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Local expression of matrix metalloproteinases, cathepsins, and their inhibitors during the development of murine antigen-induced arthritis.

Schurigt U, Stopfel N, Hückel M, Pfirschke C, Wiederanders B, Bräuer R - Arthritis Res. Ther. (2004)

Bottom Line: Proteoglycan loss analysed by safranin O staining was also strongest on days 1 and 3.Expression of most of the proteinases followed the expression of pro-inflammatory cytokines.The present study indicates that MMPs and cathepsins are important in AIA and contribute to the degradation of cartilage and bone.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pathology, Friedrich Schiller University, Jena, Germany. Uta.Schurigt@med.uni-jena.de

ABSTRACT
Cartilage and bone degradation, observed in human rheumatoid arthritis (RA), are caused by aberrant expression of proteinases, resulting in an imbalance of these degrading enzymes and their inhibitors. However, the role of the individual proteinases in the pathogenesis of degradation is not yet completely understood. Murine antigen-induced arthritis (AIA) is a well-established animal model of RA. We investigated the time profiles of expression of matrix metalloproteinase (MMP), cathepsins, tissue inhibitors of matrix metalloproteinases (TIMP) and cystatins in AIA. For primary screening, we revealed the expression profile with Affymetrix oligonucleotide chips. Real-time polymerase chain reaction (PCR) analyses were performed for the validation of array results, for tests of more RNA samples and for the completion of the time profile. For the analyses at the protein level, we used an MMP fluorescence activity assay and zymography. By a combination of oligonucleotide chips, real-time PCR and zymography, we showed differential expressions of several MMPs, cathepsins and proteinase inhibitors in the course of AIA. The strongest dysregulation was observed on days 1 and 3 in the acute phase. Proteoglycan loss analysed by safranin O staining was also strongest on days 1 and 3. Expression of most of the proteinases followed the expression of pro-inflammatory cytokines. TIMP-3 showed an expression profile similar to that of anti-inflammatory interleukin-4. The present study indicates that MMPs and cathepsins are important in AIA and contribute to the degradation of cartilage and bone.

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Detection, by gelatin zymography, of pro-matrix metalloproteinase-2 (pro-MMP-2) and pro-MMP-9 expression in joint extracts at different time points after the induction of antigen-induced arthritis. Joint extracts were prepared at days 0, 1, 3, 7 and 21 and from normal mice (N) (three individual animals per day). The protein concentration was determined by bicinchoninic acid protein assay, and equal amounts of protein were applied to each lane. The zymography was developed and stained as described in Materials and Methods. The picture shows a representative gel of three comparable experiments. S, standard (human pro-MMP-2, [72 kDa] and pro-MMP-9 [92 kDa]).
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Figure 5: Detection, by gelatin zymography, of pro-matrix metalloproteinase-2 (pro-MMP-2) and pro-MMP-9 expression in joint extracts at different time points after the induction of antigen-induced arthritis. Joint extracts were prepared at days 0, 1, 3, 7 and 21 and from normal mice (N) (three individual animals per day). The protein concentration was determined by bicinchoninic acid protein assay, and equal amounts of protein were applied to each lane. The zymography was developed and stained as described in Materials and Methods. The picture shows a representative gel of three comparable experiments. S, standard (human pro-MMP-2, [72 kDa] and pro-MMP-9 [92 kDa]).

Mentions: Zymography of total protein knee joint extracts was performed for the different time points of AIA (Fig. 5). By gelatin zymography, bands could be seen at about 105 kDa (MMP-9) and 66 kDa (MMP-2). Protein solution from arthritic knee joints showed elevated gelatinolytic activity. In contrast to mRNA studies, zymography showed an increased expression of MMP-9 (105 kDa) in the course of AIA in comparison with day 0 (Fig. 5).


Local expression of matrix metalloproteinases, cathepsins, and their inhibitors during the development of murine antigen-induced arthritis.

Schurigt U, Stopfel N, Hückel M, Pfirschke C, Wiederanders B, Bräuer R - Arthritis Res. Ther. (2004)

Detection, by gelatin zymography, of pro-matrix metalloproteinase-2 (pro-MMP-2) and pro-MMP-9 expression in joint extracts at different time points after the induction of antigen-induced arthritis. Joint extracts were prepared at days 0, 1, 3, 7 and 21 and from normal mice (N) (three individual animals per day). The protein concentration was determined by bicinchoninic acid protein assay, and equal amounts of protein were applied to each lane. The zymography was developed and stained as described in Materials and Methods. The picture shows a representative gel of three comparable experiments. S, standard (human pro-MMP-2, [72 kDa] and pro-MMP-9 [92 kDa]).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064893&req=5

Figure 5: Detection, by gelatin zymography, of pro-matrix metalloproteinase-2 (pro-MMP-2) and pro-MMP-9 expression in joint extracts at different time points after the induction of antigen-induced arthritis. Joint extracts were prepared at days 0, 1, 3, 7 and 21 and from normal mice (N) (three individual animals per day). The protein concentration was determined by bicinchoninic acid protein assay, and equal amounts of protein were applied to each lane. The zymography was developed and stained as described in Materials and Methods. The picture shows a representative gel of three comparable experiments. S, standard (human pro-MMP-2, [72 kDa] and pro-MMP-9 [92 kDa]).
Mentions: Zymography of total protein knee joint extracts was performed for the different time points of AIA (Fig. 5). By gelatin zymography, bands could be seen at about 105 kDa (MMP-9) and 66 kDa (MMP-2). Protein solution from arthritic knee joints showed elevated gelatinolytic activity. In contrast to mRNA studies, zymography showed an increased expression of MMP-9 (105 kDa) in the course of AIA in comparison with day 0 (Fig. 5).

Bottom Line: Proteoglycan loss analysed by safranin O staining was also strongest on days 1 and 3.Expression of most of the proteinases followed the expression of pro-inflammatory cytokines.The present study indicates that MMPs and cathepsins are important in AIA and contribute to the degradation of cartilage and bone.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pathology, Friedrich Schiller University, Jena, Germany. Uta.Schurigt@med.uni-jena.de

ABSTRACT
Cartilage and bone degradation, observed in human rheumatoid arthritis (RA), are caused by aberrant expression of proteinases, resulting in an imbalance of these degrading enzymes and their inhibitors. However, the role of the individual proteinases in the pathogenesis of degradation is not yet completely understood. Murine antigen-induced arthritis (AIA) is a well-established animal model of RA. We investigated the time profiles of expression of matrix metalloproteinase (MMP), cathepsins, tissue inhibitors of matrix metalloproteinases (TIMP) and cystatins in AIA. For primary screening, we revealed the expression profile with Affymetrix oligonucleotide chips. Real-time polymerase chain reaction (PCR) analyses were performed for the validation of array results, for tests of more RNA samples and for the completion of the time profile. For the analyses at the protein level, we used an MMP fluorescence activity assay and zymography. By a combination of oligonucleotide chips, real-time PCR and zymography, we showed differential expressions of several MMPs, cathepsins and proteinase inhibitors in the course of AIA. The strongest dysregulation was observed on days 1 and 3 in the acute phase. Proteoglycan loss analysed by safranin O staining was also strongest on days 1 and 3. Expression of most of the proteinases followed the expression of pro-inflammatory cytokines. TIMP-3 showed an expression profile similar to that of anti-inflammatory interleukin-4. The present study indicates that MMPs and cathepsins are important in AIA and contribute to the degradation of cartilage and bone.

Show MeSH
Related in: MedlinePlus