In situ zymographic localisation of type II collagen degrading activity in osteoarthritic human articular cartilage.
OBJECTIVE
Chondrocytic matrix metalloproteinases (MMPs) are believed to be important in osteoarthritic cartilage degradation. The cartilage lesion of osteoarthritis (OA) is focal and often progressive. During its development chondrocytes differentially up and down regulate production of mRNA for individual MMPs. This observation has potential implications for understanding the disease processes that lead to progressive cartilage loss in OA and designing appropriate targeted treatment. The complex regulation of MMP mediated effects means there is a pressing need to establish whether visualisation of MMP mRNA or protein equates to enzyme activity. The technique of in situ zymography (ISZ) offers a way of examining diseased human tissue for in vivo production of an excess of degrading enzyme over inhibitor. The primary objective of this study was to assess, and if positive follow, collagen II degrading activity in cartilage during development of the OA lesion. A secondary objective was to assess whether there was any correlation between sites of collagen II degrading activity and expression of the collagenase (MMP-13), recently implicated in type II collagen degredation in this lesion.


METHODS
Biopsied human normal and osteoarthritic cartilage, showing various degrees of damage, was examined by in situ zymography, with and without enzyme inhibitors, to establish sites of type II collagenase activity. Paired samples were probed for MMP-13 mRNA using 35S-labelled oligonucleotide probes. Comparative analyses were performed.


RESULTS
In situ zymography showed collagen II degrading activity over chondrocytes only in osteoarthritic cartilage. Distribution and amount varied with the extent of cartilage damage and position of chondrocytes, being greatest in deep cartilage and in cartilage lesions where fissuring was occurring. The enzyme causing the degradation behaved as a matrix metalloproteinase. MMP-13 mRNA expression codistributed with the type II collagenase activity.


CONCLUSIONS
In OA, chondrocytes can degrade type II collagen. The type II collagen degrading activity varies in site and amount as the cartilage lesion progresses and throughout codistributes with MMP-13 mRNA expression.
Freemont
A J
AJ
Musculoskeletal Research Group, Stopford Building, University of Manchester, Manchester M13 9PT.
Byers
R J
RJ
Taiwo
Y O
YO
Hoyland
J A
JA
D000328:Adult; D000368:Aged; D002358:Cartilage, Articular / Q000201:enzymology*; D004796:Clinical Enzyme Tests; D003094:Collagen / Q000032:analysis / Q000378:metabolism*; D017364:Collagenases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D017403:In Situ Hybridization; D008297:Male; D053509:Matrix Metalloproteinase 13; D008875:Middle Aged; D010003:Osteoarthritis / Q000201:enzymology; D012333:RNA, Messenger / Q000032:analysis
D012333:RNA, Messenger; D003094:Collagen; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13