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Three NF-kappa B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression.

Tularik Inc., South San Francisco, California 94080.

ABSTRACT

Transcription of the gene encoding the endothelial cell-leukocyte adhesion molecule (ELAM-1; E-selectin) is induced in response to various cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1. A DNase I-hypersensitive site in the 5' proximal promoter region of the E-selectin gene is observed in human umbilical vein endothelial cells only following TNF-alpha treatment, suggesting the presence of a TNF-alpha-inducible element close to the transcriptional start site. Transient transfection studies in endothelial cells demonstrated that 170 bp of upstream sequences is sufficient to confer TNF-alpha inducibility. Systematic site-directed mutagenesis of this region revealed two regulatory elements (-129 to -110 and -99 to -80) that are essential for maximal promoter activity following cytokine treatment. Protein binding studies with crude nuclear extracts and recombinant proteins revealed that the two elements correspond to three NF-kappa B binding sites (site 1, -126; site 2, 116; and site 3, -94). All three sites can be bound by NF-kappa B when used as independent oligonucleotides in mobility shift assays. However, within the context of a larger promoter fragment, sites 2 and 3 are preferentially occupied over site 1. These data are consistent with results obtained in transfection studies demonstrating that mutations in sites 2 and 3 are more detrimental than mutations within site 1. Hence, inducibility of the E-selectin gene requires the interaction of NF-kappa B proteins bound to multiple regulatory elements.

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