Relative contribution of matrix metalloprotease and cysteine protease activities to cytokine-stimulated articular cartilage degradation.
OBJECTIVE
Both matrix metalloprotease (MMP) activity and cathepsin K (CK) activity have been implicated in cartilage turnover. We investigated the relative contribution of MMP activity and CK activity in cartilage degradation using ex vivo and in vivo models.


METHODS
Bovine articular cartilage explants were stimulated with oncostatin M (OSM) 10 ng/ml and tumor necrosis factor-alpha (TNF-alpha) 20 ng/ml in the presence or absence of the broad-spectrum MMP inhibitor GM6001 and the cysteine protease inhibitor, E64. Cartilage degradation was evaluated in the conditioned medium by glycosaminoglycans (GAG), hydroxyproline, and cross-linked C-telopeptide fragments of type II collagen (CTX-II), which were compared to immunohistochemical evaluations of proteoglycans and CTX-II. We assessed MMP expression by gelatine zymography and CK expression by immunohistochemistry. In vivo, CTX-II release was measured from CK-deficient mice.


RESULTS
OSM and TNF-alpha combined induced significant (P<0.01) increase in cartilage degradation products measured by hydroxyproline and CTX-II compared to vehicle control. The cytokines potently induced MMP expression, assessed by zymography, and CK expression investigated by immunohistochemistry. Inhibition of MMP activity completely abrogated hydroxyproline and CTX-II release (P<0.01) and GAG release (P<0.05). In contrast, E64 resulted in increased CTX-II release by 100% (P<0.05) and inhibited GAG release by 30%. Up-regulation of CTX-II fragments was confirmed in vivo in CK null mice.


CONCLUSIONS
Inhibition of MMP activity reduced both proteoglycan loss and type II collagen degradation. In contrast, inhibition of cysteine proteases resulted in an increase rather than a decrease in MMP derived fragments of collagen type II degradation, CTX-II, suggesting altered collagen metabolism.
Sondergaard
B C
BC
Nordic Bioscience Diagnostics, Herlev, Denmark.
Henriksen
K
K
Wulf
H
H
Oestergaard
S
S
Schurigt
U
U
Bräuer
R
R
Danielsen
I
I
Christiansen
C
C
Qvist
P
P
Karsdal
M A
MA
D000818:Animals; D001169:Arthritis, Experimental; D015415:Biomarkers / Q000032:analysis; D002358:Cartilage, Articular / Q000187:drug effects / Q000201:enzymology*; D056657:Cathepsin K; D002403:Cathepsins / Q000172:deficiency / Q000378:metabolism; D002417:Cattle; D024042:Collagen Type I / Q000032:analysis; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology*; D004151:Dipeptides / Q000494:pharmacology; D005109:Extracellular Matrix / Q000201:enzymology / Q000276:immunology*; D006025:Glycosaminoglycans / Q000032:analysis; D006909:Hydroxyproline / Q000032:analysis; D007150:Immunohistochemistry / Q000379:methods; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D053683:Oncostatin M / Q000494:pharmacology; D020370:Osteoarthritis, Knee / Q000201:enzymology / Q000276:immunology*; D010455:Peptides / Q000032:analysis; D013268:Stimulation, Chemical; D046509:Tissue Culture Techniques; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D015415:Biomarkers; D024042:Collagen Type I; D015853:Cysteine Proteinase Inhibitors; D016207:Cytokines; D004151:Dipeptides; D006025:Glycosaminoglycans; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D010455:Peptides; D014409:Tumor Necrosis Factor-alpha; C052929:collagen type I trimeric cross-linked peptide; D053683:Oncostatin M; C032425:N-(N-(3-carboxyoxirane-2-carbonyl)leucyl)isoamylamine; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056657:Cathepsin K; C534132:Ctsk protein, mouse; D020782:Matrix Metalloproteinases; D007930:Leucine; D006909:Hydroxyproline