Inhibition of nuclear factor-κB-mediated adhesion molecule expression in human endothelial cells

The transcriptional regulatory protein nuclear factor-kappa B (NF-kappa B) 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-kappa B is found in the cytoplasm complexed with its inhibitory protein I kappa B. On activation, I kappa B is phosphorylated and degraded, thereby freeing NF-kappa B for translocation to the nucleus. We have generated populations of endothelial cells expressing wild-type and a proteolysis-resistant mutation of I kappa B that is lacking the 36 N-terminal amino acids (I kappa B Delta N) in order to examine the effects of expression of the mutated I kappa B on tumor necrosis factor-alpha (TNF-alpha)-induced E-selectin and ICAM-1 expression, Wild-type and I kappa B Delta N were introduced into primary endothelial cells using retrovirus infection followed by selection with G418. The I kappa B Delta N protein remained at untreated control levels in endothelial cells treated with TNF-alpha and also remained complexed with the NF-kappa B family member p65, Furthermore, TNF-alpha-induced NF-kappa B DNA binding activity was inhibited in the population of endothelial cells expressing I kappa B Delta N. 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 I kappa B alpha protein to prevent NF-kappa B-mediated gene expression provides a novel and specific approach for investigating the role of NF-kappa B in processes associated with adhesion molecule expression during inflammation.