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

Francisco J. Blanco,Jesús Mateos,Patricia Fernández-Puente,Lucía Lourido,Eulália Montell,Valentina Calamia,Beatriz Rocha,Cristina Ruiz-Romero,Josep Vergés

doi:10.1074/mcp.m111.013417

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.

Highlights

From the ‡Osteoarticular and Aging Research Lab, Proteomics Unit, ProteoRed/ISCIII, Rheumatology Division, INIBIC-CHU A Coruna, As Xubias 84, 15006 A Coruna, Spain, the **Medical Department, Bioiberica Pharma, Barcelona, Spain, the ‡‡CIBER-BBN, Instituto de Salud Carlos III-Madrid-Spain, and the §Catedra Bioiberica, Universidad de A Coruna, A Coruna, Spain
The abbreviations used are: OA, osteoarthritis; CS, chondroitin sulfate; FBS, fetal bovine serum; GANAB, neutral ␣-glucosidase AB; GS, glucosamine sulfate; IGF, insulin-like growth factor; IGFbinding proteins (IGFBPs), IGFbinding protein; I␬B, inhibitor ␬B; MMP1, interstitial collagenase; SILAC, stable isotope labeling with amino acids in cell culture; SOD2, mitochondrial superoxide dismutase; TGF-␤, transforming growth factor-␤
Whole cell extracts were collected 48 h after CS addition, according to a protocol previously set up by our group [11, 16]. These samples were subjected to a six-plex two-dimensional DIGE analysis, resulting in three individual images from each gel

Summary

Introduction

From the ‡Osteoarticular and Aging Research Lab, Proteomics Unit, ProteoRed/ISCIII, Rheumatology Division, INIBIC-CHU A Coruna, As Xubias 84, 15006 A Coruna, Spain, the **Medical Department, Bioiberica Pharma, Barcelona, Spain, the ‡‡CIBER-BBN, Instituto de Salud Carlos III-Madrid-Spain, and the §Catedra Bioiberica, Universidad de A Coruna, A Coruna, Spain. Beneficial effects of orally administrated CS in OA patients have been reported (14 –16), caution should be exercised in the study or use of different CS formulations, because the species or tissue of origin could result in great differences in CS structural organization or disaccharide composition. For this reason, recent meta-analysis [9] and large scale clinical trials [10] have demonstrated variable effects on OA symptoms, yielding conflicting results

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