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.

Matrix-Embedded Cytokines to Simulate Osteoarthritis-Like Cartilage Microenvironments

“…Calamia et al also reported vimentin was the most abundant protein expressed after cytokine treatment to chondrocytes. 52 Interestingly in microarray results, vimentin was 5.8-fold upregulated in the in vitro OA model compared to the OA cartilage biopsies, whereas it was 59.98-fold upregulated in the in vitro OA model compared to tissue-engineered cartilage (Fig. 8D).…”

Pharmacoproteomic Study of Three Different Chondroitin Sulfate Compounds on Intracellular and Extracellular Human Chondrocyte Proteomes

“…Nevertheless, two-dimensional gels present a number of limitations, such as the difficulties in focusing very acidic proteins (greatly represented in cartilage by the high density and anionic nature of proteoglycans). To avoid these problems and to increase the dynamic range for a more accurate protein quantification (usually, 1-2 orders of magnitude in two-dimensional electrophoresis experiments), we recently developed an alternative method based on SILAC for the quantitative analysis of chondrocytes proteome and secretome (15). The present work illustrates the successful complement of the best quantitative gel-based method up to date (DIGE) for the proteomic analysis of intracellular proteins and the SILAC approach followed by LC-MS analysis for the quantitative study of extracellular proteins.…”

“…In the case of light media, standard L-lysine and L-arginine were added, whereas in the heavy media, isotope-labeled L-lysine ( 13 C 6 ) and isotope-labeled L-arginine ( 13 C 6 , 15 N 4 ) were used. Cell expansion was carried out as described previously by our group (15). Chondrocytes were used at week 3 in primary culture (P1) when 100% of labeling was reached.…”

Secretome analysis of human articular chondrocytes unravels catabolic effects of nicotine on the joint

“…In the case of light media, standard L-lysine and L-arginine were used, while in the heavy media isotope-labelled L-lysine ( 13 C 6 ), and isotope-labeled L-arginine ( 13 C 6 , 15 N 4 ) were added at 146 mg/L for K and 28 mg/L for R. HACs expansion, cell type verification by the assessment of COL2 expression, and evaluation of complete labeling were performed as described previously by our group [24]. Cell proliferation and cell viability were tested by cell count and Trypan Blue dye exclusion.…”

“…Then, heavy and light samples were mixed 1:1, and 6 g of each mixed sample were separated onto a 10% SDS-PAGE gel. Gels were stained with Coomassie blue and the resulting lanes were cut into six slices and subjected to in-gel digestion according to previous protocols [24]. Digestion was performed overnight with 12.5 ng/L of Sequencing Grade Modified Trypsin (Promega) at 37ЊC.…”