ENDOTHELIAL INTERFERON REGULATORY FACTOR-1 COOPERATES WITH NF-KAPPA-BAS A TRANSCRIPTIONAL ACTIVATOR OF VASCULAR CELL-ADHESION MOLECULE-1

Transcription of the vascular cell adhesion molecule 1 (VCAM-1) gene i n endothelial cells is induced by lipopolysaccharide and the inflammat ory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF- alpha). Previous studies have demonstrated that tandem binding sites f or the inducible transcription factor MF-kappa B are necessary but not sufficient for full cytokine-mediated transcriptional activation. Her ein, we demonstrate that full cytokine-induced accumulation of VCAM1 t ranscript requires protein synthesis. We report the definition of a fu nctional regulatory element in the VCAM1 promoter interacting with the transcriptional activator interferon regulatory factor 1 (IRF-1). DNA -protein binding studies with endothelial nuclear extracts revealed th at IRF-1 is cytokine inducible and binds specifically to a consensus s equence motif located 3' of the TATA element. We have identified heter odimeric p65 and p50 as the NF-kappa B species binding to the VCAM1 pr omoter in TNF-alpha-activated endothelial cells. Experiments with reco mbinant proteins showed that p50/p65 and high-mobility-group I(Y) prot ein cooperatively facilitated the binding of IRF-1 to the VCAM1 IRF bi nding site and that IRF-1 physically interacted with p50 and with high -mobility-group I(Y) protein. Transient transfection assays in endothe lial cells showed that overexpressed IRF-1 resulted in superinduction of TNF-alpha-stimulated transcription. Site-directed mutations in the IRF binding element decreased TNF-alpha-induced activity and totally a bolished superinduction. Cotransfection assays in P19 embryonal carcin oma cells revealed that IRF-1 synergized with p50/p65 NF-kappa B to ac tivate the VCAM1 promoter or heterologous promoter constructs bearing isolated VCAM1 NF-kappa B and IRF binding moths. Cytokine inducibility of VCAM1 in endothelial cells utilizes the interaction of heterodimer ic p50/p65 proteins with IRF-1.