Pharmacoproteomic study of three different chondroitin sulfate compounds on intracellular and extracellular human chondrocyte proteomes

Abstract

Chondroitin sulfate (CS) is a symptomatic slow acting drug for osteoarthritis (OA) widely used for the treatment of this highly prevalent disease, characterized by articular cartilage degradation. However, little is known about its mechanism of action, and recent large scale clinical trials have reported variable results on OA symptoms. Herein, we aimed to study the modulations in the intracellular proteome and the secretome of human articular cartilage cells (chondrocytes) treated with three different CS compounds, with different origin or purity, by two complementary proteomic approaches. Osteoarthritic cells were treated with 200 μg/ml of each brand of CS. Quantitative proteomics experiments were carried out by the DIGE and stable isotope labeling with amino acids in cell culture (SILAC) techniques, followed by LC-MALDI-MS/MS analysis. The DIGE study, carried out on chondrocyte whole cell extracts, led to the detection of 46 spots that were differential between conditions in our study: 27 were modulated by CS1, 4 were modulated by CS2, and 15 were modulated by CS3. The SILAC experiment, carried out on the subset of chondrocyte-secreted proteins, allowed us to identify 104 different proteins. Most of them were extracellular matrix components, and 21 were modulated by CS1, 13 were modulated by CS2, and 9 were modulated by CS3. Each of the studied compounds induces a characteristic protein profile in OA chondrocytes. CS1 displayed the widest effect but increased the mitochondrial superoxide dismutase, the cartilage oligomeric matrix protein, and some catabolic or inflammatory factors like interstitial collagenase, stromelysin-1, and pentraxin-related protein. CS2 and CS3, on the other hand, increased a number of structural proteins, growth factors, and extracellular matrix proteins. Our study shows how, from the three CS compounds tested, CS1 induces the activation of inflammatory and catabolic pathways, whereas CS2 and CS3 induce an anti-inflammatory and anabolic response. The data presented emphasize the importance of employing high quality CS compounds, supported by controlled clinical trials, in the therapy of OA. Finally, the present work exemplifies the usefulness of proteomic approaches in pharmacological studies.

Fig. 1.

DIGE experimental design. Chondrocytes were obtained from three pathological OA cartilages and primary cultured. After the first passage, the cells were treated with 200 μg/ml of each brand of CS for 48 h. Untreated cells were used as control (CTL). Chondrocytic proteins were extracted and labeled with the corresponding Cy dyes, following the scheme shown in the table. The samples were then mixed and resolved on six independent DIGE gels. Three fluorescence images were obtained from each gel and subjected to image analysis using SameSpots software.

Fig. 2.

Representative Cy2-labeled internal standard proteome map, indicating protein spots altered in CS-treated chondrocytes. The proteins were resolved in the 3–11 (nonlinear) pH range on the first dimension and on 12% acrylamide gels on the second dimension. Proteins that exhibited a significant alteration in expression in at least one of the CS-treated samples were identified by MALDI-TOF or MALDI-TOF/TOF mass spectrometry and are listed in Table I with the same number as in the figure.

Fig. 3.

Chondrocyte intracellular pathways modulated by CS. A, predicted localization of those proteins identified as altered by pharmacological treatment. B, predicted biological process in which they are involved. C, functional distribution of those proteins that were identified as altered by almost one of the three CS compounds employed in this study. Protein abbreviations, as in Table I.

Fig. 4.

SOD2 is increased by CS1. Two-dimensional Western blot (WB) analysis of SOD2 protein levels in CS1-treated chondrocytes confirms the DIGE results. A representative blot is shown, along with the data obtained by DIGE analysis for one of the three spots corresponding to a SOD2 isoform (spot ID 1217).

Fig. 5.

Silver staining of a monodimensional gel, showing the differences in the proteome and secretome profiles of treated and untreated chondrocytes and highlighting the complementarity of the two approaches. CTL, control.

Fig. 6.

Modulation by CS of the secretome profile of chondrocytes. As indicated by the Venn diagram, each of the studied compounds induces a characteristic protein profile.

Fig. 7.

Catabolic effect of CS1. As determined by LC-MS analysis, CS1 strongly increased the expression of catabolic and inflammatory mediators (MMP1, MMP3, and PTX3). Pathway Studio software was used to highlight the pathways in which these molecules are involved and that are directly related to the OA process.