Endogenous production of reactive oxygen species is required for stimulation of human articular chondrocyte matrix metalloproteinase production by fibronectin fragments

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 μg/ml of the α5β1 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 μM 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-κB-associated proteins and blocked activation of an NF-κB 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-κB 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.