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

摘要

class="enumerated" style="list-style-type:decimal">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.