Abstract
BackgroundMitochondrion has an important role in the osteoarthritis (OA) pathology. We have previously demonstrated that the alteration of the mitochondrial respiratory chain (MRC) contributes to the inflammatory response of the chondrocyte. However its implication in the process of cartilage destruction is not well understood yet. In this study we have investigated the relationship between the MRC dysfunction and the regulation of metalloproteases (MMPs) in human normal chondrocytes in culture.MethodsHuman normal chondrocytes were isolated from human knees obtained form autopsies of donors without previous history of rheumatic disease. Rotenone, 3-Nitropropionic acid (NPA), Antimycin A (AA), Sodium azide and Oligomycin were used to inhibit the activity of the mitochondrial complexes I, II, III, IV and V respectively. The mRNA expression of MMPs -1, -3 and -13 was studied by real time PCR. The intracellular presence of MMP proteins was evaluated by western blot. The liberation of these proteins to the extracellular media was evaluated by ELISA. The presence of proteoglycans in tissue was performed with tolouidin blue and safranin/fast green. Immunohistochemistry was used for evaluating MMPs on tissue.ResultsFirstly, cells were treated with the inhibitors of the MRC for 24 hours and mRNA expression was evaluated. An up regulation of MMP-1 and -3 mRNA levels was observed after the treatment with Oligomycin 5 and 100 μg/ml (inhibitor of the complex V) for 24 hours. MMP-13 mRNA expression was reduced after the incubation with AA 20 and 60 μg/ml (inhibitor of complex III) and Oligomycin. Results were validated at protein level observing an increase in the intracellular levels of MMP-1 and -3 after Oligomycin 25 μg/ml stimulation [(15.20±8.46 and 4.59±1.83 vs. basal=1, respectively (n=4; *P<0.05)]. However, AA and Oligomycin reduced the intracellular levels of the MMP-13 protein (0.70±0.16 and 0.3±0.24, respectively vs. basal=1). In order to know whether the MRC dysfunction had an effect on the liberation of MMPs, their levels were evaluated in the supernatants. After 36 hours of stimulation, values were: MMP-1=18.06±10.35 with Oligomycin 25 μg/ml vs. basal=1, and MMP-3=8.49±4.32 with Oligomycin 5 μg/ml vs. basal=1 (n=5; *P<0.05). MMP-13 levels in the supernatants were reduced after AA 60 μg/ml treatment (0.50±0.13 vs. basal=1) and Oligomycin 25 μg/ml (0.41±0.14 vs. basal=1); (n=5; *P<0.05). The treatment of explants with Oligomycin, showed an increase in the positivity of MMP-1 and -3. Explants stimulated with AA or Oligomycin revealed a decrease in MMP-13 expression. Proteoglycan staining demonstrated a reduction of proteoglycan levels in the tissues treated with Oligomycin.ConclusionsThese results reveal that MRC dysfunction modulates the MMPs expression in human normal chondrocytes demonstrating its role in the regulation of the cartilage destruction.
Highlights
Mitochondrion has an important role in the osteoarthritis (OA) pathology
Intracellular MRC dysfunction and the regulation of metalloproteases (MMPs)-1, MMP-3 and MMP-13 expression after mitochondrial respiratory chain (MRC) dysfunction We evaluated the possible modulation at mRNA level of MMPs −1, -3 and −13 after the induction of the MRC dysfunction
We stimulated the cells at different concentrations of Antimycin A (AA) or Oligomycin according to the preliminary mRNA results
Summary
We have previously demonstrated that the alteration of the mitochondrial respiratory chain (MRC) contributes to the inflammatory response of the chondrocyte. Osteoarthritis (OA) is a pathology characterized by the destruction of the cartilage and joint dysfunction [1,2]. Our group has previously described that the activity of the mitochondrial respiratory chain (MRC) complexes II and III is reduced in human OA chondrocytes in culture compared to healthy chondrocytes [3,4]. Among other substances produced by the tissues in the OA joint, nitric oxide (NO) has an important role on the mitochondrial activity [7,8]. Our group has demonstrated that the MRC dysfunction could generate an inflammatory response in the chondrocyte with upregulation of COX-2 and PGE2 production [11,12,13]
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