TNF-alpha mediated suppression of tissue type plasminogen activator expression in vascular endothelial cells is NF-kappaB- and p38 MAPK-dependent.
BACKGROUND
Several proatherothrombotic conditions are associated with enhanced levels of circulating proinflammatory cytokines, which are believed to impair endothelial fibrinolytic capacity.


OBJECTIVE
This study aims at investigating how tumor necrosis factor (TNF)-alpha regulates endothelial gene expression of the key fibrinolytic enzyme tissue-type plasminogen activator (t-PA).


METHODS
Cultured human umbilical vein endothelial cells were pretreated with selective inhibitors of the three major inflammatory signaling pathways activated by TNF-alpha; the nuclear factor kappa-B (NF-kappaB), the p38 mitogen-activated protein kinase (p38 MAPK), and the c-jun N-terminal kinase (JNK) pathways. Following TNF-alpha stimulation, effects on t-PA gene expression were evaluated with real-time reverse transcriptase polymerase chain reaction and interactions of nuclear proteins with potential gene regulatory elements were studied with electrophoretic mobility shift assays.


RESULTS
Approximately 50% suppression of t-PA gene expression was observed after prolonged stimulation with TNF-alpha (> or =24 h). The repression was shown to be preferentially dependent on NF-kappaB activation, but also on p38 MAPK signaling. Further, we provide evidence for a TNF-alpha induced binding of NF-kappaB to the recently described kappaB site in the t-PA gene and of cyclic adenosine monophosphate response element binding protein (CREB) to the t-PA CRE-like site.


CONCLUSIONS
We conclude that TNF-alpha impairs fibrinolytic capacity in vascular endothelial cells by a NF-kappaB and p38 MAPK-dependent suppression of t-PA. This mechanism sheds a light on how inflammation contributes to the pathogenesis of cardiovascular diseases.
Ulfhammer
E
E
Clinical Experimental Research Laboratory, Sahlgrenska University Hospital/Ostra, Göteborg University, Göteborg, Sweden.
Larsson
P
P
Karlsson
L
L
Hrafnkelsdóttir
T
T
Bokarewa
M
M
Tarkowski
A
A
Jern
S
S
D001665:Binding Sites; D002318:Cardiovascular Diseases / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005342:Fibrinolysis; D005786:Gene Expression Regulation; D006801:Humans; D007249:Inflammation; D016328:NF-kappa B / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000502:physiology*; D014471:Umbilical Veins / Q000166:cytology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; D010959:Tissue Plasminogen Activator