scholarly journals Involvement of the transcription factor NF-kappaB in tubular morphogenesis of human microvascular endothelial cells by oxidative stress.

1996 ◽  
Vol 16 (8) ◽  
pp. 4231-4239 ◽  
Author(s):  
T Shono ◽  
M Ono ◽  
H Izumi ◽  
S I Jimi ◽  
K Matsushima ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Oxygen Radicals ◽  
Tumor Necrosis ◽  
Microvascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Microvascular Endothelial ◽  
Nf Kappab ◽  
Necrosis Factor

Oxygen radicals are induced under various pathologic conditions associated with neovascularization. Oxygen radicals modulate angiogenesis in cultured human microvascular endothelial cells by an unknown mechanism. Treatment of human microvascular endothelial cells for 15 min with 0.1 to 0.5 mM hydrogen peroxide (H2O2) or 100 U of tumor necrosis factor alpha per ml induced tubular morphogenesis in type I collagen gels. Gel shift assays with nuclear extracts demonstrated that H2O2 increases the binding activities of two transcription factors, NF-kappaB and AP-1, but not of Spl. Tumor necrosis factor alpha increased the binding activities of all three factors. A supershift assay with specific antibodies against JunB, JunD, and c-Jun (Jun family) showed that the antibody against c-Jun supershifted the AP-1 complex after H2O2 treatment. Coadministration of the antisense sequence of NF-kappaB inhibited H2O2-dependent tubular morphogenesis, and the antisense c-Jun oligonucleotide caused partial inhibition. The angiogenic factor responsible for H2O2-induced tubular morphogenesis was examined. Cellular mRNA levels of vascular endothelial growth factor and interleukin-8 (IL-8), but not those of transforming growth factor alpha, were increased after treatment with 0.5 mM H2O2. Coadministration of anti-IL-8 antibody inhibited tubular morphogenesis enhanced by H2O2, and IL-8 itself also enhanced the formation of tube-like structures. Treatment with antisense NF-kappaB oligonucleotide completely blocked H2O2-dependent IL-8 production by endothelial cells. The tubular morphogenesis of vascular endothelial cells after treatment with oxidative stimuli and its possible association with NF-kappaB and IL-8, is examined.

scholarly journals Acute Tumor Necrosis Factor Alpha Signaling via NADPH Oxidase in Microvascular Endothelial Cells: Role of p47phox Phosphorylation and Binding to TRAF4

2005 ◽  
Vol 25 (6) ◽  
pp. 2320-2330 ◽  
Author(s):  
Jian-Mei Li ◽  
Lampson M. Fan ◽  
Michael R. Christie ◽  
Ajay M. Shah
Keyword(s):  
Endothelial Cells ◽  
Tumor Necrosis Factor ◽  
Nadph Oxidase ◽  
Tumor Necrosis ◽  
Fold Increase ◽  
Microvascular Endothelial Cells ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Factor Alpha ◽  
Necrosis Factor

ABSTRACT Tumor necrosis factor alpha (TNF-α) receptor-associated factors (TRAFs) play important roles in TNF-α signaling by interacting with downstream signaling molecules, e.g., mitogen-activated protein kinases (MAPKs). However, TNF-α also signals through reactive oxygen species (ROS)-dependent pathways. The interrelationship between these pathways is unclear; however, a recent study suggested that TRAF4 could bind to the NADPH oxidase subunit p47phox. Here, we investigated the potential interaction between p47phox phosphorylation and TRAF4 binding and their relative roles in acute TNF-α signaling. Exposure of human microvascular endothelial cells (HMEC-1) to TNF-α (100 U/ml; 1 to 60 min) induced rapid (within 5 min) p47phox phosphorylation. This was paralleled by a 2.7- ± 0.5-fold increase in p47phox-TRAF4 association, membrane translocation of p47phox-TRAF4, a 2.3- ± 0.4-fold increase in p47phox-p22phox complex formation, and a 3.2- ± 0.2-fold increase in NADPH-dependent O2 − production (all P < 0.05). TRAF4-p47phox binding was accompanied by a progressive increase in extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38MAPK activation, which was inhibited by an O2 − scavenger, tiron. TRAF4 predominantly bound the phosphorylated form of p47phox, in a protein kinase C-dependent process. Knockdown of TRAF4 expression using siRNA had no effect on p47phox phosphorylation or binding to p22phox but inhibited TNF-α-induced ERK1/2 activation. In coronary microvascular EC from p47phox−/− mice, TNF-α-induced NADPH oxidase activation, ERK1/2 activation, and cell surface intercellular adhesion molecule 1 (ICAM-1) expression were all inhibited. Thus, both p47phox phosphorylation and TRAF4 are required for acute TNF-α signaling. The increased binding between p47phox and TRAF4 that occurs after p47phox phosphorylation could serve to spatially confine ROS generation from NADPH oxidase and subsequent MAPK activation and cell surface ICAM-1 expression in EC.

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