title: Endotoxin, but not platelet-activating factor, activates nuclear factor-kappaB and increases IkappaBalpha and IkappaBbeta turnover in enterocytes.
abstract: Bacterial endotoxin (lipopolysaccharide; LPS) and platelet-activating factor (PAF) are important triggers of bowel inflammation and injury. We have previously shown that LPS activates the transcription factor nuclear factor (NF)-kappaB in the intestine, which up-regulates many pro-inflammatory genes. This effect partly depends on neutrophils and endogenous PAF. However, whether LPS and PAF directly activate NF-kappaB in enterocytes remains controversial. In this study, we first investigated the effect of LPS and PAF on NF-kappaB activation in IEC-6 (a non-transformed rat small intestinal crypt cell line) cells, by electrophoresis mobility shift assay and supershift, and found that LPS, but not PAF, activates NF-kappaB mostly as p50-p65 heterodimers. The effect was slower than tumour necrosis factor (TNF). Both LPS and TNF induce the expression of the NF-kappaB-dependent gene inducible nitric oxide synthase (iNOS), which occurs subsequent to NF-kappaB activation. We then examined the effect of LPS and TNF on the inhibitory molecules IkappaBalpha and IkappaBbeta. We found that TNF causes rapid degradation of IkappaBalpha and IkappaBbeta. In contrast, LPS did not change the levels of IkappaBalpha and IkappaBbeta up to 4 hr (by Western blot). However, in the presence of cycloheximide, there was a slow reduction of IkappaBalpha and IkappaBbeta, which disappeared almost completely at 4 hr. These observations suggest that LPS causes slow degradation and synthesis of IkappaBalpha and IkappaBbeta and therefore activates NF-kappaBeta via at least two mechanisms: initially, through an IkappaB-independent mechanism, and later, via an increased turnover of the inhibitor IkappaB. NF-kappaBeta activation precedes the gene expression of iNOS (assayed by reverse transcription-polymerase chain reaction), suggesting that LPS up-regulates iNOS via this transcription factor.
authors.affiliation: Departments of Pediatrics (Neonatology) and Pathology, Children's Memorial Hospital, Northwestern University Medical School, Chicago, IL 60614, USA.
authors.forename: Isabelle G
authors.initials: IG
authors.lastname: De Plaen
authors.forename: Xiao-Wu
authors.initials: XW
authors.lastname: Qu
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.forename: Xiao-Di
authors.initials: XD
authors.lastname: Tan
authors.forename: Liya
authors.initials: L
authors.lastname: Wang
authors.forename: Xin-Bing
authors.initials: XB
authors.lastname: Han
authors.forename: Ranna A
authors.initials: RA
authors.lastname: Rozenfeld
authors.forename: Wei
authors.initials: W
authors.lastname: Hsueh
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002450:Cell Communication / Q000276:immunology; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D004306:Dose-Response Relationship, Immunologic; D004357:Drug Synergism; D004731:Endotoxins / Q000276:immunology*; D020895:Enterocytes / Q000276:immunology / Q000378:metabolism*; D020672:I-kappa B Proteins; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology; D016328:NF-kappa B / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010972:Platelet Activating Factor / Q000276:immunology*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D004268:DNA-Binding Proteins; D004731:Endotoxins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D010972:Platelet Activating Factor; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat