Cyclosporin A inhibition of aggrecanase-mediated proteoglycan catabolism in articular cartilage.
OBJECTIVE
To determine the effect of cyclosporin A (CSA) on aggrecanase- and matrix metalloproteinase (MMP)-mediated catabolism of proteoglycan (aggrecan) in articular cartilage explants stimulated with interleukin-1 (IL-1) in a culture system that mimics early pathologic processes associated with arthritic disease.


METHODS
Proteoglycan (glycosaminoglycan) and lactate quantification, Western immunoblot analyses of aggrecan degradation products, reverse transcription-polymerase chain reaction analyses of aggrecanase-1, aggrecanase-2 (ADAM-TS4, ADAM-TS5, respectively), MMP-1, MMP-3, MMP-13, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, and TIMP-3 messenger RNA (mRNA) expression in articular cartilage explant cultures, and electrophoretic mobility shift assay analysis of nuclear factor of activated T cells (NF-AT) transcription factor activation were used.


RESULTS
CSA inhibited, in a dose-dependent and noncytotoxic manner, aggrecanase-mediated proteoglycan catabolism and loss from IL-1-stimulated cartilage explants. There was no evidence of MMP-mediated aggrecan catabolism in this in vitro model. Treatment of articular cartilage explant cultures with 10 ng/ml of IL-1alpha up-regulated the expression of mRNA for ADAM-TS4, ADAM-TS5, MMP-1, MMP-3, and MMP-13. The expression of ADAM-TS4, ADAM-TS5, and MMP-13 was abrogated by the inclusion of 10 microM CSA in the culture medium. NF-AT activation was observed in chondrocytes but could not be inhibited by preincubation with 10 microM CSA.


CONCLUSIONS
CSA can inhibit IL-1-induced aggrecanase-mediated proteoglycan catabolism in articular cartilage explants maintained in culture for 4 days, thus demonstrating molecular mechanisms whereby CSA may be an effective therapy for degenerative joint disease.
Little
Chris B
CB
Connective Tissue Biology Laboratories, Cardiff School of Biosciences, Cardiff University, UK. LittleCB@Cardiff.ac.uk
Hughes
Clare E
CE
Curtis
Clare L
CL
Jones
Simon A
SA
Caterson
Bruce
B
Flannery
Carl R
CR
D053674:Aggrecans; D000818:Animals; D018501:Antirheumatic Agents / Q000494:pharmacology*; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000201:enzymology; D016572:Cyclosporine / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007375:Interleukin-1 / Q000494:pharmacology; D007592:Joint Diseases / Q000188:drug therapy / Q000378:metabolism; D037181:Lectins, C-Type; D008666:Metalloendopeptidases / Q000235:genetics; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011509:Proteoglycans / Q000378:metabolism*; D019714:Tissue Inhibitor of Metalloproteinases / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism
D053674:Aggrecans; D018501:Antirheumatic Agents; D004268:DNA-Binding Proteins; D016326:Extracellular Matrix Proteins; D007375:Interleukin-1; D037181:Lectins, C-Type; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011509:Proteoglycans; D019714:Tissue Inhibitor of Metalloproteinases; D014157:Transcription Factors; D016572:Cyclosporine; D010450:Endopeptidases; D008666:Metalloendopeptidases; C083641:aggrecanase