Nuclear factor kappa B is involved in lipopolysaccharide-stimulated induction of interferon regulatory factor-1 and GAS/GAF DNA-binding in human umbilical vein endothelial cells.
1. In this study we examined the signalling events that regulate lipopolysaccharide (LPS)-stimulated induction of interferon regulatory factor (IRF)-1 in human umbilical vein endothelial cells (HUVECs). 2. LPS stimulated a time- and concentration-dependent increase in IRF-1 protein expression, an effect that was mimicked by the cytokine, tumour necrosis factor (TNF)-alpha. 3. LPS stimulated a rapid increase in nuclear factor kappa B (NFkappaB) DNA-binding activity. Pre-incubation with the NFkappaB pathway inhibitors, N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK) or pyrrolidine dithiocarbamate (PDTC), or infection with adenovirus encoding IkappaBalpha, blocked both IRF-1 induction and NFkappaB DNA-binding activity. 4. LPS and TNFalpha also stimulated a rapid activation of gamma interferon activation site/gamma interferon activation factor (GAS/GAF) DNA-binding in HUVECs. Preincubation with the Janus kinase (JAK)-2 inhibitor, AG490 blocked LPS-stimulated IRF-1 induction but did not affect GAS/GAF DNA-binding. 5. Preincubation with TLCK, PDTC or infection with IkappaBalpha adenovirus abolished LPS-stimulated GAS/GAF DNA-binding. 6. Incubation of nuclear extracts with antibodies to RelA/p50 supershifted GAS/GAF DNA-binding demonstrating the involvement of NFkappaB isoforms in the formation of the GAS/GAF complex. 7. These studies show that NFkappaB plays an important role in the regulation of IRF-1 induction in HUVECs. This is in part due to the interaction of NFkappaB isoforms with the GAS/GAF complex either directly or via an intermediate protein.
Liu
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Department of Physiology & Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, G4 0NR, UK.
Paul
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MacKenzie
C J
CJ
Bryant
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Graham
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Plevin
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D000256:Adenoviridae / Q000235:genetics; D015153:Blotting, Western; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007231:Infant, Newborn; D050856:Interferon Regulatory Factor-1; D050832:Interferon-Stimulated Gene Factor 3; D007371:Interferon-gamma / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000096:biosynthesis*; D011392:Proline / Q000031:analogs & derivatives* / Q000494:pharmacology; D020033:Protein Isoforms / Q000378:metabolism; D013859:Thiocarbamates / Q000494:pharmacology; D013997:Time Factors; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020032:Tyrphostins / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology
D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D050832:Interferon-Stimulated Gene Factor 3; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009687:Nuclear Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D013859:Thiocarbamates; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; C079868:gamma interferon activation factor; C066229:prolinedithiocarbamate; D014107:Tosyllysine Chloromethyl Ketone; D007371:Interferon-gamma; D011392:Proline