title: Hydrogen peroxide mediates interleukin-1beta-induced AP-1 activation in articular chondrocytes: implications for the regulation of iNOS expression.
abstract: The pro-inflammatory cytokine interleukin-1beta (IL-1) induces articular chondrocytes to produce reactive oxygen species (ROS), including hydrogen peroxide (H2O2), which mediate some IL-1-induced responses. This study aimed at elucidating the role of ROS, particularly H2O2, in mediating IL-1-induced activation of the transcription factor activator protein-1 (AP-1) in primary cultures of articular chondrocytes. AP-1 may function either as an inducer or as a repressor of the inducible nitric oxide synthase (iNOS) gene promoter. Since we observed that AP-1 is not required for iNOS expression in chondrocytes, we also investigated whether it is a repressor of this gene. The results of electrophoretic mobility shift assays showed that both IL-1 and H2O2 activated AP-1 and that inhibition of IL-1-induced ROS production abrogated AP-1 activation. The AP-1 complexes, induced by either IL-1 or H2O2, contained c-Fos/c-Jun and c-Fos/JunD heterodimers, but IL-1 activated AP-1 with a kinetics slower than that observed with H2O2. Pre-activation of AP-1, before stimulation of the cells with IL-1, did not inhibit iNOS mRNA and protein synthesis, relative to cells treated with IL-1 alone. These results indicate that H2O2 is a major mediator of IL-1-induced AP-1 activation in articular chondrocytes and that inhibition of ROS production is an effective strategy to block this IL-1-induced response. This study also identifies c-Fos/c-Jun and c-Fos/JunD heterodimers as the AP-1 transcription factors induced by IL-1, which, although not involved in the transcriptional regulation of the iNOS gene, may be important for the regulation of other genes also relevant in arthritic diseases, namely the collagenase-1 and IL-8 genes.
authors.affiliation: Faculty of Pharmacy, Centre for Neurosciences and Cell Biology, Department of Zoology, University of Coimbra, 3004-517 Coimbra, Portugal. afmendes@ff.uc.pt
authors.forename: A Ferreira
authors.initials: AF
authors.lastname: Mendes
authors.forename: M M
authors.initials: MM
authors.lastname: Caramona
authors.forename: A P
authors.initials: AP
authors.lastname: Carvalho
authors.forename: M C
authors.initials: MC
authors.lastname: Lopes
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000201:enzymology* / Q000378:metabolism; D017364:Collagenases / Q000096:biosynthesis; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007249:Inflammation; D007375:Interleukin-1 / Q000378:metabolism*; D016209:Interleukin-8 / Q000096:biosynthesis; D007700:Kinetics; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*
chemical_list: D007375:Interleukin-1; D016209:Interleukin-8; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D017364:Collagenases; C417249:collagenase 1