Analysis and Quantitation of NF-[kappa]B Nuclear Translocation in Tumor Necrosis Factor Alpha (TNF-[alpha]) Activated Vascular Endothelial Cells

2006 ◽  
Vol 12 (03) ◽  
pp. 269-276 ◽  
Author(s):  
John W. Fuseler ◽  
Dana M. Merrill ◽  
Jennifer A. Rogers ◽  
Matthew B. Grisham ◽  
Robert E. Wolf
Keyword(s):  
Endothelial Cells ◽  
Tumor Necrosis ◽  
Nuclear Translocation ◽  
Vascular Endothelial Cells ◽  
Tnf Alpha ◽  
Vascular Endothelial ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Necrosis Factor

scholarly journals Three NF-kappa B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression.

1994 ◽  
Vol 14 (9) ◽  
pp. 5820-5831 ◽  
Author(s):  
U Schindler ◽  
V R Baichwal
Keyword(s):  
Endothelial Cells ◽  
Tumor Necrosis Factor ◽  
Binding Sites ◽  
Tumor Necrosis ◽  
Regulatory Elements ◽  
Tnf Alpha ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Necrosis Factor

Transcription of the gene encoding the endothelial cell-leukocyte adhesion molecule (ELAM-1; E-selectin) is induced in response to various cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1. A DNase I-hypersensitive site in the 5' proximal promoter region of the E-selectin gene is observed in human umbilical vein endothelial cells only following TNF-alpha treatment, suggesting the presence of a TNF-alpha-inducible element close to the transcriptional start site. Transient transfection studies in endothelial cells demonstrated that 170 bp of upstream sequences is sufficient to confer TNF-alpha inducibility. Systematic site-directed mutagenesis of this region revealed two regulatory elements (-129 to -110 and -99 to -80) that are essential for maximal promoter activity following cytokine treatment. Protein binding studies with crude nuclear extracts and recombinant proteins revealed that the two elements correspond to three NF-kappa B binding sites (site 1, -126; site 2, 116; and site 3, -94). All three sites can be bound by NF-kappa B when used as independent oligonucleotides in mobility shift assays. However, within the context of a larger promoter fragment, sites 2 and 3 are preferentially occupied over site 1. These data are consistent with results obtained in transfection studies demonstrating that mutations in sites 2 and 3 are more detrimental than mutations within site 1. Hence, inducibility of the E-selectin gene requires the interaction of NF-kappa B proteins bound to multiple regulatory elements.

Three NF-kappa B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression

1994 ◽  
Vol 14 (9) ◽  
pp. 5820-5831
Author(s):  
U Schindler ◽  
V R Baichwal
Keyword(s):  
Endothelial Cells ◽  
Tumor Necrosis Factor ◽  
Binding Sites ◽  
Tumor Necrosis ◽  
Regulatory Elements ◽  
Tnf Alpha ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Necrosis Factor

Transcription of the gene encoding the endothelial cell-leukocyte adhesion molecule (ELAM-1; E-selectin) is induced in response to various cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1. A DNase I-hypersensitive site in the 5' proximal promoter region of the E-selectin gene is observed in human umbilical vein endothelial cells only following TNF-alpha treatment, suggesting the presence of a TNF-alpha-inducible element close to the transcriptional start site. Transient transfection studies in endothelial cells demonstrated that 170 bp of upstream sequences is sufficient to confer TNF-alpha inducibility. Systematic site-directed mutagenesis of this region revealed two regulatory elements (-129 to -110 and -99 to -80) that are essential for maximal promoter activity following cytokine treatment. Protein binding studies with crude nuclear extracts and recombinant proteins revealed that the two elements correspond to three NF-kappa B binding sites (site 1, -126; site 2, 116; and site 3, -94). All three sites can be bound by NF-kappa B when used as independent oligonucleotides in mobility shift assays. However, within the context of a larger promoter fragment, sites 2 and 3 are preferentially occupied over site 1. These data are consistent with results obtained in transfection studies demonstrating that mutations in sites 2 and 3 are more detrimental than mutations within site 1. Hence, inducibility of the E-selectin gene requires the interaction of NF-kappa B proteins bound to multiple regulatory elements.

scholarly journals Regulation of thrombomodulin by tumor necrosis factor-alpha: comparison of transcriptional and posttranscriptional mechanisms

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 542-550 ◽  
Author(s):  
SR Lentz ◽  
M Tsiang ◽  
JE Sadler
Keyword(s):  
Endothelial Cells ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis Factor Alpha ◽  
Posttranscriptional Regulation ◽  
Tumor Necrosis ◽  
Vascular Endothelial Cells ◽  
Necrosis Factor Alpha ◽  
Subsequent Decrease ◽  
Factor Alpha ◽  
Necrosis Factor

Abstract The procoagulant properties of cultured vascular endothelial cells are enhanced in response to inflammatory cytokines such as tumor necrosis factor-alpha (TNF). A major component of this response is a reduction in expression of thrombomodulin, a cell surface cofactor for the activation of protein C. Regulation of thrombomodulin expression by TNF has been reported to occur through multiple mechanisms. To determine the relative roles of transcriptional and posttranscriptional regulation, the effect of TNF on the turnover of thrombomodulin protein and mRNA was examined in human and bovine endothelial cells. Quantitative nuclease S1 protection assays showed a 70% to 90% reduction in thrombomodulin mRNA within 4 hours of the addition of 1.0 nmol/L TNF to the culture medium. The decrease in thrombomodulin mRNA resulted from inhibition of transcription, followed by rapid degradation of thrombomodulin transcripts (t1/2 less than or equal to 3 hours). In pulse-chase incubations, thrombomodulin synthesis decreased parallel with mRNA, but the rate of degradation of radiolabeled thrombomodulin was not significantly altered by TNF. Human thrombomodulin was degraded with a t1/2 of 8.2 +/- 2.4 hours (SD) or 7.5 +/- 1.3 hours (SD) in the absence or presence of TNF, respectively. We conclude that TNF acts primarily to inhibit thrombomodulin transcription. The subsequent decrease in activity results from the inherent instability of thrombomodulin mRNA and protein in these cells, and not from the regulation of thrombomodulin degradation.

scholarly journals Tumor necrosis factor-alpha modulation of glycoprotein Ib alpha expression in human endothelial and erythroleukemia cells

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 153-161 ◽  
Author(s):  
V Rajagopalan ◽  
DW Essex ◽  
SS Shapiro ◽  
BA Konkle
Keyword(s):  
Endothelial Cells ◽  
Tumor Necrosis Factor ◽  
Protein Expression ◽  
Tumor Necrosis Factor Alpha ◽  
Tumor Necrosis ◽  
Mrna Level ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Necrosis Factor

Abstract Glycoprotein Ib alpha (GpIb alpha) is a platelet membrane Gp that binds von Willebrand factor and mediates platelet adhesion to subendothelium. We have found both GpIb alpha mRNA and protein in human umbilical vein endothelial cells (HUVEC). In previously published work we reported that combined treatment with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) markedly increased the GpIb alpha mRNA level in HUVEC. We have now documented that TNF-alpha alone induces GpIb alpha mRNA and protein expression, studied the kinetics of this response, and investigated potential mechanisms of the TNF-alpha effect. GpIb alpha mRNA induction by TNF-alpha is detectable as early as 2 hours after exposure to this cytokine, and reaches a maximal level after 20 to 24 hours. Using a nuclear run-on assay we found that GpIb alpha gene transcription is increased approximately 10-fold after 2 hours of TNF-alpha treatment. Furthermore, using two monoclonal antibodies that recognize different epitopes of GpIb alpha, we found that the protein expression in endothelial cells is markedly increased by TNF-alpha. Interleukin-1 (IL-1) and the phorbol ester phorbol myristate acetate, which mimic many effects of TNF-alpha on endothelial cells, have no effect on endothelial or human erytholeukemia (HEL)-cell GpIb alpha mRNA. TNF-alpha treatment for 24 hours increases the HEL cell GpIb alpha mRNA level approximately fourfold, showing a time- and dose-dependent effect similar to that seen in HUVEC. TNF-alpha-induced GpIb alpha mRNA and protein synthesis may play a role in mediating platelet or other cell interaction with activated endothelium. Unlike other endothelial pro-thrombotic and pro-adhesive proteins induced by TNF-alpha, GpIb alpha is not induced by IL-1 treatment, which suggests a novel pathway for induction of this protein.

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