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.
Conclusion: 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.