title: Proteasome inhibition leads to NF-kappaB-independent IL-8 transactivation in human endothelial cells through induction of AP-1.
abstract: IL-8 is an important mediator of leukocyte trafficking and activation, participating in tumor angiogenesis, inflammatory processes and coronary atherosclerosis. Under flow conditions IL-8, in conjunction with MCP-1, triggers the firm adhesion of monocytes to the vascular endothelium. While previous studies have suggested the requirement of NF-kappaB for IL-8 secretion by endothelial cells, we investigated the possibility of IL-8 transactivation under conditions of NF-kappaB suppression. Inhibition of the proteasome by MG-132 or lactacystin completely blocked TNF-alpha-induced IkappaBalpha degradation as well as NF-kappaB activity in human arterial endothelial cells. Surprisingly, basal secretion of IL-8 protein was eight- to tenfold induced by proteasome inhibitors, while MCP-1 expression was, as expected, completely down-regulated. IL-8 was up-regulated at the transcriptional level, and promoter studies proved a more than ninefold induction of transcription factor AP-1 activity to be the cause of increased IL-8 transcription. Mutation of the AP-1 binding site in an IL-8 promoter construct completely abrogated this effect, while mutation of the NF-kappaB motif did not influence IL-8 transactivation by proteasome inhibitors. With DNA binding assays we found a seven- to eightfold induction of phosphorylated c-Jun and hence JNK kinase activity under MG-132 treatment. Induction of JNK kinase appeared independent of the cell type, even in tumor cell lines not responding to proteasome inhibitors. Since neither inactivation of p53 in wild-type p53 cells nor reintroduction of functional p53 into p53(-/-) cells affected MG-132-inducible IL-8 secretion, a direct influence of p53 on IL-8 regulation could be excluded. These results show that proteasome inhibitors can not only lead to functional AP-1 induction by enhanced c-Jun phosphorylation, but also transactivate the IL-8 gene in human endothelial cells despite complete suppression of NF-kappaB activity.
authors.affiliation: Department of Cardiology, University of Tübingen, School of Medicine, Tübingen, Germany.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Hipp
authors.forename: Carmen
authors.initials: C
authors.lastname: Urbich
authors.forename: Petra
authors.initials: P
authors.lastname: Mayer
authors.forename: Jörg
authors.initials: J
authors.lastname: Wischhusen
authors.forename: Michael
authors.initials: M
authors.lastname: Weller
authors.forename: Michael
authors.initials: M
authors.lastname: Kracht
authors.forename: Ioakim
authors.initials: I
authors.lastname: Spyridopoulos
mesh_terms: D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000502:physiology; D016328:NF-kappa B / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex; D018808:Transcription Factor AP-1 / Q000096:biosynthesis*; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000502:physiology
chemical_list: D016209:Interleukin-8; D009097:Multienzyme Complexes; D016328:NF-kappa B; D018808:Transcription Factor AP-1; D016159:Tumor Suppressor Protein p53; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex