Transcriptional regulation of the intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells. Essential roles of a variant NF-kappa B site and p65 homodimers.
Intercellular adhesion molecule-1 (ICAM-1) is greatly up-regulated on endothelial cells at sites of inflammation and is involved in leukocyte attachment and extravasation. Previously, we had shown that the ICAM-1 gene expression in human umbilical vein endothelial cells (HUVECs) was transcriptionally regulated by tumor necrosis factor-alpha (TNF-alpha) (Wertheimer, S. J., Myers, C. L., Wallace, R. W., and Parks, T. P. (1992) J. Biol. Chem. 267, 12030-12035). In the present investigation, TNF-alpha-induced transcription was found to be initiated exclusively at two sites, 40 and 41 base pairs upstream of the translation start site. Deletion analysis of the 5' regulatory region of the ICAM-1 gene revealed a 92-base pair sequence which was both necessary and sufficient to confer TNF-alpha responsiveness to a linked luciferase reporter gene in transient transfection assays. This TNF-alpha-responsive region contained a variant NF-kappa B site at -187 to -178, which when mutated, completely abolished ICAM-1 promoter activation by TNF-alpha, interleukin-1 beta, and lipopolysaccharide. Two inducible nuclear protein complexes bound to the ICAM-1 kappa B and were identified as the NF-kappa B p65 homodimer and p65/p50 heterodimer. Overexpression of p65, but not p50, transactivated the ICAM-1 promoter in a kappa B site-dependent manner in HUVECs. In addition, p65-mediated transactivation was suppressed by co-expression of p50. Our results suggest that cytokine activation of the ICAM-1 promoter in HUVECs may critically depend on p65 homodimers binding to a variant kappa B site.
Ledebur
H C
HC
Department of Inflammatory Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877.
Parks
T P
TP
D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051996:Transcription Factor RelA; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009838:Oligodeoxyribonucleotides; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1