DNA‐DEPENDENT PROTEIN KINASE IS CRITICAL FOR VCAM‐1 EXPRESSION UPON TNF TREATMENT THROUGH PHOSPHORYLATION OF p50 NF‐kappa B: A CRITICAL INVOLVEMENT IN VASCULAR AND LUNG INFLAMMATION

Abstract

NF‐κB is critical for vascular cell adhesion molecule 1 (VCAM‐1) expression during vascular inflammation. We show that DNA‐dependent protein kinase (DNA‐PK) is required for VCAM‐1 expression in response to TNF‐α in an established cell line and in primary endothelial cells. DNA‐PK deficiency, achieved by DNA‐PKcs heterozygosity, reduced VCAM‐1 expression in the aorta in an animal model of septic shock and blocked airway neutrophilia in an animal model of acute lung injury. Interestingly, such effect unravels a novel function for DNA‐PK independent of its role in immune competence. Phosphorylation and nuclear translocation of p65 NF‐κB (p65) was insufficient for VCAM‐1 expression in response to TNF‐α. The requirement for p50 NF‐κB (p50) was associated with its interaction with and phosphorylation by DNA‐PK, which appears to be dominant over the requirement for p65 activation. Additionally, DNA‐PK activity appeared to increase the κB‐site binding ability of p50/p50 and p50/p65 dimers to VCAM‐1 promoter. Serine 20 in p50 was found to be phosphorylated by DNA‐PK. Re‐establishing wild‐type p50, but not its S20/A mutant, in p50‐knockout cells reversed VCAM‐1 expression. Together, these results elucidate a novel mechanism for the regulation of VCAM‐1 expression by DNA‐PK through phosphorylation of p50 and its critical involvement in vascular and lung inflammation.