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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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Related in: MedlinePlus

Proteoglycan presence in tissues treated with MRC inhibitors. Cartilage explants were treated with Oligomycin 5 μg/mL (A) or AA 20 μg/mL (B) for 72 h and proteoglycanes were stained with touidine blue. Safranine/fast-green was also used to detect proteoglycans with Oligomycin (C). The figure represents an experiment of 3.
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Figure 7: Proteoglycan presence in tissues treated with MRC inhibitors. Cartilage explants were treated with Oligomycin 5 μg/mL (A) or AA 20 μg/mL (B) for 72 h and proteoglycanes were stained with touidine blue. Safranine/fast-green was also used to detect proteoglycans with Oligomycin (C). The figure represents an experiment of 3.

Mentions: MMPs degrade the cartilage affecting the proteoglycan integrity and quantity. We evaluated the levels in tissue sections with toulidin blue staining. Results showed that Oligomycin 5 μg/ml produced a decrease in the proteoglycan quantity (Figure 7A). Figure 7B shows tissue explants treated with the complex III inhibitor (AA 20 μg/ml) for 72 h. Results indicated that the levels of proteoglycans did not change with AA. However, Oligomycin 5 μg/mL reduced proteoglycan quantities as safranin staining shows in Figure 7C (25.00±9.82 vs. basal=32.03±8.97).


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)

Proteoglycan presence in tissues treated with MRC inhibitors. Cartilage explants were treated with Oligomycin 5 μg/mL (A) or AA 20 μg/mL (B) for 72 h and proteoglycanes were stained with touidine blue. Safranine/fast-green was also used to detect proteoglycans with Oligomycin (C). The figure represents an experiment of 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Proteoglycan presence in tissues treated with MRC inhibitors. Cartilage explants were treated with Oligomycin 5 μg/mL (A) or AA 20 μg/mL (B) for 72 h and proteoglycanes were stained with touidine blue. Safranine/fast-green was also used to detect proteoglycans with Oligomycin (C). The figure represents an experiment of 3.
Mentions: MMPs degrade the cartilage affecting the proteoglycan integrity and quantity. We evaluated the levels in tissue sections with toulidin blue staining. Results showed that Oligomycin 5 μg/ml produced a decrease in the proteoglycan quantity (Figure 7A). Figure 7B shows tissue explants treated with the complex III inhibitor (AA 20 μg/ml) for 72 h. Results indicated that the levels of proteoglycans did not change with AA. However, Oligomycin 5 μg/mL reduced proteoglycan quantities as safranin staining shows in Figure 7C (25.00±9.82 vs. basal=32.03±8.97).

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