Mechanism of the tumor necrosis factor α-mediated induction of endothelial tissue factor

Abstract

This study examines the regulation of the human tissue factor (TF) promotor in vitro and in vivo. Transient transfeetions were performed in bovine aortic endothelial cells to investigate the role of two fundamentally different AP-1 sites and a closely located NF-κB site in the human TF promotor. The NF-κB site is functionally active, since overexpression of NF- κB(p65) resulted in induction of TF mRNA and activity. Promotor analysis showed that NF-κB induction was dependent on the integrity of the region from base pair -188 to -181. Overexpression of Jun/Fos resulted in TF induction of transcription and protein/activity. Functional studies revealed that the proximal AP-1 site, but not the distal, was inducible by Jun/Fos heterodimers. The distal AP-1 site, which has a G → A switch at position 4, was inducible by Jun homodimers. Electrophoretic mobility shift assays, using extracts of tumor necrosis factor α (TNFα)stimulated bovine aortic endothelial cells, demonstrated TNFα-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 proteins of the Jun and ATF family, was observed. Consistently, this site only marginally participates in TNFα induction. Functional studies with TF promotor plasmids confirmed that deletion of the proximal AP-1 or the NF-κB site decreased TNFα-mediated TF induction to a higher extend than loss of the distal AP-1 site. However, integrity of both AP-1 sites and the NF-κB site was required for optimal TNFα stimulation. The relevance of these in vitro data was confirmed in vivo in a mouse tumor model. Expression plasmids for a dominant negative Jun mutant or I-κB were packaged in liposomes. When either mutated Jun or I-κB were injected intravenously 48 h before TNFα, a reduction in TNFα-mediated TF expression in the tumor endothelial cells was observed. Simultaneously, fibrin/fibrinogendeposition decreased and free blood flow could be restored. Thus, TNFα-induced up-regulation of endothelial cell TF depends on a concerted action of members of the bZIP and NF-κB family.