In the present work, we examined the utility of a pharmacoproteomic approach for analyzing the putative intracellular targets of glucosamine (GS) and chondroitin sulphate (CS) in cartilage cells. Using proteomic techniques, we studied the influence of these compounds, both alone and in combination, on the molecular biology of chondrocytes challenged with the proinflammatory cytokine IL-1β.
The conditions used in this study represent supraphysiological levels of both drugs and cytokine. These concentrations, however, are included in the range of in vitro
concentrations used by other laboratories, thus facilitating the comparison with other studies [27
]. In our work, we chose them according to the bibliography, where a very wide range of both glucosamine and chondroitin sulfate have been used on different cell types and tissues [29
]. We tested different concentrations of both drugs in the standardization step of the proteomic analysis (CS from 10 to 200 μg/ml and GS from 1 to 10 mM), and selected the highest concentrations in order to better unravel the molecular mechanisms that are modulated by these compounds. Moreover, in the case of glucosamine it is important to emphasize that its pharmacokinetic is modulated by the levels of glucose in the culture medium, as it utilizes glucose transporters to be taken up by the cells [31
]. Since our cells are grown under high levels of glucose (DMEM, containing 25 mmol/l glucose), it is necessary to use high concentrations of glucosamine in order to appreciate its effect in the presence of high glucose. The molecular mechanisms driven with these high amounts of both drugs might not be comparable to their classical oral administration, but they can mimic a direct delivery into the joint. In this sense, it has been recently proposed that intra-articular administration of CS may provide an immediate contact with the synoviocytes and chondrocytes, as is the case in cellular culture models [33
]. Furthermore, a recent study performed on cartilage explants shows how cyclic preloading significantly increased tissue PG content and matrix modulus when they are directly supplemented with high concentrations of the combination of GS and CS (500 μg/ml and 250 μg/ml, respectively), resulting in a reduction of matrix damage and cell death following an acute overload [34
All the mentioned limitations are inherent to in vitro studies, and also highlight the screening utility of proteomic approaches. Given the high complexity of these kinds of studies (and specifically the present one, in which five different conditions are evaluated), it is essential to be reminded how these approaches aim to screen for differences between the conditions that are being compared, opening the door for subsequent more exhaustive verification studies of some of these changes (which would allow both the inclusion of more samples to be analyzed and the performance of time-course or dose-response experiments). As a proof of the act, in this work (and based on their previously described relationship with OA pathogenesis) we selected one protein that was increased (GRP78) by the drug treatment and one that was decreased (SOD2), and performed orthogonal studies on them to verify their alteration.
Despite their limitations, several in vitro
studies have previously shown how CS and GS could moderate some aspects of the deleterious response of chondrocytes to stimulation with IL-1β. In chondrocyte cultures, GS and CS diminish the IL-1β-mediated increase of metalloproteases, [35
] the expression of phospholipase A2 [37
] and cyclooxygenase-2, [39
] and the concentrations of prostaglandin E2
]. They also reduce the concentration of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and IL-1β, in joints, [41
] and systemic and joint concentrations of nitric oxide [42
] and reactive oxygen species (ROS) [43
]. All these studies showed similar results for both molecules, mainly related to their anti-inflammatory effect, while the results obtained by our pharmacoproteomic approach highlight the different molecular mechanisms affected by GS or CS. It is essential to point out that our study has been performed with chondrocytic intracellular extracts. In this context, it is difficult to identify proteins that are known to be secreted by the chondrocytes, such as metalloproteinases, cytokines or aggrecanases, which have been the focus of a recent mRNA-based analysis [44
], or hyaluronan synthases, which have been newly found to be increased by CS in synoviocytes [45
]. All these were also described to be modulated by GS in a previous transcriptomic study [10
]. However, detection of this type of proteins in intracellular fractions by shotgun proteomics is not easily achievable because they are mainly delivered to the extracellular space after their synthesis, being those small amounts that are retained inside the cells masked by other typical cytoplasmic proteins which are more abundant [13
]. Given the high dynamic range of proteins in biological systems, this problem is inherent to global screening proteomic experiments, and is only solvable employing hypothesis-driven proteomics strategies (targeted proteomics).
As mentioned before, this study is focused on the investigation of the intracellular mechanisms modulated by CS and GS, which are the background for ulterior putative changes of ECM turnover. In our work, 25% 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, whereas CS affects mainly energy production (31%) and metabolic pathways (13%) by decreasing the expression levels of 10 proteins (Figure ). Bioinformatic analysis using Pathway Studio 6.1 software (Ariadne Genomics, Rockville, MD, USA) enabled the characterization of the biological association networks related to these differentially expressed chondrocytic proteins. A simplified picture of their interactions is showed in Figure . Using this analysis, we identified the biochemical pathways that may be altered when chondrocytes are treated with GS and CS.
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 (more ...)
Most of the proteins modulated by GS belong to the complex homeostatic signalling pathway known as the unfolded protein response (UPR). The UPR system is involved in balancing the load of newly synthesized proteins with the capacity of the ER to facilitate their maturation. Dysfunction of the UPR plays an important role in certain diseases, particularly those involving tissues like cartilage that are dedicated to extracellular protein synthesis. The effect of GS on molecular chaperones and the role of protein disulfide isomerases (PDIs) in the maturation of proteins related with cartilage ECM structure have been described [46
]. PDIA3 (GRP58) is a protein on the ER that interacts with the lectin chaperones, calreticulin and calnexin, to modulate the folding of newly synthesized glycoproteins [47
], whereas PDIA1 (prolyl 4-hydroxylase subunit beta) constitutes a structural subunit of prolyl 4-hydroxylase, an enzyme that is essential for procollagen maturation [48
]. The marked GS-mediated increase of these proteins in chondrocytes points to an elevation in ECM protein synthesis, which might be also hypothesized by the detected increase in Type IV Collagen (COL6A1, essential for chondrocyte anchoring to the pericellular matrix [49
]) synthesis caused by GS.
Finally, GS remarkably increases another UPR-related protein, GRP78 (BiP), a fact that we confirmed both at transcript and protein levels. This protein is localized in the ER, and has been previously identified as an RA autoantigen [50
], which was subsequently characterized by its anti-inflammatory properties through the stimulation of an anti-inflammatory gene program from human monocytes and the development of T-cells that secrete regulatory cytokines such as IL-10 and IL-4 [51
]. In a previous work, we found an increase of this protein in OA chondrocytes, which might be a consequence of heightened cellular stress [14
]. A number of previous reports have described the positive modulation of GS on ER proteins, including GRP78 expression [52
], but this is the first time that such modulation was found to arise from GS treatment in chondrocytes; thus interestingly suggesting an specific mechanism of action for the putative anti-inflammatory effect of GS in OA.
On the other hand, most proteins modulated by CS are proteins related to metabolism and energy production. It is remarkable that all except one (an enolase isoform) were decreased. In this group, we identified seven out of the 10 enzymes that directly participate in the glycolysis pathway (aldolase, triose phosphate isomerase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase, enolase and pyruvate kinase). This suggests that, while IL-1β treatment tends to elevate glycolytic energy production ([15
] and our observations), it is then lowered by CS (which reduces five of these enzymes) and by the combination of both drugs (which reduces all seven glycolytic enzymes). The decrease of Neutral alpha-glucosidase AB (or glucosidase II, GANAB), only caused by CS alone (Figure ), and two other metabolism-related proteins (AK1C2 and UGDH), points also to a reduction of cellular metabolism. GANAB is an ER-enzyme that has profound effects on the early events of glycoprotein metabolism, and has been recently proposed as biomarker for detecting mild human knee osteoarthritis [53
Interestingly, only four proteins were found to be modulated by GS and CS combination but not by either of the drugs alone, whereas we observed a quantitative synergistic effect of the combination in more than a half (55%) of the altered proteins (Table ). One of the proteins whose decrease by both drugs alone was significant and furthermore powered by their combination is the redox-related protein SOD2. This protein, the mitochondrial superoxide dismutase, has substantial relevance in stress oxidative pathways and in cytokine-related diseases, such as OA [54
]. We found SOD2 to be upregulated by IL-1β ([15
] and our observations), and downregulated by GS and CS treatment, both at the transcriptional and protein levels (Real Time-PCR and Western blotting). Supporting our findings, other authors have recently reported the role of GS in counteracting the IL-1β-mediated increase of inducible nitric oxide synthase (iNOS) and the decrease of heme oxygenase, and indicated that the influence of GS and CS on oxidative stress is a possible mechanism of action for its protective effect on chondrocytes [55