Subject(s)
Cartilage, Articular/metabolism , Gene Expression Regulation/physiology , Laminin/physiology , Matrix Metalloproteinase 3/genetics , Osteoarthritis/metabolism , DNA Primers/chemistry , Humans , Immunohistochemistry , Matrix Metalloproteinase 3/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
OBJECTIVE: Sphingosine-1-phosphate (S1P) is a messenger molecule, with important functions in inflammation and wound healing. The present study was performed to elucidate a possible role of S1P signaling in articular chondrocytes. METHODS: Human and bovine primary chondrocytes were cultured in monolayer. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect S1P receptor mRNA. Proliferation of S1P stimulated chondrocytes was measured by 3H-thymidine uptake. Supernatants of cultured bovine chondrocytes stimulated with S1P alone or in combination with interleukin-1beta (IL-1beta) were tested for nitric oxide (NO) formation and expression of inducible nitric oxide synthase (iNOS). Matrixmetalloprotease-13 (MMP-13) and aggrecanase-1 (ADAMTS-4) were evaluated using real-time PCR. Glycosaminoglycan (GAG) loss from bovine cartilage explants was evaluated using the dimethylene blue method. RESULTS: S1P1, S1P2 and S1P3 but not S1P4 and S1P5 receptor mRNA were detected in human and bovine chondrocytes. S1P dose dependently induced proliferation in bovine and human chondrocytes. S1P significantly reduced NO formation and iNOS mRNA and protein expression, both in un-stimulated and IL-1beta stimulated bovine chondrocytes. Furthermore, S1P dose dependently inhibited IL-1beta induced expression of ADAMTS-4 and MMP-13 and diminished IL-1beta mediated GAG depletion from cartilage explants. CONCLUSION: These results suggest that S1P provides an anti-catabolic signal in articular chondrocytes.