LIPOPOLYSACCHARIDE-INDUCED NF-KAPPA-B ACTIVATION IN HUMAN ENDOTHELIAL-CELLS INVOLVES DEGRADATION OF I-KAPPA-B-ALPHA BUT NOT I-KAPPA-B-BETA(1)

We studied the signal transduction pathways involved in NF-kappa B act ivation and the induction of the cytoprotective A20 gene by lipopolysa ccharide (LPS) in human umbilical vein endothelial cells (HUVEC), LPS induced human A20 mRNA expression with a maximum level 2 h after stimu lation. The proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucina l-H (ALLN) and the tyrosine kinase inhibitor herbimycin A (HMA) blocke d A20 mRNA expression and partially inhibited NF-kappa B DNA-binding a ctivity induced by LPS treatment, LPS induced I kappa B alpha degradat ion at 30-60 min after treatment, but did not induce I kappa B beta de gradation 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 degr adation, indicating that newly synthesized proteins induced by LPS wer e not involved in LPS-stimulated I kappa B alpha degradation, LPS-indu ced I kappa B alpha degradation was inhibited by ALLN, confirming that ALLN inhibits NF-kappa B activation by preventing I kappa B alpha deg radation. Of note, HMA also inhibited LPS-induced I kappa B alpha degr adation. However, tyrosine phosphorylation of I kappa B alpha itself w as not elicited by LPS stimulation, suggesting that tyrosine phosphory lation of a protein(s) upstream of I kappa B alpha is required for sub sequent 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-sen sitive kinase(s) upstream of I kappa B alpha. (C) 1998 Academic Press.