SP1 IS A COMPONENT OF THE CYTOKINE-INDUCIBLE ENHANCER IN THE PROMOTEROF VASCULAR CELL-ADHESION MOLECULE-1

Transcription of the vascular cell adhesion molecule-1 (VCAM-1) gene i n endothelial cells is induced by the inflammatory cytokines interleuk in-1 beta, tumor necrosis factor-alpha, and lipopolysaccharide. Previo us studies demonstrated that the cytokine-response region in the VCAM1 promoter contains binding sites for the transcription factors nuclear factor-kappa B (NF-kappa B) and interferon regulatory factor-1. Using a saturation mutagenesis approach, we report that the cytokine induci ble enhancer consists of these previously characterized elements and a novel region located 3' of the NF-kappa B sites. Electrophoretic mobi lity shift assays and DNase I footprint studies with endothelial nucle ar extracts and recombinant protein revealed that the transcriptional activator Sp1 interacts with this novel element in a specific manner. Transient transfection assays using vascular endothelial cells reveale d that site-directed mutations in the Sp1 binding element decreased tu mor necrosis factor-alpha-induced activity of the VCAM1 promoter. The cytokine-induced enhancer of the VCAM1 gene requires constitutively bo und Sp1 and induced heterodimeric NF-kappa B for maximal promoter acti vity.