Inhibition of nuclear factor-kappaB-mediated adhesion molecule expression in human endothelial cells.
The transcriptional regulatory protein nuclear factor-kappaB (NF-kappaB) participates in the control of gene expression of many modulators of the inflammatory and immune responses, including the adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1). NF-kappaB is found in the cytoplasm complexed with its inhibitory protein IkappaB. On activation, IkappaB is phosphorylated and degraded, thereby freeing NF-kappaB for translocation to the nucleus. We have generated populations of endothelial cells expressing wild-type and a proteolysis-resistant mutation of IkappaB that is lacking the 36 N-terminal amino acids (IkappaBdeltaN) in order to examine the effects of expression of the mutated IkappaB on tumor necrosis factor-alpha (TNF-alpha)-induced E-selectin and ICAM-1 expression. Wild-type and IkappaBdeltaN were introduced into primary endothelial cells using retrovirus infection followed by selection with G418. The IkappaBdeltaN protein remained at untreated control levels in endothelial cells treated with TNF-alpha and also remained complexed with the NF-kappaB family member p65. Furthermore, TNF-alpha-induced NF-kappaB DNA binding activity was inhibited in the population of endothelial cells expressing IkappaBdeltaN. That population of cells was also refractory to upregulation of E-selectin and ICAM-1 after treatment with TNF-alpha. The use of a truncated IkappaBalpha protein to prevent NF-kappaB-mediated gene expression provides a novel and specific approach for investigating the role of NF-kappaB in processes associated with adhesion molecule expression during inflammation.
Lockyer
J M
JM
Department of Medicine, Tulane University Medical School, New Orleans, La 70112-2699, USA. Lockyer.Jean@tmc.tulane.edu
Colladay
J S
JS
Alperin-Lea
W L
WL
Hammond
T
T
Buda
A J
AJ
D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004355:Drug Stability; D019040:E-Selectin / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics / Q000502:physiology*; D010641:Phenotype; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D051841:Transcription Factor RelB; D014157:Transcription Factors; D014161:Transduction, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D015815:Cell Adhesion Molecules; D019040:E-Selectin; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; C497593:RELB protein, human; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D051841:Transcription Factor RelB; D004247:DNA