Abstract 13252: DNA-Dependent Protein Kinase (DNA-PK) Regulates TNF-Induced VCAM-1 Expression Independently of its Function in DNA Repair: A Novel Role of DNA-PK and its Involvement in Athrogenesis
Vascular cell adhesion molecule (VCAM)-1 plays a dominant role in the initiation of atherosclerosis. Our group has recently reported that the DNA-dependent protein kinase (DNA-PK) regulates TNF-induced expression of VCAM-1 in glioblastoma cells via phosphorylation of p50NF-kppaB at serine-20. Such modification is crucial for the interaction of the transcription factor with p65NF-kappa B and VCAM-1 gene promoter occupancy. In this study, we examined the role of DNA-PK in VCAM-1 expression in vascular cells and the mechanism by which TNF-kappa activates the kinase. DNA-PK is required for TNF-induced VCAM-1 expression in human vein (HUVEC) and aortic (HAEC) endothelial cells. Inhibition of DNA-PK kinase activity blocked p50NF-kappa B phosphorylation at serine-20 using newly-generated antibodies targeting the phosphorylated form of the protein. Inhibition of DNA-PK markedly reduced monocyte adhesion to TNF-stimulated endothelial cells strongly suggesting a potential involvement in atherosclerosis. Indeed, phosphorylated p50 NF-kappa B at serine-20 was detected in human atherosclerotic plaques in. Interestingly, TNF-induced VCAM-1 expression occurred independently of DNA breaks/repair, as TNF treatment caused no double-stranded breaks (DSB) as assessed by comet assay or phosphorylation of histone H2AX(gamma), a DNA repair marker. Additionally, VP-16, a DNA damaging agent, did not induce VCAM-1 expression in our experimental system. Furthermore, knocking-down of Ku70, a protein required for the DNA repair function of DNA-PK, did not block TNF-induced VCAM-1 expression. We further found that p38-MAPK, but not MEK, appeared to be key to the regulation of TNF-induced VCAM-1 expression by DNA-PK. p38-MAPK pathway inhibitor, SB203580, suppressed phosphorylation of DNA-PKcs after TNF treatment. Altogether, our data unravel a novel function for DNA-PK in regulation of NF-kappa B-dependent gene independently of its established function in DNA repair. It may provide a novel therapeutic target for atherosclerosis.
- © 2012 by American Heart Association, Inc.