The objective of the present study was to determine if reactive oxygen species (ROS) are required as secondary messengers for fibronectin fragment-stimulated matrix metalloproteinase (MMP) production in human articular chondrocytes. Cultured cells were stimulated with 25 microg/ml of the alpha5beta1 integrin-binding 110-kDa fibronectin fragment (FN-f) in the presence and absence of various antioxidants including Mn(III) tetrakis(4-benzoic acid)porphyrin (MnTBAP). FN-f stimulation significantly increased intracellular levels of ROS in articular chondrocytes. Pretreatment of cells with 250 microM MnTBAP or 40 mM N-acetyl-L-cysteine, but not inhibitors of nitric oxide synthase, completely prevented FN-f-stimulated MMP-3, -10, and -13 production. MnTBAP also blocked FN-f-induced phosphorylation of the MAP kinases and NF-kappaB-associated proteins and blocked activation of an NF-kappaB promoter-reporter construct. Overexpression of catalase, superoxide dismutase, or glutathione peroxidase also inhibited FN-f-stimulated MMP-13 production. Preincubation of chondrocytes with rotenone, an inhibitor of the mitochondrial electron transport chain, or nordihydroguaiaretic acid (NDGA), a selective 5-lipoxygenase inhibitor, partially prevented FN-f-stimulated MMP-13 production and decreased MAP kinase and NF-kappaB phosphorylation. These results show that increased production of ROS but not nitric oxide as obligatory secondary messengers in the chondrocyte FN-f signaling pathway leads to the increased production of MMPs, including MMP-13.