title: Lipopolysaccharide-induced NF-kappaB activation in human endothelial cells involves degradation of IkappaBalpha but not IkappaBbeta.
abstract: We studied the signal transduction pathways involved in NF-kappaB 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-kappaB DNA-binding activity induced by LPS treatment. LPS induced IkappaBalpha degradation at 30-60 min after treatment, but did not induce IkappaBbeta degradation up to 120 min. In contrast, TNF-alpha rapidly induced IkappaBalpha degradation within 5 min and IkappaBbeta degradation within 15 min. Cycloheximide did not prevent LPS-induced IkappaBalpha degradation, indicating that newly synthesized proteins induced by LPS were not involved in LPS-stimulated IkappaBalpha degradation. LPS-induced IkappaBalpha degradation was inhibited by ALLN, confirming that ALLN inhibits NF-kappaB activation by preventing IkappaBalpha degradation. Of note, HMA also inhibited LPS-induced IkappaBalpha degradation. However, tyrosine phosphorylation of IkappaBalpha itself was not elicited by LPS stimulation, suggesting that tyrosine phosphorylation of a protein(s) upstream of IkappaBalpha is required for subsequent degradation. We conclude that in HUVEC, LPS induces NF-kappaB-dependent genes through degradation of IkappaBalpha, not IkappaBbeta, and propose that this degradation is induced in part by HMA-sensitive kinase(s) upstream of IkappaBalpha.
authors.affiliation: Division of Hematology, Department of Medicine, Seattle, Washington 98195, USA.
authors.forename: K
authors.initials: K
authors.lastname: Zen
authors.forename: A
authors.initials: A
authors.lastname: Karsan
authors.forename: T
authors.initials: T
authors.lastname: Eunson
authors.forename: E
authors.initials: E
authors.lastname: Yee
authors.forename: J M
authors.initials: JM
authors.lastname: Harlan
mesh_terms: D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004730:Endothelium, Vascular; D006801:Humans; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D004268:DNA-Binding Proteins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D003513:Cycloheximide; D017871:Calcium-Calmodulin-Dependent Protein Kinases