REGULATION OF INTERCELLULAR-ADHESION MOLECULE-1 GENE BY TUMOR-NECROSIS-FACTOR-ALPHA IS MEDIATED BY THE NUCLEAR FACTOR-KAPPA-B HETERODIMERS P65 P65 AND P65/C-REL IN THE ABSENCE OF P50/

Human intercellular adhesion molecule-1 (ICAM-1) plays an important ro le in immune responses as the major specific ligand for the beta 2-int egrins LFA-1 and Mac-1. During the inflammatory process, ICAM-1 expres sion is stimulated by various proinflammatory cytokines, We have exami ned the mechanisms of transcriptional control involved in the stimulat ion of ICAM-1 gene expression by tumor necrosis factor-alpha (TNF-alph a) and by the nuclear factor-kappa B (NF-kappa B) family of transcript ion factors in the Ad5-transformed human embryonal kidney cell line 29 3. A proximal site (5'-TTGGAAATTCC-3') mapping at position -228 from t he ATG and known to mediate TNF-alpha responsiveness in endothelial ce lls is also critical for TNF-alpha responsiveness in 293 cells. Howeve r, unlike endothelial cells, electrophoretic mobility shift assays, us ing whole-cell extracts prepared from TNF-alpha-treated cells, showed that TNF-alpha induces the formation of a specific kappa B binding com plex, mainly composed of NF-kappa B subunits RelA and c-Rel. Electroph oretic mobility shift assays done with 293 cells transfected with p50, p65, or both subunits showed that p50 only has a weak ability to bind the proximal ICAM-1 NF-kappa B site, Another element exhibiting seque nce homology with NF-kappa B binding sites and located at position -54 0 relative to the mRNA cap site was found to be involved in the basal activity of the ICAM-1 promoter, is not required for TNF-alpha respons iveness, and does not bind NF-kappa B subunits. Whereas transactivatio n of the ICAM-1 promoter by p65 requires the proximal NF-kappa B site, deletion mutant analysis showed that p50 and, to a greater extent, p5 2 transactivate reporter plasmids lacking NF-kappa B sites, suggesting the presence of other p50/p52 responsive element(s).