COOPERATIVE BINDING AND SYNERGISTIC ACTIVATION BY RELA AND C EBP-BETAON THE INTERCELLULAR-ADHESION MOLECULE-1 PROMOTER/

Intercellular adhesion molecule-1 (ICAM-1) is upregulated on numerous cell types in response to inflammatory cytokines. Tumor necrosis facto r-alpha(TNF-alpha) activates the ICAM-1 promoter through a variant nuc lear factor-kappa B (NF-kappa B) site at -187/-178 bp upstream of the transcription start site. In this investigation, we provide biochemica l and functional evidence that an adjacent CCAAT/enhancer binding prot ein (C/EBP) site and this variant NF-kappa B site define a composite e lement for activation of the ICAM-1 promoter in certain cell lines. We detected an endogenous TNF-alpha-inducible DNA-protein complex in nuc lear extracts from A549, HeLa, and EVC304 cells that contained both Re lA and C/EBP beta but not other family members. Complex formation requ ired intact C/ EBP and NF-kappa B sites and was absolutely dependent o n translocation of RelA into the nucleus. Complex formation and cooper ative binding were also demonstrated using recombinant proteins, and a s above, both binding sites were necessary. Interestingly, the RelAIC/ EBP beta complex was not detected in either Jurkat or Raji cells, indi cating cell type specificity. Functional studies with various reporter gene constructs revealed that both binding sites were required for ma ximal activation of the ICAM-1 promoter in response to TNF-alpha and f or synergistic activation by RelA and C/EBP beta. This is the first de tailed analysis of how RelA and C/EBPP function to regulate ICAM-1 exp ression, and this study has important implications for how this gene i s activated in specific cell types.