Pharmacoproteomic study of the effects of chondroitin and glucosamine sulfate on human articular chondrocytes

Abstract

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 μg/mL, and then stimulated with interleukin-1β (IL-1β) 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-1β 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 IL1β-stimulated human articular chondrocytes.

Figure 1

Representative two-dimensional electrophoresis (2-DE) map of human articular chondrocyte proteins obtained in this work. Proteins were resolved in the 3 to 11 (non linear) pH range on the first dimension, and on 10% T gels on the second dimension. The 35 mapped and identified spots are annotated by numbers according to Table 1.

Figure 2

Subcellular localization (A) and functional distribution (B) of the GS- and/or CS-modulated proteins identified by proteomics. Database searches were used to classify these 35 proteins according to their subcellular localization and cellular function. Based on these characteristics, the proteins were assigned into six groups.

Figure 3

Proteins modulated similarly and differently by GS-, CS- and GS+CS-treatment in IL-1β-treated human articular chondrocytes. Proteins in the yellow circle are modulated by GS, proteins in the green circle are modulated by CS, and proteins in the white circle are modulated by the combination treatment. Upregulated proteins are indicated in red and downregulated proteins are in black (*two different isoforms; ^#the same isoform).

Figure 4

The 78 kDa glucose-regulated protein precursor (GRP78) is increased by GS alone and in combination with CS. A. Overexpression values of GRP78 determined by real-time polymerase chain reaction (PCR) analysis of cultured human articular chondrocytes treated with interleukin-1β (IL-1β) plus GS and/or CS (n = 6, P < 0.05*). B. Western blot analysis of GRP78 protein levels in treated chondrocytes. A representative blot is shown, along with the numeric data obtained by densitometry analysis of the blots (n = 4, P < 0.05*).

Figure 5

Mitochondrial superoxide dismutase (SOD2) is decreased by GS alone and in combination with CS. A. Underexpression values of SOD2 determined by real-time polymerase chain reaction (PCR) analysis on cultured human articular chondrocytes treated with interleukin-1β (IL-1β) plus GS and/or CS (n = 4, P < 0.05*). B. Western blot analysis of SOD2 protein levels in treated chondrocytes. A representative blot is shown, along with the numeric data obtained by densitometry analysis of the blots (n = 7, P < 0.05*).

Figure 6

Pathways and networks related to chondrocyte proteins identified by proteomics as altered by GS and/or CS. Pathway Studio software was used to map the identified proteins into characterized human pathways and networks that associate proteins based on known protein-protein interactions, mRNA expression studies and other previously described biochemical interactions. Abbreviations are shown as in Table 1. Most of the proteins modulated by GS belong to the unfolded protein response (UPR) system, while CS seems to affect mainly energy production (glycolysis) and metabolic pathways.