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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
Author(s) -
Liu Li,
Paul Andrew,
MacKenzie Christopher J,
Bryant Clare,
Graham Anne,
Plevin Robin
Publication year - 2001
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0704404
Subject(s) - umbilical vein , microbiology and biotechnology , pyrrolidine dithiocarbamate , biology , interferon regulatory factors , transcription factor , signal transduction , nf κb , biochemistry , in vitro , gene
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). 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)‐α. LPS stimulated a rapid increase in nuclear factor kappa B (NFκB) DNA‐binding activity. Pre‐incubation with the NFκB pathway inhibitors, N‐α‐tosyl‐ L ‐lysine chloromethyl ketone (TLCK) or pyrrolidine dithiocarbamate (PDTC), or infection with adenovirus encoding IκBα, blocked both IRF‐1 induction and NFκB DNA‐binding activity. LPS and TNFα 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. Preincubation with TLCK, PDTC or infection with IκBα adenovirus abolished LPS‐stimulated GAS/GAF DNA‐binding. Incubation of nuclear extracts with antibodies to RelA/p50 supershifted GAS/GAF DNA‐binding demonstrating the involvement of NFκB isoforms in the formation of the GAS/GAF complex. These studies show that NFκB plays an important role in the regulation of IRF‐1 induction in HUVECs. This is in part due to the interaction of NFκB isoforms with the GAS/GAF complex either directly or via an intermediate protein.British Journal of Pharmacology (2001) 134 , 1629–1638; doi: 10.1038/sj.bjp.0704404