Medicine

Human mesenchymal stem cells (hMSCs), residing in bone marrow as well as in the synovial lining of joints, can be triggered to differentiate toward chondrocytes. Thus, hMSCs harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we applied for the first time the stable isotope labeling by amino acids in cell culture (SILAC) technique for the quantitative analysis of protein modulation during the chondrogenic differentiation process of hMSCs. First, we have standardized the metabolic labeling procedure on MSCs isolated from bone marrow (hBMSCs), and we have assessed the quality of chondrogenesis taking place in these conditions. Then, chondrogenic differentiation was induced on these labeled cells, and a quantitative proteomics approach has been followed to evaluate protein changes between two differentiation days. With this strategy, we could identify 622 different proteins by LC-MALDI-TOF/TOF analysis and find 65 proteins whose abundance was significantly modulated between day 2 and day 14 of chondrogenesis. Immunohistochemistry analyses were performed to verify the changes on a panel of six proteins that play different biological roles in the cell: fibronectin, gelsolin, vimentin, alpha-ATPase, mitochondrial superoxide dismutase, and cyclophilin A. All of these proteins were increased at day 14 compared to day 2 of chondrogenic induction, thus being markers of the enhanced extracellular matrix synthesis, cell adhesion, metabolism, and response to stress processes that take place in the early steps of chondrogenesis. Our strategy has allowed an additional insight into both specific protein function and the mechanisms of chondrogenesis and has provided a panel of protein markers of this differentiation process in hBMSCs.

Chondrocytes are widely used as an in vitro model of cartilage diseases such as osteoarthritis (OA). As the unique residents of mature cartilage, they are responsible of the synthesis and release of proteins essential for a proper tissue turnover. In this work, the stable isotope labeling with amino acids in cell culture (SILAC) technique has been standardized in primary human articular chondrocytes (HACs) for quantitative proteomic analyses. Then, it has been employed to study those protein modifications caused by the proinflammatory cytokine Interleukin-1beta (IL-1β), a well-known OA mediator, in these cells. Quantitative analysis of the IL-1β-treated HACs proteome revealed a global increase in cellular chaperones concurrent with a down-regulation of the actin cytoskeleton. HACs secretome analysis led to the identification and quantification of 115 proteins and unveiled the effects of the cytokine on the cartilage extracellular matrix metabolism. Among those modulated proteins, three protein clusters were found to be remarkably increased by IL-1β: proinflammatory mediators and proteases, type VI collagen and proteins known to bind this molecule, and proteins related with the TGF-beta pathway. On the other hand, secretion of aggrecan, two vitamin K-dependent proteins, and thrombospondin, among others, was strongly reduced. Altogether, these data demonstrate the usefulness of metabolic labeling for quantitative proteomics studies in HACs, show the complementarity of intracellular proteome and secretome analyses, and provide a comprehensive study of the IL-1β-mediated effects on these cells. Proteins identified in the secretome approach have a potential use as biomarkers or therapeutic targets for OA.