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 leukocyt e 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-al pha) (Wertheimer, S. J., Myers, C. L., Wallace, R. W., and Parks, T. P . (1992) J. Biol. Chem. 267, 12030-12035). In the present investigatio n, TNF-alpha-induced transcription was found to be initiated exclusive ly at two sites, 40 and 41 base pairs upstream of the translation star t site, Deletion analysis of the 5' regulatory region of the ICAM-1 ge ne revealed a 92-base pair sequence which was both necessary and suffi cient to confer TNF-alpha responsiveness to a linked luciferase report er gene in transient transfection assays, This TNF-alpha-responsive re gion contained a variant NF-kappa P site at -187 to -178, which when m utated, completely abolished ICAM-1 promoter activation by TNF-alpha, interleukin-1 beta, and lipopolysaccharide. Two inducible nuclear prot ein complexes bound to the ICAM-1 kappa B site and were identified as the NF-kappa D p65 homodimer and p65/p50 heterodimer. Overexpression o f p65, but not p50, transactivated the ICAM-1 promoter in a kappa B si te-dependent manner in HUVECs. In addition, p65-mediated transactivati on was suppressed by co-expression of p50. Our results suggest that cy tokine activation of the ICAM-1 promoter in HUVECs may critically depe nd on p65 homodimers binding to a variant kappa B site.