Transcription of endothelial-leukocyte adhesion molecule-1 (E-selectin or ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) is induced by the inflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha). The positive regulatory domains required for maximal levels of cytokine induction have been defined in the promoters of all three genes. DNA binding studies reveal a requirement for nuclear factor-kappa B (NF-kappa B) and a small group of other transcriptional activators. The organization of the cytokine-inducible element in the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human interferon-beta gene in that both promoters require NF-kappa B, activating transcription factor-2 (ATF-2), and high mobility group protein I(Y) for induction. Based on this structural similarity, a model has been proposed for the cytokine-induced E-selectin enhancer that is similar to the stereospecific complex proposed for the interferon-beta gene promoter. In these models, multiple DNA bending proteins facilitate the assembly of higher order complexes of transcriptional activators that interact as a unit with the basal transcriptional machinery. The assembly of unique enhancer complexes from similar sets of transcriptional factors may provide the specificity required to regulate complex patterns of gene expression and correlate with the distinct patterns of expression of the leukocyte adhesion molecules.

SummaryThe vascular cell adhesion molecule 1 (VCAM-1) is a 110-kD member of the immunoglobulin gene superfamily expressed on the surface of interleukin 1/$-or tumor necrosis factor a (TNF)-stimulated endothelial cells. The cell surface protein functions as an inducible adhesion receptor for circulating mononuclear leukocytes and some tumor cells. We have previously characterized the genomic organization of the VCAMI gene and described its chromosomal localization. In this report, the promoter of the VCAM1 gene is characterized. New transcription of the VCAMI gene occurred when endothelial cells were treated with TNF. Fusion plasmids containing the 5' flanking sequence of the VCAM1 gene and the chloramphenicol acetyltransferase reporter gene were used to identify c/s-acting sequences that direct the cytokine-induced transcription. When transfected into bovine aortic endothelial cells, constructs containing 755 bp of the 5' flanking sequence were induced by TNE Within the cytokine-responsive region of the core promoter were functional NF-rB and GATA elements. Upstream of the core promoter, the VCAM1 5' flanking sequence contained a negative regulatory activity. NF-rB-mediated activation of VCAM1 gene expression may lead to endothelial expression of a mononuclear leukocyte adhesion molecule associated with initial events in the development of an atherosclerotic lesion.

SummaryStructural analysis of the promoters of several endothelial genes induced at sites of inflammatory or immune responses reveals binding sites for the transcription factor nuclear factor KB (NF-KB). Endothelial cells express transcripts encoding the pS0/p105 and p65 components of NF-IcB and the tel-related proto-oncogene c-re/; steady state levels of these transcripts are transiently increased by tumor necrosis factor ce (TNF-ce). Western blotting revealed that stimulation of endothelial calls with TNF-ce resulted in nuclear accumulation of the p50 and p65 components of NF-KB. Ultraviolet crosslinking and immunoprecipitation demonstrated binding of the p50 and p65 components of NF-KB to the E-selectin xB site. Endothelial cells express an inhibitor of NF-gB activation, IgB-ce (MAD-3). Protein levels of this inhibitor fall rapidly after TNF-cx stimulation. In parallel, p50 and p65 accumulate in the nucleus and RNA transcript levels for IgB-o~ are dramatically upreguhted. Recombinant p65 stimulates expression of E-sdectin promoter-reporter constructs. IKB-ce inhibits p65 or TNF-ce-stimulated E-sdectin promoter-reporter gene expression in transfected endothelial cells. The NF-gB and IKB-ce system may be an inducible regulatory mechanism in endothelial activation.

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