) The resulting messenger RNA (mRNA) exits the nucleus into the cytosol, where it stimulates ribosomal production of the specific proteins encoded by the particular genes.

This step is rate-limited and probably substrate-limited as well. Although the expression of target mRNA is rapidly upregulated (within minutes of cell stimulation), the actual production of the proteins encoded by those genes can take an hour or more to get going, and several more hours (as many as 16 hours) to peak. [11,20] This “post-nuclear” lag time, between nuclear transcription and protein secretion, may provide a window of opportunity for interference with, or modulation of, the cell’s response.

2. Dynamic feedback loops are built-in and are amenable to alteration.

(i) The activation and nuclear binding of NFkB stimulates the production of its principal inhibitor, IkB, forming a neat little negative feedback or autoregulatory loop and ensuring that upregulation of NFkB is normally a transient, or at least limited, event. [1,3]

(ii) Reactive oxygen species (ROS) play an important role in NFkB expression in most cell types studied. However, the interaction is complex, with both positive- and negative-feedback loops occurring among NFkB, ROS, and other signal transducers. [16] In short, ROS can activate, be activated by, or be inhibited by NFkB, depending on the circumstances, including (especially?) the redox state within the cell and its immediate environs.

For example, when ROS are overexpressed, NFkB is activated and produces various gene products (ferritin heavy chain, manganese superoxide dismutase, etc.) that block further generation or the activity of ROS. [16] As further evidence, various antioxidants are documented to downregulate NFkB expression. [11,16,18,19]

Thus, there are multiple points at which the expression of NFkB may be influenced — facilitated or impeded — by pharmaceutical and even nutraceutical interventions.

Example: Osteoarthritis and NFkB

The NFkB proteins are found in virtually all mammalian cell types studied, including synoviocytes, chondrocytes, osteocytes, endothelial and vascular smooth muscle cells, fibroblasts and fibrocytes, and the various classes of immunocytes. So, it can be expected that NFkB plays a central role in joint health. And as NFkB is a crucial regulatory protein involved in the inflammatory process, it should not be surprising that the NFkB system is persistently upregulated in chronic inflammatory states such as osteoarthritis (OA). [17,21-27]

What isn’t so obvious, however, is why NFkB is persistently upregulated in OA, when inflammation is intended to be a transient, self-limiting, and ultimately reparative process. It is clear that multiple inflammatory mediators, representing multiple signalling pathways, are chronically upregulated or overexpressed in osteoarthritic cartilage [24] — but why?

Research into atherosclerosis may shed some light on this aberrant process. Whereas high shear stress in endothelial cells causes a transient upregulation in NFkB activity, low but more persistent or multidirectional shear stress, such as occurs at arterial bifurcations and in curved sections of large arteries, causes a persistent increase in NFkB activation. Evidently, low shear stress enhances IKK activity via the induction of ROS, an effect that is blocked by certain antioxidants. [28]

Assuming that the pathogenesis and/or progression of OA involves pathomechanics, [29] and most patients with OA must continue to use the arthritic joint to some extent, it is likely that low but persistent or abnormally directed mechanical stress — or simply normal load that now exceeds the reduced capacity of the compromised tissue — is experienced by components of the osteoarthritic joint and may contribute to persistent NFkB activation. In support of this concept, mechanical compression has been documented to cause a significant increase in the expression of 5-lipoxygenase and leukotriene B4 by articular cartilage explants [30] — both of which are products of NFkB activation. [3]

Looking beyond the specific components and events, a wide-angle view of OA reveals a destructive cycle of inflammation and damage to the cells and extracellular matrix of the articular complex. Panning out even further, we see progressive loss of muscle mass and even bone density from chronic disuse, which renders the joint even more vulnerable to further damage under what would ordinarily be normal loads. If this destructive spiral is to be halted through (bio)chemical means, then the ideal target would be the central (i.e., upstream) regulatory proteins involved in the inflammatory response and in cell, tissue, and organismal homeostasis: NFkB.

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References

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14. Kumar S, Mehta K (2012) Tissue transglutaminase constitutively activates HIF-1 promoter and nuclear factor-kB via a non-canonical pathway. PloS One 7(11): e49321.

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23. Pilichou A, Papassotiriou I, Michalakakou K, et al. (2008) High levels of synovial fluid osteoprotegerin (OPG) and increased serum ratio of receptor activator of nuclear factor-kappa B ligand (RANKL) to OPG correlate with disease severity in patients with primary knee osteoarthritis. Clin Biochem 41(9): 746–749.

24. Attur M, Dave M, Abramson SB, et al. (2012) Activation of diverse eicosanoid pathways in osteoarthritic cartilage. Bull NYU Hosp Jt Dis 70(2): 99–108.

25. Ellabban AS, Kamel SR, Ahmed SS, et al. (2012) Receptor activator of nuclear factor kappa B ligand serum and synovial fluid level. A comparative study between rheumatoid arthritis and osteoarthritis. Rheumatol Int 32(6): 1589–1596.

26. Wu L, Huang X, Li L, et al. (2012) Insights on biology and pathology of HIF-1/-2, TGF/BMP, Wnt/-catenin, and NF-kB pathways in osteoarthritis. Curr Pharm Des 18(22): 3293-3312.

27. Zupan J, Komadina R, Marc J (2012) The relationship between osteoclastogenic and anti-osteoclastogenic pro-inflammatory cytokines differs in human osteoporotic and osteoarthritic bone tissues. J Biomed Sci 19:28.

28. Mohan S, Koyoma K, Thangasamy A, et al. (2006) Low shear stress preferentially enhances IKK activity through selective sources of ROS for persistent activation of NF-kB in endothelial cells. Am J Physiol Cell Physiol 292: C362–C371.

29. Felson DT (2013) Osteoarthritis as a disease of mechanics. Osteoarthritis Cartilage 21(1): 10–15.

30. Fermor B, Haribabu B, Weinberg JB, et al. (2001) Mechanical stress and nitric oxide influence leukotriene production in cartilage. Biochem Biophys Res Commun 285(3): 806–810.

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