Inhibition of TNF-alpha-induced NF-kappaB activation and IL-8 release in A549 cells with the proteasome inhibitor MG-132.
The working hypothesis of the studies described herein was that inhibition of proteasome-mediated IkappaB degradation would inhibit TNF-alpha-induced nuclear factor-kappaB (NF-kappaB) activation, interleukin-8 (IL-8) gene transcription, and IL-8 protein release in A549 cells. Mutational analysis of the 5' flanking region of the IL-8 gene confirmed that an intact NF-kappaB site is necessary for TNF-alpha-induced IL-8 gene transcription. The addition of TNF-alpha to A549 cells resulted in rapid loss of IkappaB from the cytoplasm of cells, associated with a corresponding increase in NF-kappaB-binding activity in nuclear extracts from the cells. However, pretreatment of the cells with the proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132, 10 microM) reversed the effects of TNF-alpha on IL-8 release from A549 cells (as determined with an enzyme-linked immunosorbent assay [ELISA]) and on IL-8 gene transcription (as determined with reporter-gene assays). MG-132 reversed the effects of TNF-alpha on IkappaB degradation as determined by Western blot analysis. IkappaB phosphorylation and ubiquination were not altered by MG-132, which implies that the effects of MG-132 were secondary to proteasome inhibition. MG-132 also reversed the increase in NF-kappaB binding in nuclear extracts from TNF-alpha-treated cells. These studies show that inhibition of proteasome-mediated IkappaB degradation results in inhibition of TNF-alpha induced IL-8 production in A549 cells by limiting NF-kappaB-mediated gene transcription.
Division of Pulmonary Medicine, Children's Hospital Research Foundation, Cincinnati, Ohio, USA. FIEDM0@CHMCC.ORG
M A
MA
Fiedler
K
K
Wernke-Dollries
J M
JM
Stark
D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000187:drug effects*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology*; D009097:Multienzyme Complexes / Q000187:drug effects*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051841:Transcription Factor RelB; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014452:Ubiquitins / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D016209:Interleukin-8; D007976:Leupeptins; D009097:Multienzyme Complexes; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; C497593:RELB protein, human; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D014452:Ubiquitins; D051841:Transcription Factor RelB; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde