Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion

We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM- 1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombi n resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell su rface expression and in ICAM-1-dependent endothelial adhesivity toward poly morphonuclear leukocytes. Transient transfection of endothelial cells Kith ICAM-1 promoter luriferase reporter gene (ICAM-1LUC) constructs indicated t hat deletion of upstream NF-kappa B site (-533 bases from translation start site had no effect on thrombin responsiveness, whereas mutation/deletion o f downstream NF-kappa B site (-223 bases from the translation start site) p resented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. KF-kappa B-directed luciferase activity increased similar to 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were expos ed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM -1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1. mimicked thrombin's action in inducing ICAM-1 expression. These data indica te that thrombin activates endothelial ICAM-1 expression and polymorphonucl ear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activa tion.