ANALYSIS OF THE EFFECT OF CHONDROITIN SULFATE ON CARTILAGE EXTRACELLULAR MATRIX BY METABOLIC LABELLING OF HUMAN ARTICULAR CHONDROCYTES

Background: Chondroitin sulfate (CS) is widely used in the treatment of mild and moderate osteoarthritis (OA). However, its mechanism of action remains largely unknown. Objectives: To standardize the stable isotope labelling by amino acids in cell culture (SILAC) technique on human articular chondrocytes and study the effect of CS in normal chondrocytes stimulated with interleukin-1β (IL1β) and in osteoarthritic chondrocytes. Methods: The basis of SILAC is the incorporation of stable isotope labelled amino acids into the whole proteome. When these labelled (heavy) analogues are supplied to cultured cells instead of their natural (light) forms, they are incorporated into all newly synthesized proteins. Chondrocytes released from cartilage by enzymatic digestion were recovered and plated at low density in SILAC DMEM, lacking lysine and arginine. In the case of light media, we supplemented it with the standard amino acids, while in the heavy media isotope-labelled L-lysine (C6) and L-arginine (C6N4) were employed. When complete incorporation of the heavy isotope was achieved, normal chondrocytes were treated with CS 200μg/mL and then stimulated with IL1β 5ng/mL, while osteoarthritic chondrocytes were treated with CS 200μg/mL alone. 48 hours later, conditioned media were collected and their proteins were concentrated and quantified. Heavy and light protein samples were mixed 1:1 and then subjected to enzymatic digestion and liquid chromatography/mass spectrometry (LC/MS) analysis. Protein identification and quantitative comparison between the heavy and light proteomes were therefore carried out with Protein Pilot software. Results: Collection and LC/MS analysis of the chondrocyte secreted proteins allowed the identification of 63 different proteins. Interestingly, the most abundant protein was cartilage glycoprotein 39, which has been previously related with OA pathogenesis. Moreover, most of the proteins identified are cartilage ECM components, such as collagens, lumican, proteoglycans or the cartilage oligomeric matrix protein, which clearly demonstrates the usefulness of secretome analysis for the study of ECM metabolism. We could also detect a high number of growth factors, matrix remodelling proteins, structural proteins and cytokines. Using normal chondrocytes, we analyzed the effect of CS on IL1β treated cells, and found how CS decreased a number of proteins involved in the inflammatory response like IL6, MMP1 and TNF-inducible gene 6 protein, which were previously upregulated by IL1β when compared to basal condition. In OA chondrocytes, CS seems to modulate mainly structural proteins like decorin, lumican and fibronectin. Decorin is a component of the connective tissue, binds to type I collagen fibrils, interacts with fibronectin and plays a role in matrix assembly and cartilage function. CS upregulates the expression level of all these ECM components. Conclusion: We have standardized the SILAC technique for the proteomic analysis of human primary cartilage cells, and we have applied this technology for studying the effect of CS on the chondrocyte secretome. The obtained information will increase knowledge about OA pathogenesis and therapy, and already indicates those cellular pathways that are regulated by CS, pointing out the effectiveness of this compound as anti-inflammatory and structure-modifying drug. Disclosure of Interest: None declaredCitation: Annals of the Rheumatic Diseases, volume 69, supplement 3, year 2010, page 335Session: Cartilage and synovial-like fibroblasts (Poster Presentations )