Chondrocyte-mediated catabolism of aggrecan: aggrecanase-dependent cleavage induced by interleukin-1 or retinoic acid can be inhibited by glucosamine.
A rat chondrosarcoma cell line and bovine cartilage explants have been used to study the control of aggrecan degradation by chondrocytes treated with interleukin-1 (IL-1) or retinoic acid (RA). Aggrecan fragment analysis with anti-neo-epitope antibodies suggests that aggrecanase (an as yet unidentified enzyme) is the only aggrecan-degrading proteinase active in these cultures. With rat cells, aggrecanase converts the aggrecan core protein into two major G1-domain-bearing products (60 kDa with a C-terminal Glu-373, and 220 kDa with a C-terminal Glu-1459). Both products were quantified on a standardized Western analysis system with a G1-specific antibody. Immunoblots were analysed by scanning densitometry and the sensitivity, linearity and reproducibility of the assay were established. With rat cells the aggrecanase response to IL-1 was optimal at about 2 mM glutamine, but was progressively inhibited at higher concentrations, with about 90% inhibition at 10 mM glutamine. Such inhibition by glutamine was not, however, observed with bovine explants. On the other hand, marked inhibition of aggrecanase-dependent cleavage was observed with both rat cells and bovine explants when d(+)-glucosamine was included at concentrations above 2 mM. Inhibition was apparently not due to cytotoxicity or interference with IL-1 signalling, since biosynthetic activity was not inhibited and inhibition of the aggrecanase response was also obtained when RA was used as the catabolic stimulator. Possible mechanisms for the inhibition of the aggrecanase response by glucosamine in chondrocytes treated with IL-1 or RA are discussed.
Sandy
J D
JD
Biochemistry Section, Research Department, Shriners Hospital for Children, 12502 North Pine Drive, Tampa, FL 33612, USA. johnsandy@mindspring.com
Gamett
D
D
Thompson
V
V
Verscharen
C
C
D053674:Aggrecans; D000818:Animals; D002356:Cartilage / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D005944:Glucosamine / Q000494:pharmacology*; D005973:Glutamine / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology*; D037181:Lectins, C-Type; D011509:Proteoglycans / Q000378:metabolism*; D051381:Rats; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
C508321:Acan protein, rat; D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D007375:Interleukin-1; D037181:Lectins, C-Type; D011509:Proteoglycans; D005973:Glutamine; D014212:Tretinoin; D010450:Endopeptidases; C083641:aggrecanase; D005944:Glucosamine