Transcription factor decoy for nuclear factor-kappaB inhibits tumor necrosis factor-alpha-induced expression of interleukin-6 and intracellular adhesion molecule-1 in endothelial cells.
BACKGROUND
Several cytokines and adhesion molecules released from endothelium play an important role in inflammation, immune responses, and probably atherogenesis.


OBJECTIVE
To determine whether the transcription factor nuclear factor-kappaB mediated expression of these genes involved in the inflammatory response of endothelial cells to tumor necrosis factor-alpha, by using transcription factor decoy oligodeoxynucleotides.


METHODS
We first transfected fluorescein isothiocyanate (FITC)-labeled double-stranded oligodeoxynucleotides into endothelial cells by a cationic liposome-mediated method of gene transfer. We then confirmed that the decoy oligodeoxynucleotides could block binding of nuclear factor-kappaB to its specific cis element effectively. In addition, we transfected the reporter gene chloramphenicol acetyltransferase driven by three repeated nuclear factor-kappaB binding sequences in the promoter and enhancer region.


RESULTS
FITC-labeled oligodeoxynucleotides were detected in the nuclei of approximately 70% of the total cells. Tumor necrosis factor--stimulated expression of chloramphenicol acetyltransferase was partially inhibited by transfection of nuclear factor-kappaB decoy oligodeoxynucleotides, but not by transfection of scrambled oligodeoxynucleotides. Also nuclear factor-kappaB decoy oligodeoxynucleotides but not scrambled oligodeoxynucleotides inhibited tumor necrosis factor-induced expression of interleukin-6 and intracellular adhesion molecule-1 both at the messenger RNA and at protein level (assessed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay).


CONCLUSIONS
Our results demonstrate that nuclear factor-kappaB decoy oligodeoxynucleotides transfected by cationic liposome method inhibited tumor necrosis factor--induced expression of interleukin-6 and intracellular adhesion molecule-1 in endothelial cells.
Tomita
N
N
Department of Geriatric Medicine, Osaka University Medical School, Suita, Japan.
Morishita
R
R
Tomita
S
S
Yamamoto
K
K
Aoki
M
M
Matsushita
H
H
Hayashi
S
S
Higaki
J
J
Ogihara
T
T
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004730:Endothelium, Vascular / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D007249:Inflammation / Q000209:etiology / Q000517:prevention & control; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics*; D015850:Interleukin-6 / Q000235:genetics*; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D017931:DNA Primers; D015850:Interleukin-6; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D015500:Chloramphenicol O-Acetyltransferase; D016650:Fluorescein-5-isothiocyanate