Aggrecanolysis in human osteoarthritis: confocal localization and biochemical characterization of ADAMTS5-hyaluronan complexes in articular cartilages.
OBJECTIVE
Human osteoarthritis (OA) is characterized by aggrecanase-mediated depletion of cartilage aggrecan. We have examined the abundance, location and some biochemical properties of the six known aggrecanases (A disintegrin and metalloproteinase with thrombospondin-like motifs 1 (ADAMTS1) 4, 5, 8, 9 and 15) in normal and OA human cartilages.


METHODS
Formalin-fixed, ethylenediamine tetraacetic acid (EDTA)-decalcified sections of full-depth cartilage from human OA tibial plateaus and normal control samples were studied by confocal imaging. Probes included specific antibodies to aggrecanases and two aggrecan epitopes, as well as biotinylated hyaluronan binding protein (HABP) for hyaluronan (HA) visualization. Cartilage extracts were analyzed by Western blot for the individual proteinases and aggrecan fragments.


RESULTS
ADAMTS5 was present in association with cells throughout normal cartilage and was markedly increased in OA, particularly in clonal groups in the superficial and transitional zones, where it was predominantly co-localized with HA. Consistent with the confocal analysis, a high molecular weight complex of ADAMTS5 and HA was isolated from human OA cartilage by isotonic salt extraction and chromatography on Superose 6. The complex eluted with an apparent molecular size of about 2x10(6) and contained major ADAMTS5 forms of 150, 60, 40 and 30kDa. The yield of most forms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was markedly enhanced by prior digestion of the complex with either Streptomyces hyaluronidase or chondroitinase ABC.


CONCLUSIONS
ADAMTS5 abundance and distribution in human OA cartilages is consistent with a central role for this enzyme in destructive aggrecanolysis. HA-dependent sequestration of ADAMTS5 in the pericellular matrix may be a mechanism for regulating the activity of this proteinase in human OA cartilage.
Plaas
A
A
Department of Internal Medicine, College of Medicine, University of South Florida, Tampa, FL, USA. aplaas@gmail.com
Osborn
B
B
Yoshihara
Y
Y
Bai
Y
Y
Bloom
T
T
Nelson
F
F
Mikecz
K
K
Sandy
J D
JD
D051722:ADAM Proteins / Q000378:metabolism*; D000368:Aged; D053674:Aggrecans / Q000378:metabolism*; D002358:Cartilage, Articular / Q000378:metabolism*; D019902:Chondrocytes / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D005260:Female; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods; D008875:Middle Aged; D010003:Osteoarthritis / Q000378:metabolism
D053674:Aggrecans; D006820:Hyaluronic Acid; D010450:Endopeptidases; D051722:ADAM Proteins; C083641:aggrecanase