NF-KB septic shock, viral infections and diseases of

NF-KB family and regulation

The transcription factor NF-kB can be found in all
cell types (Oeckinghaus, Ghosh, 2009). NF-KB family is involved in all cell
reactions to stimuli, such as stress, cytokines, free radicals, ultraviolet
irradiation and attack from bacterial or bacterial antigens. Thus, they control
a range of genes in response to changes in the environment. NF-kB plays a key role
in regulating the immune response to infections and its dysfunctions have been
linked to cancer (such as multiple myeloma), inflammatory processes, autoimmune
diseases, septic shock, viral infections and diseases of the immune system (Oeckinghaus,
Ghosh, 2009). NF-?B
is an important regulator of proinflammatory gene expression (Tak and
Firestein, 2001). Transcription of proteins linked to the inflammatory
processes is controlled through NF-KB. Furthermore, this transcription factor
has a role in the physiology of the bone, skin, central nervous system and
embryonic development, The NF-KBNF-kB is also considered to be involved in
synaptic plasticity and memory processes. Since NF-KB family controls all these
processes, a dysregulation of NF-KB pathway can cause tremendous diseases, e.g.
arthritis, immunodeficiency, autoimmunity or cancer. In mature B cells,
macrophages, neurons and vascular smooth muscles, or in unhealthy cells like
tumor cells, NF-KB is constitutive (Oeckinghaus, Ghosh, 2009). Studies in
knockout mice have shown that a dysfunction in the expression of RelA causes
embryonic lethality and liver degeneration, while dysfunctions in p50 and RelB
cause immunodeficiency, such as abnormal mitogen responses and antibody
production (Tak and Firestein, 2001). RelB is implicated in the development and
differentiation of dendritic cells, and a mutation disrupting relB impairs
antigen presentation.
C-Rel and p52 have a role in the immune function. P50/p52 double knockout mice
exhibit impaired development of osteoclasts and B cells. P50 homodimers
activate IL-1–induced collagenase expression in synoviocytes, binding to a critical
NF-?B–like binding site. Both p50 and p65 play a role in constitutive IL-6
production in rheumatoid arthritis (RA) synovial fibroblasts, whereas p65
activation by thrombin regulates ICAM-1 expression in endothelial cells.
Heterodimers of p50 and p65 have a role in activation of inflammatory genes by
IL-1 or TNF-? in human monocytes, and these effects are blocked by the anti-inflammatory
cytokine IL-10 (Tak and Firestein, 2001).

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NF-kB activity is influenced at multiple levels and
the control activity of this transcription factor is highly regulated. Post-translational
modifications of various core components of this signaling pathway are involved (Oeckinghaus,
Ghosh, 2009).  Five proteins compose the
NF-KB family: p65 (RelA), RelB, c-Rel, p105/p50 (NF-KB1), and p100/52 (NF-KB2).
These proteins associate each other forming homodimers or heterodimers and the
Rel homology domain, a shared 300 amino acid long domain, is required for
dimerization, as well as DNA binding, nuclear translocation and interaction
with IkBs (inhibitor of NF-KB). NF-KB regulates its regulators (IKBa, P105 OR
a20) itself (Oeckinghaus, Ghosh, 2009). This generates in turn an
auto-regulatory feedback loops. IkBs (IkBa, IkBb and IkB1, or p100 and p105)
retain NF-kB in an inactive form in the cytosol in a dimeric form, so that the
IkB proteins have a crucial role in signal responsiveness. Upon stimulation,
BcI-3 and IkBz can be induced. These two proteins regulate the activity of the
dimers of NF-Kb after the nuclear translocation. The IkB kinase complex
phosphorylates IkB proteins degrade the inhibitors, and then NF-kB migrates to
the nucleus and the transcription of target genes is stimulated (Oeckinghaus,
Ghosh, 2009). I?Ba has a role in the transient activation of NF-kB, it works binding
the p50-p65 heterodimer and the p50 homodimer, although it does not inhibit DNA
binding activity of the latter (Tak and Firestein, 2001). I?Bb has a role in the
sustained activation. It
inhibits p50-p65 more strongly than it inhibits p50-RelB and p50-c-Rel
complexes, whereas I?Ba has similar effects on these complexes (Tak and
Firestein, 2001). For the regulation of NF-KB, degradation of IkBs and nuclear
translocation are important steps, whereas it needs something more to elicite a
maximal NF-kB response. Post-translational modifications such as phosphorylation
by protein kinase A (PKA) is critical for p65 driven gene expression of many
target genes (Oeckinghaus, Ghosh, 2009).

Synthesis of cytokines (e.g. TNF-?, IL-1?, IL-6, and
IL-8), is mediated by NF-?B, as is the expression of cyclooxygenase 2 (Tak and
Firestein, 2001). In patients with RA and osteoarthritis, IKK in primary
fibroblast-like synoviocytes isolated from synovium were studied. In these
cells, immunoreactive IKK protein is abundant and IKK-? and IKK-? are
constitutively expressed at the mRNA level. IKK function in these cells can be greatly enhanced by
TNF-? and IL-1, leading to degradation of endogenous I?B? and nuclear
translocation of NF-?B. Activation of this pathway and the consequent induction
of IL-6, IL-8, ICAM-1, and collagenase-1 expression depends specifically on
IKK-?. Thus,
transfection with adenoviral constructs encoding an IKK-? dominant-negative
mutant prevents TNF?–mediated NF-?B nuclear translocation and proinflammatory
gene expression in synoviocytes, whereas dominant-negative IKK-?mutant has no
effect (Tak and Firestein, 2001).

Inflammation

A coordinate activation of signaling pathways
characterise the inflammatory response. This turns in the regulation of the
expression of pro-inflammatory and anti-inflammatory mediators (Lawrence,
2009). To activate NF-kB, phosphorylation, ubiquitination and proteolysis of
IkB are required (Oeckinghaus, Ghosh, 2009). Then, NF-kB is transferred to the
nucleus and here it binds to specific sequences of the promoters. Cytokines are
usually synthesised de novo and not
stored (Blackwell and Christman, 1997). Thus, after an inflammatory stimulus,
the cytokine genes are transcripted and NF-kB is crucial for this step since
this transcription factor upregulates the transcription of a specific set of
cytokine genes. There
are many factors that determine the production of cytokine in response to an
inflammation. According to Blackwell and Christman (1997), NF-kB has an
interaction with the glucocorticoid receptor. Moreover, rate of
posttranscriptional RNA processing, mRNA stability, and translation efficiency
have a role in the network of cytokines. The p65 subunit of NF-kB and NF-IL6
directly associate with each other and cooperatively transactivate IL-6 gene
and IL-8 gene expression. Inflammation is usually associated with their coordinated
production and action, e.g. in the sepsis syndrome the bacterial endotoxin and
other toxic products stimulate rapid production of TNF-a and IL-1b (Matsusaka et
al., 1993). The p65 and p 50 homodimers, and the p50-p65
heterodimers can recognise the motif GGGATTTCC of the IL-6 gene. IL-6 and IL-8
are cytokines secreted by cells such as macrophages in response to other cytokines
after an infection (Blackwell and Christman, 1997). To activate the IL-6 gene,
p50 and p65 need the presence of NF-IL6, otherwise the bond of the NF-KB
subunits with the gene is absent. An increase in the amount of p50 inhibits the
cooperative effect of p65. Moreover, it acts with NF-IL6 to stimulate the
expression of IL-6, even though p65 is the most effective subunit (Matsusaka et
al., 1993).  

The transcriptional activation of IL-8 and IL-6
requires a
cooperative binding with the transcription factor NF–IL6 (Blackwell and
Christman, 1997). In
addition to the IL-6 gene,
for regulation of many immune response and acute-phase response of the genes
IL-8, TNF-a, IL-1,8, granulocyte colony stimulating factor, immunoglobulin K
light chain, and serum amyloid A-, the NF-KB binding sites are important (Matsusaka et
al., 1993). The IL-6 production is constitutively activated by the
transactivator protein Tax of the human T-lymphotrophic leukemia virus 1. The
protein Tax activates NF-KB that causes in part the constitutive production of
IL-6. Furthermore, this protein synergistically increases the activation of the
IL-6 transcription by NF-IL6. In the study made by Matsusaka et al. (1993) it was suggested that
through the protein Tax, the activation of these two factors can mediate the
constitutive expression of IL-6. Through the NF-kB and the NF-IL6 binding
sites, the transcription of the IL-8 gene is stimulated by the X product of
hepatitis B virus. The viral expression can be regulated by NF-IL6 and NF-KB
that bind the regulatory regions. Furthermore, transcriptional factors
controlling genes encoding inflammatory cytokines, including NF-KB and NF-IL6,
are constitutively activated in other pathological conditions such as
malignancies and autoimmune diseases since constitutive expression of inflammatory
cytokines are observed in these cases (Matsusaka et al., 1993).

Chromatin and NF-kB
influence each other

One of the key regulatory mechanism is the dynamic alteration of the chromatin
state. The chromatin environment at stimulus-responsive NF-?B sites is a major
determinant in transcription factor binding (Bhatt and Ghosh, 2014). The
chromatin state is also influenced by NF-kB through a variety of mechanisms.
The chromatin is recruited modifying co-activator complexes such as p300, the
competitive eviction of negative chromatin modifications, and the recruitment
of components of the general transcriptional machinery. In their work, Bhatt
and Ghosh (2014) explain how the induction of the human interferon-b (IFNB)
gene in response to viral infection is an example of transcription factor
synergy. The regulation of the
IFN? enhanceosome is an example of transcriptional regulation through the
combinatorial control of multiple transcriptional factors. To summarise, three transcription factors (NF-kB, IRF3 / IRF7
and ATF-2 / c-JUN) associate each other and allow the expression of this gene,
which is dependent on the stimulus. The first event is the binding of NF-kB to
the PRDII element stored in the promoter, which in turn facilitates the recruitment
of IRF and ATF-2 / c-Jun (Bhatt and Ghosh, 2014). These transcription factors
are assembled at the promoter and they have a role as a platform for the
sequential recruitment of the PCAF chromatin modifying complex, the p300/CBP
acetyltransferase, and subsequently the SWI/SNF chromatin remodeling complexes (Bhatt and Ghosh, 2014).  SWI/SNF remodels the downstream nucleosome
that encompasses the TATA box, thus allowing TBP binding and subsequent
pre-initiation complex assembly. With the concerted work of these factors the presence
of a chromatin barrier can be resolved (Bhatt and Ghosh, 2014).