br The involvement of DNA PK in the inflammatory

The involvement of DNA-PK in the inflammatory response Protein complex, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) regulates many genes involved in mammalian immune and inflammatory responses, apoptosis, cell proliferation, and differentiation in response to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral Phosphate Colorimetric Assay Kit [75], [76], [77]. Activated NF-κB unmasks the nuclear translocation and binding of NF-κB to specific κB consensus sequences in the chromatin as well as the activation of specific subsets of genes. Moreover, incorrect regulation of NF-κB has been linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development. For example, expression of VCAM-1 is critical for the initiation and progression of inflammatory diseases, such as asthma and arthrosclerosis [78], [79], as well as cancer [80]. The expression of VCAM-1 and other adhesion molecules plays an important role in both the recruitment of Th2 cells and the accumulation of eosinophils in allergic inflammatory foci [81]. Boulares group [82] has demonstrated that the NF-κB1/p50 NF-κB, a family member of type I NF-κB, is an excellent substrate for DNA-PK, which-dependent phosphorylation of NF-κB1 at serine 20 appears to enhance its binding to DNA as either a homodimer or a heterodimer with p65 NF-κB and the ability of NF-κB1 to drive the transcription of a number of additional NF-κB1-dependent genes, such as VCAM-1. Mutation of serine 20 completely eliminated phosphorylation of NF-κB1 by DNA-PK, and re-establishing wild type p50 NF-κB1, but not its serine 20/alanine mutant, in NF-κB1−/− fibroblasts reversed VCAM-1 expression after TNF treatment, demonstrating the importance of the serine 20 phosphorylation site in the induction of VCAM-1 expression. Furthermore, DNA-PK is also involved in the proinflammatory response initiated by CD158d signals. CD158d, also known as the killer cell immunoglobulin like receptor 2 DL4 (KIR2DL4), is a member of the killer cell immunoglobulin (Ig)-like receptor (KIR) family and is found in all natural killer (NK) cells and in some T cells. Unlike other KIRs, CD158d resides in Rab5-containing early endosomes and signals from this intracellular location [83]. Soluble ligand such as soluble HLA-G or an antigen-binding fragment (Fab) of an antibody to CD158d, is endocytosed by CD158d and induces a unique proinflammatory and proangiogenic response in NK cells [83]. Activation of resting human NK cells by CD158d stimulates the secretion of cytokines, but not cytotoxicity [84], [85]. The transcriptional response to CD158d signals includes the expression of cytokines, such as IL-1b, IL-6, tumor necrosis factor-a (TNF-a), and IL-23 and chemokines, such as IL-8, macrophage inflammatory protein-3a (MIP-3a), MIP-1d, and MIP-1a. Long group reported [86] that this CD158d-initiated proinflammatory response is mediated by the cascade activation of DNA-PKcs, Akt and NF-kB. In response to soluble agonist antibody or soluble HLA-G, CD158d interacts with DNA-PKcs in Rab5-containing endosomes. The CD158d signaling recruits Akt to endosomes and stimulates the DNA-PKcs-dependent phosphorylation of Akt at Ser473. In turn, Akt leads to the activation of NF-kB to trigger a proinflammatory response [87], [88], [89], [90]. However, in the proinflammatory response, both Ku subunits were not present in the CD158d-associated proteins identified by MS [86], which was consistent with the in vitro study data from the other independent group [91]. Since DNA-PK activates Akt through the phosphorylation of same residue of Ser473 in response to ionizing radiation [90], [92], [93], this phosphorylation may trigger the proinflammatory response, which may explain, at least partially, how ionizing radiation initiates inflammatory response in tissue.