Survival of an organism is dependent on its ability to rapidly and effectively respond to adverse changes in its environment. Eukaryotic cells possess a number of distinct signal transduction pathways that couple environmental stimuli to specific changes of gene expression. One such pathway is the transcription factor NF-KappaB (Nuclear Factor-KappaB), which is implicated in the regulation of many genes that code for mediators of apoptosis, viral replication, tumorigenesis, various autoimmune diseases and inflammatory responses (Ref.1). NF-KappaB is composed of homo- and heterodimeric complexes of members of the Rel (NF-KappaB) family: p50, p65 (RelA), c-Rel, p52 and RelB. The most common and best-characterized form of NF-KappaB is the p65/p50 heterodimer. Each dimer combination exhibits differences in DNA binding affinity and transactivation potential. The activation of NF-KappaB is thought to be part of a stress response as it is activated by a variety of stimuli that include growth factors, cytokines, such as TNF (Tumor Necrosis Factor) and IL-1 (Interleukin-1) lymphokines, components of bacterial cell walls, such as LPS (Lipopolysaccharide), UV, pharmacological agents, and stress (Ref.2).
Depending on the stimulus, the mechanism of activation involves overlapping and nonoverlapping steps. Among all the stimuli, perhaps the most is known about the mechanism by which TNF
activates NF-KappaB. In its inactive form, NF-KappaB is sequestered in the cytoplasm, bound by members of the I-KappaB family of inhibitor proteins. The activity of NF-KappaB is regulated by inhibitory proteins, which include I-KappaB-Alpha, I-KappaB-Beta, I-KappaB-Gamma, I-KappaB-Epsilon, Bcl3, p105, and p100. I-KappaB proteins are phosphorylated by I-KappaB Kinase complex consisting of at least three proteins, IKK-Alpha, IKK-Beta, and IKK-Gamma (Ref.3).
NF-KappaB pathway involves the interaction of the ligand with its receptor at the cell surface (TNFR), which then recruits a protein called TRADD (TNF Receptor-Associated Death Domain). This protein binds to TRAF2 (TNF Receptor-Associated Factor-2), which activates RIP (Receptor-Interacting Protein). RIP interacts with MEKK
(Mitogen-Activated Protein Kinase Kinase) and NIK (NF-KappaB-Inducing Kinase) to phosphorylate and activate the IKK (I-KappaB-Alpha kinase complex). The IKK complex phosphorylates I-KappaB-Alpha, which leads to ubiquitination and then leads to the degradation of I-KappaB-Alpha by the proteosome, resulting in the translocation of NF-KappaB to the nucleus. In the nucleus it binds to its consensus sequence (5-GGGACTTTC-3) and positively regulates the transcription of genes involved in immune and inflammatory responses, cell growth control, and apoptosis. Genes encoding cytokines, cytokine receptors, cell adhesion molecules, chemoattractant proteins, and growth regulators are positively regulated by NF-KappaB (Ref.4). TRAF2 also interacts with A20, a zinc finger protein whose expression is induced by agents that activate NF-KappaB. A20 functions to block TRAF2-mediated NF-KappaB activation
IL-1 has similar downstream affects through NF-KappaB, including immunoregulation, proinflammatory, and hematopoietic activities. IL-1 induced signaling is mediated through association of IL-1 receptors with adaptor proteins, such as IRAK
(IL-1 Receptor-Associated Kinases) and MyD88 (Myeloid Differentiation Primary Response Gene-88). Activation of NF-KappaB by bacterial LPS promotes the upregulation of proinflammatory cytokines. Following recognition of LPS, the adapter protein MyD88 is recruited to the cytoplasmic domain of TLR4 (Toll Like Receptor-4). MyD88 contains a highly conserved DD (Death Domain) that facilitates its interaction with another DD-containing signaling molecule, IRAK. Following recruitment to MyD88, IRAK undergoes rapid autophosphorylation and dissociation from the signaling complex. Phosphorylated IRAK subsequently interacts with TRAF6, initiating the activation of a kinase cascade involving NIK and IKK. Activation of this cascade culminates in the phosphorylation and degradation of the I-KappaB, enabling NF-KappaB to translocate to the nucleus and promote new gene expression (Ref.5). NF-KappaB-dependent gene expression involves a growing family of proteins termed transcriptional coactivators that probably function by facilitating or bridging the sequence-specific activators to the basal transcriptional machinery and altering chromatin structure. The p65 component of NF-KappaB binds to the coactivator CBP (cyclic AMP response element binding protein [CREB]-binding protein) and its structural homolog p300. Phosphorylation of p65 by PKA (Protein Kinase-A) stimulates NF-KappaB-dependent gene expression by enhancing p65 association with CBP. Recent observations have shown that levels of the CBP homolog, p300, are limiting relative to those of p65 and that competition for CBP may regulate p65 transactivation. Deacetylation of p65 through specific interactions with HDAC3 (Histone Deacetylase-3) promotes effective binding to newly synthesized I-kappaB-Alpha, which subsequently leads to I-KappaB-Alpha dependent nuclear export. Overexpression of the p110 catalytic subunit of PI3K (Phosphoinositide-3 Kinase) also induces p65 -mediated transactivation and that the specific PI3K inhibitor LY294002 represses this process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also induces p65/RelA-mediated transactivation. NF-KappaB pathways provide many targets for developing specific drugs to treat inflammatory diseases. Inappropriate activation of NF-KappaB has been linked to inflammatory events associated with autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, atherosclerosis, and AIDS.