title: Metabolic labeling of chondrocytes for the quantitative analysis of the interleukin-1-beta-mediated modulation of their intracellular and extracellular proteomes.
abstract: 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.
authors.affiliation: Unidad de Investigación del Envejecimiento Osteoarticular INIBIC-Complejo Hospitalario Universitario A Coruña, A Coruña, Spain.
authors.forename: Valentina
authors.initials: V
authors.lastname: Calamia
authors.forename: Beatriz
authors.initials: B
authors.lastname: Rocha
authors.forename: Jesús
authors.initials: J
authors.lastname: Mateos
authors.forename: Patricia
authors.initials: P
authors.lastname: Fernández-Puente
authors.forename: Cristina
authors.initials: C
authors.lastname: Ruiz-Romero
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Blanco
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D002853:Chromatography, Liquid; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism*; D020543:Proteome; D060888:Real-Time Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D053583:Interleukin-1beta; D020543:Proteome; D016212:Transforming Growth Factor beta