Mechanism of the Tumor Necrosis Factor α-mediated Induction of Endothelial Tissue Factor

Angelika Bierhaus,Youming Zhang,Youhua Deng,Nigel Mackman,Peter Quehenberger,Michael Haase,Thomas Luther,Martin Müller,Hubert Böhrer,Johannes Greten,Eike Martin,Patrick A Baeuerle,Rüdiger Waldherr,Walter Kisiel,Reinhard Ziegler,David M Stern,Peter P Nawroth

doi:10.1074/jbc.270.44.26419

Abstract

This study examines the regulation of the human tissue factor (TF) promotor in vitro and in vivo. Transient transfections were performed in bovine aortic endothelial cells to investigate the role of two fundamentally different AP-1 sites and a closely located NF-kappa B site in the human TF promoter. The NF-kappa B site is functionally active, since overexpression of NF-kappa B(p65) resulted in induction of TF mRNA and activity. Promoter analysis showed that NF-kappa B induction was dependent on the integrity of the region from base pair -188 to -181. Over-expression 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 inductible by Jun homodimers. Electrophoretic mobility shift assays, using extracts of tumor necrosis factor alpha (TNF alpha)-stimulated bovine aortic 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 proteins of the Jun and ATF family, was observed. Consistently, this site only marginally participates in TNF alpha induction. Functional studies 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, integrity 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 confirmed in vivo in a mouse tumor model. Expression plasmids for a dominant negative Jun mutant or I-kappa B were packaged in liposomes. When either mutated Jun or I-kappa B were injected intravenously 48 h before TNF alpha, a reduction in TNF alpha-mediated TF expression in the tumor endothelial cells was observed. Simultaneously, fibrin/fibrinogen deposition decreased and free blood flow could be restored. Thus, TNF alpha-induced up-regulation of endothelial cell TF depends on a concerted action of members of the bZIP and NF-kappa B family.

Highlights

This study examines the regulation of the human tissue factor (TF) promotor in vitro and in vivo
When bovine aortic endothelial cells (BAEC) were cotransfected with AP-1 and NF-␬B(p65), an additive effect in TF induction was observed in all systems tested (Fig. 1)
Members of the NF-␬B and the AP-1/bZIP family have been reported to be involved in the lipopolysaccharide- and cytokine-mediated TF induction in monocytes [15,16,17,18, 62] and porcine endothelial cells [11]

Summary

Introduction

This study examines the regulation of the human tissue factor (TF) promotor in vitro and in vivo. The proximal AP-1 site in the human (and the mouse) TF promotor contains the core of the consensus sequence [11, 15, 16] and thereby represents a high affinity site for AP-1 binding This indicates that different AP-1-like proteins may be involved in the regulation of TF expression in. If the human model [15,16,17,18] is relevant, inhibition of NF-␬B activation might lead to increased c-Fos transcription [20, 21] and thereby to AP-1-mediated TF induction To resolve this issue, we studied the role of both AP-1 sites and their cooperative action with the NF-␬B site in the human TF promotor

Methods

Results

Conclusion