Effects of glucocorticoids and interleukin-1 beta on expression and activity of aggrecanases in equine chondrocytes.
OBJECTIVE
To determine effects of interleukin (IL)-1 beta and glucocorticoids on total glycosaminoglycan (GAG) loss and aggrecanase-mediated matrix degradation in equine cartilage.


METHODS
Cartilage from 24 equine cadavers free of sepsis and musculoskeletal disease.


METHODS
Effects of IL-1 beta, IL-1 beta with glucocorticoids (dexamethasone and triamcinolone, 10(-6) and 10(-7)M), and glucocorticoids alone on degradation of equine articular and nasal cartilage explants were assessed by measuring GAG release in media and GAG content in cartilage. Aggrecanase-mediated cleavage within the interglobular domain at Glu373-Ala374 was evaluated via western blot analysis and ELISAs. Steady-state mRNA concentrations of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4, and ADAMTS5 were assessed by use of real-time reverse transcriptase PCR assay (cartilage explants) and northern blot analysis (cell culture).


RESULTS
IL-1 beta increased GAG release and aggrecanase activity (11-fold). The MMP-3, MMP-13, and ADAMTS4 mRNA were upregulated with IL-1 beta, whereas ADAMTS5 mRNA was increased (13-fold), but significantly less than ADAMTS4 mRNA (27-fold), suggesting a role for both ADAMTS4 and ADAMTS5 in degradation of cytokine-stimulated cartilage. Despite downregulation of MMP-3 and MMP-13 mRNA, glucocorticoids did not alter GAG degradation. A further increase in aggrecanase activity was detected with ELISAs and western blot analysis, whereas ADAMTS4 mRNA was downregulated and ADAMTS5 mRNA was maintained or upregulated.


CONCLUSIONS
MMP-3, MMP-13, and ADAMTS4 were regulated differently than ADAMTS5. Glucocorticoids increased aggrecanase activity despite down-regulation of ADAMTS4 mRNA, suggesting a major role of ADAMTS5. Effects of glucocorticoids on aggrecanase activity have important implications in terms of treatment.
Busschers
Evita
E
Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348, USA. e.busschers@vc-someren.nl
Holt
Jeff P
JP
Richardson
Dean W
DW
D051722:ADAM Proteins / Q000378:metabolism; D000818:Animals; D002356:Cartilage; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000201:enzymology; D003907:Dexamethasone / Q000008:administration & dosage / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005938:Glucocorticoids / Q000008:administration & dosage / Q000494:pharmacology; D006736:Horses; D053583:Interleukin-1beta / Q000008:administration & dosage / Q000494:pharmacology*; D045726:Metalloproteases / Q000235:genetics / Q000378:metabolism*; D046509:Tissue Culture Techniques; D014221:Triamcinolone / Q000008:administration & dosage / Q000494:pharmacology*
D005938:Glucocorticoids; D053583:Interleukin-1beta; D014221:Triamcinolone; D003907:Dexamethasone; D045726:Metalloproteases; D051722:ADAM Proteins