MECHANISM OF THE TUMOR-NECROSIS-FACTOR ALPHA-MEDIATED INDUCTION OF ENDOTHELIAL TISSUE FACTOR

This study examines the regulation of the human tissue factor (TF) pro motor in vitro and in vivo. Transient transfections were performed in bovine aortic endothelial cells to investigate the role of two fundame ntally different AP-1 sites and a closely located NF-kappa B site in t he human TF promotor, The NF-kappa B site is functionally active, sinc e overexpression of NF-kappa B(p65) resulted in induction of TF mRNA a nd activity, Promotor analysis showed that NF-kappa B induction was de pendent on the integrity of the region from base pair -188 to -181. Ov erexpression of Jun/Fos resulted in TF induction of transcription and protein/activity. Functional studies revealed that the proximal AP-1 s ite, but not the distal, was inducible by Jun/Fos heterodimers. The di stal AP-1 site, which has a G --> A switch at position 4, was inducibl e by Jun homodimers. Electrophoretic mobility shift assays, using extr acts of tumor necrosis factor alpha (TNF alpha)-stimulated bovine aort ic endothelial cells, demonstrated TNF alpha-inducible binding to the proximal AP-1 site, comprising JunD/Fos heterodimers. At the distal AP -1 site, only minor induction of binding activity, characterized as pr oteins of the Jun and ATF family, was observed. Consistently, this sit e only marginally participates in TNF alpha induction. Functional stud ies with TF promotor plasmids confirmed that deletion of the proximal AP-1 or the NF-kappa B site decreased TNF alpha-mediated TF induction to a higher extend than loss of the distal AP-1 site. However, integri ty of both AP-1 sites and the NF-kappa B site was required for optimal TNF alpha stimulation. The relevance of these in vitro data was confi rmed in vivo in a mouse tumor model. Expression plasmids for a dominan t negative Jun mutant or I-KB were packaged in liposomes. When either mutated Jun or I-KB were injected intravenously 48 h before TNF alpha, a reduction in TNF alpha-mediated TF expression in the tumor endothel ial cells was observed. Simultaneously, fibrin/fibrinogen deposition d ecreased and free blood flow could be re stored, Thus, TNF alpha-induc ed up-regulation of endothelial cell TF depends on a concerted action of members of the bZIP and NF-kappa B family.