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Mitochondrial respiratory chain dysfunction modulates metalloproteases -1, -3 and -13 in human normal chondrocytes in culture.

Cillero-Pastor B, Rego-Pérez I, Oreiro N, Fernandez-Lopez C, Blanco FJ - BMC Musculoskelet Disord (2013)

Bottom Line: 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.These results reveal that MRC dysfunction modulates the MMPs expression in human normal chondrocytes demonstrating its role in the regulation of the cartilage destruction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Rheumatology Division, INIBIC-Hospital Universitario A Coruña, A Coruña, Spain.

ABSTRACT

Background: Mitochondrion 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.

Methods: Human 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.

Results: Firstly, 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.

Conclusions: These results reveal that MRC dysfunction modulates the MMPs expression in human normal chondrocytes demonstrating its role in the regulation of the cartilage destruction.

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MMP-1 intracellular protein levels in chondrocytes after MRC dysfunction. A) Human chondrocytes were cultured in 6 well plates, in basal conditions with AA or Oligomycin for 24 h and intracellular proteins were detected by western blot. Data were expressed as a ratio (basal=1) represented as mean ± SE of 4 independent experiments (*P<0.05). B) Example of a representative experiment.
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Figure 2: MMP-1 intracellular protein levels in chondrocytes after MRC dysfunction. A) Human chondrocytes were cultured in 6 well plates, in basal conditions with AA or Oligomycin for 24 h and intracellular proteins were detected by western blot. Data were expressed as a ratio (basal=1) represented as mean ± SE of 4 independent experiments (*P<0.05). B) Example of a representative experiment.

Mentions: We evaluated the possible modulation at mRNA level of MMPs -1, -3 and -13 after the induction of the MRC dysfunction. According to the bibliography, we used Rotenone 10 and 50 μg/ml to inhibit the MRC complex I, NPA 0.5 and 10 mM to inhibit the MRC complex II, Antimycin A (AA) 20 and 60 μg/ml to inhibit the complex III, Sodium azide 2 and 25 mM to inhibit the complex IV and Oligomycin 5 and 100 μg/ml to inhibit the activity of the complex V. After 24 hours of treatment, we analyzed the mRNA expression of MMPs -1, -3 and -13 as Figure 1 shows. Oligomycin 5 μg/ml produced a tendency in the increase of MMP-1 and -3 expression (Figure 1A, 1B) to 68.10±39.9 and 60.13±29.7 vs. basal=1, respectively (n=9). On the other hand, the inhibition of the complex III with AA 20 μg/ml, produced a decrease in the MMP-13 mRNA expression to 0.34±0.2 vs. basal=1 (Figure 1C). To confirm these results at protein level, we evaluated the intracellular protein expression of these MMPs by western blot (Figures 2, 3 and 4). We stimulated the cells at different concentrations of AA or Oligomycin according to the preliminary mRNA results. The positive control used was IL-1β 5 ng/ml. The treatment of chondrocytes with the inhibitor of complex V (Oligomycin 2.5, 5, 10 and 25 μg/ml) after 24 hours produced an increase in the MMP-1 levels (Figure 2A). The levels increased significantly up to 12.20±3.24 and 15.20±8.46 vs. basal=1, Oligomycin 10 and 25 μg/ml respectively, (n=4; *P<0.05). Figure 2B represents an experiment of 4. As we expected, AA did not induce the MMP-1 modulation according to the mRNA results. In a similar way, MMP-3 was only induced by Oligomycin. Figure 3A shows these levels: at 24 h 5.65±2.08 and 4.59±1.83 vs. basal=1 for the concentrations of 10 and 25 μg/ml, respectively (n=4; *P<0.05). Figure 3B represents an experiment of 4. As we expected, AA did not induce the modulation of MMP-1. MMP-13 decreased after treatment with AA 40 μg/ml and Oligomycin 25 μg/ml (0.70±0.16 and 0.3±0.24 vs. basal=1; n=4; *P<0.05) (Figure 4A and 4B).


Mitochondrial respiratory chain dysfunction modulates metalloproteases -1, -3 and -13 in human normal chondrocytes in culture.

Cillero-Pastor B, Rego-Pérez I, Oreiro N, Fernandez-Lopez C, Blanco FJ - BMC Musculoskelet Disord (2013)

MMP-1 intracellular protein levels in chondrocytes after MRC dysfunction. A) Human chondrocytes were cultured in 6 well plates, in basal conditions with AA or Oligomycin for 24 h and intracellular proteins were detected by western blot. Data were expressed as a ratio (basal=1) represented as mean ± SE of 4 independent experiments (*P<0.05). B) Example of a representative experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750811&req=5

Figure 2: MMP-1 intracellular protein levels in chondrocytes after MRC dysfunction. A) Human chondrocytes were cultured in 6 well plates, in basal conditions with AA or Oligomycin for 24 h and intracellular proteins were detected by western blot. Data were expressed as a ratio (basal=1) represented as mean ± SE of 4 independent experiments (*P<0.05). B) Example of a representative experiment.
Mentions: We evaluated the possible modulation at mRNA level of MMPs -1, -3 and -13 after the induction of the MRC dysfunction. According to the bibliography, we used Rotenone 10 and 50 μg/ml to inhibit the MRC complex I, NPA 0.5 and 10 mM to inhibit the MRC complex II, Antimycin A (AA) 20 and 60 μg/ml to inhibit the complex III, Sodium azide 2 and 25 mM to inhibit the complex IV and Oligomycin 5 and 100 μg/ml to inhibit the activity of the complex V. After 24 hours of treatment, we analyzed the mRNA expression of MMPs -1, -3 and -13 as Figure 1 shows. Oligomycin 5 μg/ml produced a tendency in the increase of MMP-1 and -3 expression (Figure 1A, 1B) to 68.10±39.9 and 60.13±29.7 vs. basal=1, respectively (n=9). On the other hand, the inhibition of the complex III with AA 20 μg/ml, produced a decrease in the MMP-13 mRNA expression to 0.34±0.2 vs. basal=1 (Figure 1C). To confirm these results at protein level, we evaluated the intracellular protein expression of these MMPs by western blot (Figures 2, 3 and 4). We stimulated the cells at different concentrations of AA or Oligomycin according to the preliminary mRNA results. The positive control used was IL-1β 5 ng/ml. The treatment of chondrocytes with the inhibitor of complex V (Oligomycin 2.5, 5, 10 and 25 μg/ml) after 24 hours produced an increase in the MMP-1 levels (Figure 2A). The levels increased significantly up to 12.20±3.24 and 15.20±8.46 vs. basal=1, Oligomycin 10 and 25 μg/ml respectively, (n=4; *P<0.05). Figure 2B represents an experiment of 4. As we expected, AA did not induce the MMP-1 modulation according to the mRNA results. In a similar way, MMP-3 was only induced by Oligomycin. Figure 3A shows these levels: at 24 h 5.65±2.08 and 4.59±1.83 vs. basal=1 for the concentrations of 10 and 25 μg/ml, respectively (n=4; *P<0.05). Figure 3B represents an experiment of 4. As we expected, AA did not induce the modulation of MMP-1. MMP-13 decreased after treatment with AA 40 μg/ml and Oligomycin 25 μg/ml (0.70±0.16 and 0.3±0.24 vs. basal=1; n=4; *P<0.05) (Figure 4A and 4B).

Bottom Line: 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.These results reveal that MRC dysfunction modulates the MMPs expression in human normal chondrocytes demonstrating its role in the regulation of the cartilage destruction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Rheumatology Division, INIBIC-Hospital Universitario A Coruña, A Coruña, Spain.

ABSTRACT

Background: Mitochondrion 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.

Methods: Human 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.

Results: Firstly, 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.

Conclusions: These results reveal that MRC dysfunction modulates the MMPs expression in human normal chondrocytes demonstrating its role in the regulation of the cartilage destruction.

Show MeSH
Related in: MedlinePlus