Dataset Information

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 ?g/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.

SUBMITTER: Calamia V

PROVIDER: S-EPMC3433908 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Calamia Valentina V Fernández-Puente Patricia P Mateos Jesús J Lourido Lucía L Rocha Beatriz B Montell Eulália E Vergés Josep J Ruiz-Romero Cristina C Blanco Francisco J FJ

Molecular & cellular proteomics : MCP 20111227 6

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 di ...[more]

PMID: 22203690

Similar Datasets

Project description:INTRODUCTION:Chondroitin sulfate (CS) and glucosamine sulfate (GS) are symptomatic slow-acting drugs for osteoarthritis (OA) widely used in clinic. Despite their widespread use, knowledge of the specific molecular mechanisms of their action is limited. The aim of this work is to explore the utility of a pharmacoproteomic approach for the identification of specific molecules involved in the pharmacological effect of GS and CS. METHODS:Chondrocytes obtained from three healthy donors were treated with GS 10 mM and/or CS 200 ?g/mL, and then stimulated with interleukin-1? (IL-1?) 10 ng/mL. Whole cell proteins were isolated 24 hours later and resolved by two-dimensional electrophoresis. The gels were stained with SYPRORuby. Modulated proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry. Real-time PCR and Western blot analyses were performed to validate our results. RESULTS:A total of 31 different proteins were altered by GS or/and CS treatment when compared to control. Regarding their predicted biological function, 35% of the proteins modulated by GS are involved in signal transduction pathways, 15% in redox and stress response, and 25% in protein synthesis and folding processes. Interestingly, CS affects mainly energy production (31%) and metabolic pathways (13%), decreasing the expression levels of ten proteins. The chaperone GRP78 was found to be remarkably increased by GS alone and in combination with CS, a fact that unveils a putative mechanism for the reported anti-inflammatory effect of GS in OA. On the other hand, the antioxidant enzyme superoxide dismutase 2 (SOD2) was significantly decreased by both drugs and synergistically by their combination, thus suggesting a drug-induced decrease of the oxidative stress caused by IL-1? in chondrocytes. CONCLUSIONS:CS and GS differentially modulate the proteomic profile of human chondrocytes. This pharmacoproteomic approach unravels the complex intracellular mechanisms that are modulated by these drugs on IL1?-stimulated human articular chondrocytes.

Project description:INTRODUCTION: The pathogenesis of osteoarthritis (OA) is characterized by the production of high amounts of nitric oxide (NO), as a consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines. NO donors represent a powerful tool for studying the role of NO in the cartilage in vitro. There is no consensus about NO effects on articular cartilage in part because the differences between the NO donors available. The aim of this work is to compare the metabolic profile of traditional and new generation NO donors to see which one points out the osteoarthritic process in the best way. METHODS: Human healthy and OA chondrocytes were isolated from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with NO donors (NOC-12 or SNP). NO production was evaluated by the Griess method, and apoptosis was quantified by flow cytometry. Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities by enzymatic assay, mitochondrial membrane potential (??m) by JC-1 using flow cytometry, and ATP levels were measured by luminescence assays. Glucose transport was measured as the uptake of 2-deoxy-[(3)H]glucose (2-[(3)H]DG). Statistical analysis was performed using the Mann-Whitney U test. RESULTS: NOC-12 liberates approximately ten times more NO2- than SNP, but the level of cell death induced was not as profound as that produced by SNP. Normal articular chondrocytes stimulated with NOC-12 had reduced activity from complexes I, III y IV, and the mitochondrial mass was increased in these cells. Deleterious effects on ??m and ATP levels were more profound with SNP, and this NO donor was able to reduce 2-[(3)H]DG levels. Both NO donors had opposite effects on lactate release, SNP diminished the levels and NOC-12 lead to lactate accumulation. OA chondrocytes incorporate significantly more 2-[(3)H]DG than healthy cells. CONCLUSIONS: These findings suggest that the new generation donors, specifically NOC-12, mimic the OA metabolic process much better than SNP. Previous results using SNP have to be considered prudently since most of the effects observed can be induced by the interactions of secondary products of NO.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure