Function of NF-kB
Nuclear factor NF-?B encompasses an important family of which plays a central role in immunological processes by inducing expression of a variety of genes involved in inflammatory responses. NF-?B appears to be a master switch, or control point, for the expression of a large number of proinflammatory genes, including several cytokines, chemokines and adhesion molecules. It is a rapidly-induced stress responsive transcription factor that functions to intensify the transcription of a variety of genes including COX-2 and is linked to the .
The members of the NF-B family include p50 (NF-kB1), p52 (NF-kB2), p65 (RelA), RelB, v-Rel, and C-Rel. In cellsf, NF-kB exists as homo or heterodimers with distinct DNA binding specificity. A heterodimer composed of p50 and p65 subunits is the most common dimer. p65 homodimers have different binding specificites from p50/p65 heterodimers. p65 homodimers preferentially bind GGGRNTTTCC which is different form the p50 homdimer consensus sequence (GGGGATYCCC), and the DNA motifs that bind p65 homodimers bu tnot p50 homodimers do not bind p50/065 hterodimers well.
RelA, RelB, and C-Rel-partnering with p50-each play unique and complementary roles in the process of myeloid.
NF-kB is also a key factor for the pathogenesis of allograft rejection. Thus pharmacological agents which inhibit its activation are currently being considered as in transplantation therapies. The most important procedures of allograft rejection, including ischemia/reperfusion injury, recipient T cell activation and DC maturation are regulated by NF-kB.
Given the role of NF-?B in cell proliferation and survival, it is not surprising that constitutive NF-?B signaling has been implicated in oncogenesis. Chromosomal amplification and rearrangements of genes encoding NF-?B family members have been identified in many hematolymphoid and solid tumors, including NHL, multiple myeloma, and carcinomas of the lung and breast.
NF-kB Structure
NF-kB is dimeric transcription factor generated from combinations of 5 family members Rel (c-Rel), p65 (Rel A), RelB, p50/p105 (NFkB1), and p52/p100 (NFkB2) all of which contain an evolutionary conserved Rel homology domain that includes a DNA binding and dimerization motifs as well as the nuclear localization signal. The Rel homology domain is necessary for DNA binding, nuclear translocation, and protein-protein interactions. The consensus kB DNA binding sequence is 5-GGGRNNYYCC-3′ where R is purine and Y is pyrimidine.
Rel-A, c-Rel and Rel-B also have carboxy-terminal transactivating domains. In contrast to p65, RelB, and c-Re. the p50 and p52 subunits lack a transactivation domain and are produced either by processing of the respective precursors NF-kB1 (p105) and NF-kB2 (p100) or by a cotranslational mechanisms.
These proteins are widely expressed and regulate transcription by binding to decameric sequences ?B motifs that control transcription, particularly of proteins involved in immune and inflammatory responses.
NF-?B Dimers are kpet in cytoplasms by ikB family
As homo or heterodimers they are held latently in the cytoplasm bound to inhibitory proteins, called IkB. The IkB family are ankyrin-repeat containing proteins consisting of IkBalpha, IkBbeta, IkBepsilon, IkBl, the NF-kB precursors p105 and p100 and Bcl3.
Bcl-3 interacts with the intrinsically inactive p50 and p52 homodimers in the nucleus and is not degraded after cellular stimulation. Depending on phosphorylation and concentration of Bcl-3, it can act either as an IkB, dissociating p50 and p52 homodimers from DNA or as a coactivator that is recruited to DNA by p50 and p52 homodimers. Additionally, Bcl-3 regulates the subcellular location of p50 homodimers. The overexpression of Bcl-3 in pro-B cells results in augmented amounts of p50 homodimers in the nucleus, while internally induced Bcl-3 causes an export of p50 homodimers from the nucleus to the cytoplasm in petatocytes.
How is NF-?B molecularly turned on?
Overview: NF-?B is present in the cytosol as a heterotrimer usually consisting of its p50 and p65 subunits bound to its inhibitory protein IkB. In response to cell stimulation, a multi-subunit protein kinase, the kB kinase (IKK), is rapidly activated and phosphorylates two serine in the N-terminal regulatory domains of the IkBs, causing a dissociation of the NFkB-IkB? complex. The inducible phosphorylation of IkB is mediated by three identified IkB kinases (IKK?, ?, and ?). Phosphorylation of IkB can also occur by PKC. Phosphorylation of IkB by PKC or IkB kinase (IKK) results in its degradation and dissociation from the NF-?B complex. Upon the phosphorylation and subsequent degreadation of IkB, NF-?B activates and translocates to the nucleus, where it activates transcription from kB sites.
A variety of stress stimuli, including lipopolysaccharide (LPS), tumor necrosis factor ?, IL-1, ceramide, phorbol myristate acetate (PMA), hydrogen preoxide and ultraviolet light have been identified to cause the activation and nuclear translocation of NF-?B .
In macrophages, (NF)-?B can be activated by exposure to LPS as well as by inflammatory cytokines (TNF-? and IL-1) and viral infections. Mature DCs express high levels of the (NF)-?? family of transcription factors and signaling by members of the TNF-? family, such as CD40 and RANK, results in activation of (NF)-?B.
Phosphorylation and activation of IKK: Activation of NF-?B through TNFR family members is mediated by the TRAF molecules. TRAFs are adaptor proteins that promote intracellular signal transduction linked to NF-?B activation by binding to receptors and recruiting other proteins to form an activate signaling complex that ultimately triggers the activation of the of the I?? kinase complex (IKK). IKK then phosphorylates 2 serine residues in the N-terminus of I??, allowing for the ubiquitinization and degradation of I??, and release of NF-?B. NF-?B is then able to translocate to the nucleus where it stimulates the transcription of a wide variety of genes, including cytokines, cell adhesion molecules, and acute phase response proteins, which are involved in proliferation and survival as well as the inflammatory response.
Six distinct TRAF molecules have been identified, termed TRAF1 through TRAF6. Signal transduction via TRAF2, TRAF5, and TRAF6 is linked to NF-?B activation. TRAF2, and TRAF5 bind to many of the same receptors, including TNFRII, CD30, CD40, and RANK, and can also transduce signals from EBV LMP-1. TRAF6 binds to a more restricted group of receptors that includes CD40 and RANK.
Phosphorylation and Degradation of IkB: The activation of NF-?B by extracellular inducers depends on the phosphorylation and subsequent degradation of IkB proteins. Activation of NF-?B is achieved through the action of a family of serine/threonine kinases known as IkB kinase (IKK). The IKK contains two catalytic subunits IKKalpha and IKKbeta and a regulatory/adaptor protein NEMO (also called IKKy). The IKKalpha and IKKbeta phosphorylate IkB proteins and members of the NF-?B family. All IkB proteins contain two conserved serine residues within their N terminal region, which are phosphorylated by IKK. These phosphorylation events lead to the immediate polyubiquitination of IkB proteins and rapid degradation by the proteasomal pathway. Inhibitors of IKK have long been sought as specific regulators of NF-?B.
Translocation of NF-kB to the Nucleus: Early in DC maturation, three types of dimers are translocated to the nucleus, p65/p50, c-Rel/p50 and p50/p50, p50 being the common subunit. Accordingly, mice lacking p50 or the p105 precursor are unable to effectively clear Listeria monocytogenes are are more susceptible to S. pneumoniae.
The major form of NF-?B that is rapidly induced after stimulation is the RelA/p50 complex, and this dimer is what is commonly referred to as NF-?B. NF-?B is normally sequestered in the cytoplasm of nonstimulated cells and must be translocated into the nucleus to function. The subcellular location of NF-?B is controlled by a family of inhibitory proteins knowns as I?Bs, which bind NF-?B and mask its nuclear localization signal thereby preventing its uptake into the nucleus.
Alternative NF-kB Activation Pathways:
Ik-B-independent Pathways: There is accumulating evidence that NF-kB is also subject to an IkB-independent level of regulation. Increasing evidence suggests that signaling pathways, which by themselves are not involved in nuclear translocation of NF-kB, are needed for its transcriptional activity. For example, neither p38 nor c-Jun-N-terminal-kinase (JNK) directly phosphorylates NF-kB. However, inhibition of either pathway (using dominant negative mutants, antisense RNA or specific inhbitors) strongly reduces transcritpion from both NF-kB-dependent cytokine promoters and promtoers controlled by synthetic NF-kB bnding sites. Neither nuclear translocation nor DNA binding of NF-kB were affected by the inhibition of these MAP kinase pathways, suggesting that p38 and JNK exert their effects by enhancing the transactivation function of NF-kB. (p38 MAP kinase inhibitor SB203580 does not interfere with induce nuclear translocation and DNA binding of NF-kB, but significantly inhibits NF-kB-dependent gene expression) Moreover, selective activation of the NF-kB pathway in transfected cells only weakly induces transcription of the Il-8 gene, which contains an essential NF-kB-binding site. For maximal IL-8 transcription, a co-stimulatory signal from the JNK pathway is required.
There is strong evidence that the phosphatidylinositol-3 (PI3) kinase pathway is involved in IkB-independent regulation of NF-kB. Although the PI3 kinase inhibitors wortmannin and LY 294002 have no effect on inducible NF-kB nuclear translocation, LY294002 prevents the IL-1 induced increase in p65 phosphorylation.
Regulation of NF-kB
NF-kB activity is regulated by a family of cytoplasmic inhibitory proteins, inhibitor of k-B (IkBs), which intereact with NF-kB and prevent its nuclear translocation.
Inducers of NF-kB
NF-kB mediated signaling is induced by a multiplicity of external stimuli (cytokines/growth factors, bacterial or viral products, preapoptotic or necrotic stimuli, UV-irradiation, reactive oxygen species, and other) acting through different signal transduction pathways and causing phosphorylation of IkBs, their subsequent proteolytic degradation, and release of active NF-kB for nuclear translocation.
Measuring NF-kB
NF-kB binding: P50 and RelB DNA binding can be detected by ELISA using a Mercurity transfactor p50 kit (clontech, CA).
NK-kB inhibitor BAY11 7082: Bay has been shown to block TNFalpha stimulated NFkB translocation through inhibition of IkB?.