North Leigh, Oxon, UK
Introduction
Nuclear factor kappa B (NFB) is a rapid response transcription factor that works to ensure survival of all cells that might be subjected to environmental stress, injury, inflammation or immune reactions. NF
B activation leads to control of genes for the expression of cytokines and chemokines, immunoreceptors, cell adhesion molecules, growth factors or acute phase proteins. Thus, directly or indirectly, NF
B controls a wide variety of biological responses, in particular those that are part of innate and adaptive immunity. Cellular expression of NF
B is constitutive in B and T lymphocytes, in monocytes and neurones, or it is inducible in other cells.
NFB structure and function
NFB was discovered by Sen and Baltimore (1986) in mature B cells as a nuclear transcription factor, which binds to an element in the
-immunoglobin light chain enhancer. Soon it was found that NF
B occurs in most cells as an active cytoplasmic form, consisting of two subunits of 50 kDa and 65 kDa that are bound to an inhibitor protein termed I
B. The p65 is RelA, which has a conserved amino-terminal segment called the Rel homology domain. This is responsible for the I
B interaction or after activation for dimerization, nuclear translocation and DNA binding. It contains a nuclear localization signal which facilitates translocation of NF
B into the nucleus.
Five members of mammalian NFB/Rel proteins have been identified [1,2]. The group II proteins include RelA (p65), RelB and c-Rel. The group I proteins include p50 (NF
B-1) formed by proteolysis of precursor protein p105, and p52 (NF
B-2) formed by proteolysis of precursor p100.
So, NFB is a heterogeneous collection of DNA binding dimers, homodimers and heterodimers, composed of various combinations of the NF
B/Rel multigene family [1,2]. NF
B (p65/p50) is maintained in the cytoplasm of cells through interaction with the inhibitor protein I
B. Inducing factors act on I
B kinase (IKK), with two catalytic subunits IKK
and IKKß that are able to phosphorylate I
B. The phosphorylated inhibitor unit is tagged by ubiquitin for subsequent proteolysis. The freed NF
B complex is thus able to translocate into the nucleus (Figure 1
).
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There has to be good control over such a powerful and broadly determining system. Proteins named B-Ras1 and
Bras2 are identified, that interact with the inhibitor proteins I
B
and I
Bß and decrease their rate of degradation. They explain how I
Bß degrades slower than I
B
. Furthermore, heat shock proteins can boost I
B
levels.
Many negative feedback loops ensure that activation of NFB is transient and self-limiting [6]. NF
B itself induces the gene for the inhibitor I
B
(Table 2
), so allowing its rapid resynthesis following degradation. Yet when the cytokines IL-1ß and TNF
are being produced, there is a positive feedback loop to sustain their output [6]. TNF
production in macrophages is dependent on NF
B.
Upstream activation of the IB kinase complex
The IB kinase complex consists of IKK
/ß and IKK
(Nemo) [2]. As in Figure 2
, signals from the TNF-receptor will activate NIK, NF
B inducing kinase, and signals from the IL-1 receptor activate TAK-1, an MAPKK kinase. Thus, this level of subreceptor signalling is closely linked to the initiation of cascades [7] through MAPKK enzymes (also called MEKKs) and the cascade becomes MAPKK which activates MAPK. Specificity, in these apparently overlapping pathways, is conferred by means of scaffold proteins. The small GTPases like RhoA, Rac1 and cdc42, whose function is to reconstruct the cytoskeleton, activate the I
B kinase complex through MEKK1. Protein kinase (PK) C
is another activator. In T lymphocytes, calcineurin and Raf-1, PKD and PKC
[8] lead to NF
B activation.
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Measuring NFB
A nuclear extract of the relevant cell type can be used for blot or gel studies. One can show NFB p50 by western blot analysis. The electrophoretic mobility assay (EMSA) measures the ability of active NF
B to bind to specific DNA sequences. Changes in the mobility of DNA probes containing
B sites can be assessed when incubated with nuclear extract. The nuclear extracts are mixed with double stranded 32-dATP oligonucleotide carrying the decameric NF
B binding site. Electrophoresis through 5% polyacrylamide gel is carried out. When nuclei have been activated by a cytokine like IL-1, autoradiography shows that NF
B p50 has shifted. Confirmation of the specificity is provided by incubation with specific antibody against Rel/NF
B proteins that identify a super-shift. Control lanes are run in which (i) unlabelled probe serves as cold competitor and (ii) mutated oligonucleotides are used as specific DNA competitor [13]. EMSA is specific and reproducible but only semiquantitative. It is expensive and time consuming and radioactivity is used.
Alternatively, a Biacor optical biosensor can be used to determine activated NFB in a few minutes. Biotinylated NF
B sense and antisense consensus sequences are hybridized and captured onto a streptavidin-coated sensor chip. Nuclear extract is passed over the captured sequence and, when activated NF
B is present, a signal is generated.
Immunofluorescence microscopy is used to show that p50 has translocated from the cytosol into nuclei in IL-1 activated endothelial cells [13]. For leucocytes, flow cytometry will detect bound NFB within nuclei. With laser scanning cytometry the presence of p50 in cytoplasm and nuclei is detected with FITC-labelled antibody by measuring green fluorescence. Large numbers of cells can be analysed rapidly. Hernandez Presa et al. [14] have used non-radioactive probes for immunohistochemistry in paraffin wax embedded tissues.
NFB directed genes that are induced by TNF
have been analysed by RNase protection assays to show that MCP-1 is induced within 2 h and then builds up. MIP-2 is induced at 2 h but then is suppressed, and Rantes appears after 8 h.
Pathophysiological activation of NFB
Inflammation: glomerulonephritides
As is apparent from the list of genes that are activated (Table 2), NF
B must play a vital role in acute and chronic inflammations. NF
B mediates the release of chemokines that bring phagocytes, lymphocytes and natural killer cells to sites of inflammation and the release of the cytokines that activate them. The EMSA assay has been used to demonstrate how NF
B is involved in macrophage activation [15], and how NF
B induced upregulation of antiapoptotic proteins helps their survival. Clearly this is pertinent to glomerulonephritides. Th-1 lymphocytes depend on NF
B for their functioning, whereas Th-2 cells do not [16]. The process is so relevant to inflammatory arthritis that inhibition of p50 NF
B is being pursued as a possible therapeutic strategy.
Reactive oxygen species (ROS) released intracellularly are regarded as second messengers for activation of NFB [10]. In an inflammatory or allergic focus, phagocytes will be releasing ROS and the glutathione content of adjacent cells will be lowered. Under such circumstances the action of ceramide is enhanced [17] and leads to NF
B activation. Phagocytes may release chloramines which cause activation of NF
B in T cells, for example. Yet if they release taurine-chloramine, the effect is anti-inflammatory! Mesangial cells are not well protected against products of oxidation like 4-hydroxynonenal [18], which partly explains the liability to glomerosclerosis.
A central role of NFB activation in endothelial, mesangial and epithelial cells will account for expression of adhesion molecules ICAM-1 and VCAM-1 that aid influx of leucocytes, for release of the pro-inflammatory cytokines IL-1ß, TNF
and IL-6, for release of chemokines MCP-1, MIP
/ß and IL-8, and for induction of enzymes iNOS, Cox-2 and phospholipase A2 [19]. All of this is integral to glomerulonephritis.
Release of chemokines is determined in various ways. The promoter region of the MCP-l gene has an AP-1 transcription factor binding site. There and in the enhancer regions are B binding sites and Sp-1 binding sites. Thus induction of MCP-1 can depend on the cooperative action of NF
B and AP-1 [20]. Rantes chemokine expression requires cooperativity of NF
B with interferon regulatory factor.
The abnormally glycosylated IgA that circulates in IgA nephropathies will activate NFB of human mesangial cells [21]. That and the release of PDGF are downregulated by ACE inhibitors [21]. The florid expression of fibrin in lupus nephritis hinges on PAI-1 expression that is in part determined by NF
B, albeit mainly by TGFß [22].
Proteinuria and tubulo-interstitial injury
Albumin that is reabsorbed by proximal tubular cells activates NFB so that there is then release of chemokines MCP-1 [23] and Rantes. The attraction of inflammatory cells into the renal interstitium [23] and their release of cytokines, in particular fibrokine TGFß, presages medullary fibrosis and decline of renal function. Inhibition of NF
B protects against proteinuria induced interstitial injury [24].
Angiotensin II and vascular damage
Angiotensin II produces vascular damage by various means. Both AT1 and AT2 receptors stimulate NFB, and NF
B inhibition ameliorates damage. As well as ACEI/ATRA therapy, use of statins is protective [25].
Ischaemia-reperfusion injury
Ischaemia-reperfusion injury of the renal tubules, as happens during cardiopulmonary bypass or during exposure to lipopolysaccharide, results in the release of TNF by a NF
B-mediated reaction [26]. Both superoxide anions and nitric oxide play their part in the damage. Significant tubular injury must be mediated by iNOS, whose activation is NF
B mediated, because it does not occur in iNOS knockout mice [27]. It has been assumed that neutrophils were the source of ROS following ischaemia-reperfusion but the new evidence is that monocyte-macrophages predominate [28]. Undoubtedly, oxidative stress is the reason for NF
B activation, because it occurs in so many other tissues, as demonstrated in right atrial tissue taken at cardiopulmonary bypass [29]. The oxidative stress-induced NF
B activation is not associated with I
B
degradation (cf. Figure 1
).
Infections
When there is cystitis or pyelonephritis, release of lipopolysaccharide from bacteria will cause NFB activation and chemokine and cytokine release [30]. Bacterial cell walls via their outer membrane proteins (OMPs) activate Toll 2/4 receptors (Figure 2
) and thereby NF
B. In unusual infections like leptospirosis, NF
B plays a major role in the inflammatory process [31]. In SIRs or septic shock the NF
B of circulating leucocytes is elevated in rough proportion to the APACHE score of the patients. NF
B determines the expression of tissue factor and so the liability to DIC [32]. If there is repeated exposure to small doses of endotoxin, as might happen in intensive care unit patients, NF
B p65/50 of leucocytes becomes p50/50 and there is endotoxin tolerance. In rats that condition prevents neutrophil-induced lung inflammation [33].
Diabetes-related pathophysiology
High glucose generates ROS in mesangial cells, and they upregulate NFB and AP-1 as well as their expression of TGFß [34]. Oxidized LDL activates NF
B in endothelial and mesangial cells via the intermediary of ROS [35]. High glucose activates NF
B in vascular smooth muscle cells too [36]. Advanced glycation end products (AGEs) act as ligands for receptor RAGE, a novel member of the immunoglobulin superfamily, whose ligation creates ROS and activates NF
B. Expression of adhesion molecules ICAM-1/VCAM-1 is relevant to diabetic vascular disease. In experimental diabetic nephropathy there is enhanced activity of tissue transglutaminase (M. El-Nahas, unpublished data) and that could be a NF
B-mediated process.
Alcoholism
Clinically relevant ethanol concentrations potentiate TNF-inducible NF
B. Acetaldehyde is known to activate NF
B [37,38]. High alcohol intake is certainly associated with glomerulonephritides and HIV liability.
Dehydration: renal papillary damage
In response to water deprivation Cox2 cyclooxygenase is increased in renal medullary interstitial cells. Water deprivation and hypertonicity activate NFB and the consequent expression of Cox2 favours the survival of interstitial cells under hypertonic conditions. The implication is that inhibition of Cox2 could predispose to NSAID-induced papillary damage [39].
Notes
Correspondence and offprint requests to: E. N. Wardle, 21 Common Road, North Leigh, Oxon OX8 6RD, UK.
References
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