title: Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion.
abstract: 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 thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 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 exposed 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 indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation.
authors.affiliation: Department of Pharmacology, College of Medicine, University of Illinois, Chicago 60612, USA. ARahman@uic.edu
authors.forename: A
authors.initials: A
authors.lastname: Rahman
authors.forename: K N
authors.initials: KN
authors.lastname: Anwar
authors.forename: A L
authors.initials: AL
authors.lastname: True
authors.forename: A B
authors.initials: AB
authors.lastname: Malik
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D019281:Dimerization; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D009504:Neutrophils / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000378:metabolism / Q000502:physiology; D020218:Response Elements / Q000187:drug effects; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D051996:Transcription Factor RelA; D014158:Transcription, Genetic / Q000187:drug effects; D014471:Umbilical Veins
chemical_list: D004268:DNA-Binding Proteins; D016328:NF-kappa B; D012333:RNA, Messenger; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D051996:Transcription Factor RelA; D018799:Intercellular Adhesion Molecule-1; D013917:Thrombin