We studied the signal transduction pathways involved in NF-kappa B activation and the induction of the cytoprotective A20 gene by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVEC), LPS induced human A20 mRNA expression with a maximum level 2 h after stimulation. The proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) and the tyrosine kinase inhibitor herbimycin A (HMA) blocked A20 mRNA expression and partially inhibited NF-kappa B DNA-binding activity induced by LPS treatment, LPS induced I kappa B alpha degradation at 30-60 min after treatment, but did not induce I kappa B beta degradation up to 120 min. In contrast, TNF-cu rapidly induced I kappa B alpha degradation within 5 min and I kappa B beta degradation within 15 min, Cycloheximide did not prevent LPS-induced I kappa B alpha degradation, indicating that newly synthesized proteins induced by LPS were not involved in LPS-stimulated I kappa B alpha degradation, LPS-induced I kappa B alpha degradation was inhibited by ALLN, confirming that ALLN inhibits NF-kappa B activation by preventing I kappa B alpha degradation. Of note, HMA also inhibited LPS-induced I kappa B alpha degradation. However, tyrosine phosphorylation of I kappa B alpha itself was not elicited by LPS stimulation, suggesting that tyrosine phosphorylation of a protein(s) upstream of I kappa B alpha is required for subsequent degradation. We conclude that in HUVEC, LPS induces NF-kappa B dependent genes through degradation of I kappa B alpha, not I kappa B beta, and propose that this degradation is induced in part by HMA-sensitive kinase(s) upstream of I kappa B alpha. (C) 1998 Academic Press.
