RESUMEN
OBJECTIVE: The lipid mediator sphingosine 1-phosphate (S1P) is found in the synovial fluid of osteoarthritis (OA) patients. S1P protects bovine cartilage by counteracting the effects of interleukin-1ß (IL-1ß). This study was undertaken to examine the interaction of S1P and IL-1ß in human OA chondrocytes. METHODS: Human cartilage was obtained from patients undergoing total knee joint replacement. Chondrocytes were cultured in monolayer and treated with IL-1ß and S1P. Expression of S1P receptor subtypes and genes involved in cartilage degradation was evaluated using real-time polymerase chain reaction, immunohistochemistry, and Western blotting. S1P signaling was evaluated using inhibitors of S1P receptors and small interfering RNA (siRNA) knockdown of the S1P2 receptor. Phosphorylation of MAP kinases and NF-κB in response to IL-1ß and S1P was detected by Western blotting. RESULTS: S1P2 was identified as the most prevalent S1P receptor subtype in human OA cartilage and chondrocytes in vitro. S1P reduced expression of inducible nitric oxide synthase (iNOS) in IL-1ß-treated chondrocytes. Reduction of ADAMTS-4 and matrix metalloproteinase 13 expression by S1P correlated with S1P2 expression. Pharmacologic inhibition of the S1P2 receptor, but not the S1P1 and S1P3 receptors, abrogated the inhibition of iNOS expression. Similar results were observed using siRNA knockdown. S1P signaling inhibited IL-1ß-induced phosphorylation of p38 MAPK. CONCLUSION: In human chondrocytes, S1P reduces the induction of catabolic genes in the presence of IL-1ß. Activation of the S1P2 receptor counteracts the detrimental phosphorylation of p38 MAPK by IL-1ß.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Interleucina-1beta/farmacología , Lisofosfolípidos/farmacología , Esfingosina/análogos & derivados , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Cartílago Articular/metabolismo , Cartílago Articular/patología , Células Cultivadas , Condrocitos/metabolismo , Condrocitos/patología , Antagonismo de Drogas , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Interleucina-1beta/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación , Procolágeno N-Endopeptidasa/metabolismo , ARN Interferente Pequeño/genética , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Esfingosina/farmacología , Receptores de Esfingosina-1-Fosfato , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: FTY720 (Fingolimod) is a novel immunosuppressive drug investigated in clinical trials for organ transplantation and multiple sclerosis. It acts as a functional sphingosine-1-phosphate (S1P) receptor antagonist, thereby inhibiting the egress of lymphocytes from secondary lymphoid organs. As S1P is able to prevent IL-1beta induced cartilage degradation, we examined the direct impact of FTY720 on cytokine induced cartilage destruction. METHODS: Bovine chondrocytes were treated with the bioactive phosphorylated form of FTY720 (FTY720-P) in combination with IL-1beta or TNF-alpha. Expression of MMP-1,-3.-13, iNOS and ADAMTS-4,-5 and COX-2 was evaluated using quantitative real-time PCR and western blot. Glycosaminoglycan depletion from cartilage explants was determined using a 1,9-dimethylene blue assay and safranin O staining. RESULTS: FTY720-P significantly reduced IL-1beta and TNF-alpha induced expression of iNOS. In contrast FTY720-P increased MMP-3 and ADAMTS-5 mRNA expression. Furthermore depletion of glycosaminoglycan from cartilage explants by IL-1beta and TNF-alpha was significantly enhanced by FTY720-P in an MMP-3 dependent manner. CONCLUSIONS: Our results suggest that FTY720 may enhance cartilage degradation in pro-inflammatory environment.