Proteomic analysis of human osteoarthritic chondrocytes reveals protein changes in stress and glycolysis

Ruiz-Romero, Cristina; Carreira, V.; Rego, Ignacio; Remeseiro, Silvia; López-Armada, M.J.; Blanco, Francisco J.

doi:10.1002/pmic.200700249

Cited by 111 publications

(107 citation statements)

References 32 publications

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“…Proteomic analysis of normal and OA chondrocytes has revealed changes in stress-related proteins (Ruiz-Romero et al 2008). Yamasaki et al (2006) reported resistance to ER stress-induced apoptosis in rheumatoid arthritic synovial cells.…”

Section: Discussionmentioning

confidence: 99%

Advanced Osteoarthritis in Humans Is Associated With Altered Collagen VI Expression and Upregulation of ER-stress Markers Grp78 and Bag-1

Nugent

Speicher

Gradisar

et al. 2009

J Histochem Cytochem.

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; and Summa Health Systems, Akron, Ohio (IG) S U M M A R Y To test the hypothesis that a perturbation of endoplasmic reticulum (ER) function is involved in the pathogenesis of osteoarthritis (OA), articular cartilage was isolated from non-OA patients secondary to resection of osteo-or chondrosarcomas. Intra-joint samples of minimal and advanced osteoarthritic cartilage were isolated from patients undergoing total knee arthroplasty and scored for disease severity. Glucose-regulated protein-78 (grp78) and bcl-2-associated athanogene-1 (bag-1) were detected via immunofluorescence as markers of non-homeostatic ER function. Additionally, the expression of type VI collagen and its integrin receptor, NG2, was determined to examine cartilage matrix health and turnover. There was an upregulation of grp78 in advanced OA, and variable expression in minimal OA. Non-OA cartilage was consistently grp78 negative. The downstream regulator bag-1 was also upregulated in OA compared with normal cartilage. Collagen VI was mainly cellassociated in non-OA cartilage, with a more widespread distribution observed in OA cartilage along with increased intracellular staining intensity. The collagen VI integral membrane proteoglycan receptor NG2 was downregulated in advanced OA compared with its patientmatched minimally involved cartilage sample. These results suggest that chondrocytes exhibit ER stress during OA, in association with upregulation of a large secreted molecule, type VI collagen. (J Histochem Cytochem 57:923-931, 2009) K E Y W O R D S osteoarthritis cartilage ER stress bag-1 grp78 collagen VI NG2 OSTEOARTHRITIS (OA) IS A PROGRESSIVE DISEASE, with changes in chondrocyte gene expression and extracellular matrix (ECM) composition occurring before macroscopic structural damage is observed (Hamerman 1989). Articular chondrocytes in OA exhibit a hypermetabolic phenotype, with studies describing highly proliferative clonal chondrocytes, along with the upand downregulation of ECM molecules such as aggrecan

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Section: Discussionmentioning

confidence: 99%

Advanced Osteoarthritis in Humans Is Associated With Altered Collagen VI Expression and Upregulation of ER-stress Markers Grp78 and Bag-1

Nugent

Speicher

Gradisar

et al. 2009

J Histochem Cytochem.

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“…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 then subjected to a six-plex two-dimensional DIGE analysis, resulting in three individual images from each gel (each one corresponding to Cy2-, Cy3-, and Cy5-labeled samples).…”

Section: Analysis Of the Effect Of Different Cs Compounds On Chondrocmentioning

confidence: 99%

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

Calamia

Fernández-Puente

Mateos

et al. 2012

Molecular & Cellular Proteomics

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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. Molecular & Cellular Proteomics

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“…Nine chondrocyte pellets from nine different donors of each group were mixed to form a "mixed pool." 12,13 Pooled mixture was solubilized by vortex and 1 h incubation with gentle agitation in 500 L of an IEF-compatible lysis buffer containing 8.4 M urea, 2.4 M thiourea, 5% CHAPS, 1% carrier ampholytes (IPG buffer pH 3-10 nonlinear (NL), Amersham Biosciences, Uppsala, Sweden), 0.4% Triton X-100, and 2 mM DTT. For protein quantification, 10 L of the protein extract was diluted 10× with water and precipitated for at least 1 h with 0.02% sodium deoxycholate and 10% TCA.…”

Section: Protein Sample Preparationmentioning

confidence: 99%

REST corepressor (CoREST) repression induces phenotypic gene regulation in advanced osteoarthritic chondrocytes

Xiao

et al. 2010

Journal Orthopaedic Research

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Alternations in cartilage chondrocyte phenotype characteristic by the decreased type II collagen and aggrecan together with increased type X collagen synthesis serve as a beacon for osteoarthritis progression. However, little is known about the underlying molecular mechanisms. The current study seeks to discover molecules that involved in osteoarthritic chondrocytes phenotype regulation. Differential proteomics was generated with two-dimensional gel electrophoresis between normal articular cartilage (NAC) and advanced osteoarthritic cartilage (AOC). Those differentially expressed proteins were identified by mass spectrometry. The down-regulation of a neuronal silencer, the REST corepressor (CoREST) in AOC, was verified by Western blot. CoREST silencing was performed in primarily cultured NAC chondrocytes with specific siRNA to reveal the possible involvement of CoREST repression in chondrocyte phenotypic genes modulation. Ninteen differentially expressed proteins were screened and identified. Among these proteins, CoREST, HHL, and zinc finger protein 155 were estimated to be possible gene modulators. CoREST protein level was verified to be down-regulated by 69.5% (p < 0.001) in AOC. In response to CoREST knock-down by 64.8% (p < 0.001) in NAC chondrocytes, the gene expression level of the chondrocyte terminal differentiation marker gene, collagen X was found to be up-regulated by 40.0% (p = 0.017), whereas the chondrocyte differentiation phenotypic genes, collagen II and aggrecan were down-regulated by 71.4% (p < 0.001) and 57.6% (p < 0.001), respectively. The results indicate that the silencing of CoREST by siRNA transfection in NAC may reflect CoREST repression in AOC, which results in phenotypic genes modulation and suggests a homeostatic role of this transcription factor in articular chondrocyte. © Osteoarthritis (OA) is a disease characteristic of progressive cartilage degeneration and abrasion. The central pathogenesis underlying cartilage degeneration is the imbalance between cartilage matrix proteins synthesis and catabolism, 1 whereby the pathobiology of osteoarthritic chondrocytes is of a central concern. Aside from the increased degradative enzyme activity, the phenotype alternations of chondrocytes in advanced OA cartilage lead to hypoactive anabolism, which serves as an important mechanism for OA progression. 1The normal articular chondrocytes are characteristic of phenotypic type II collagen and aggrecan expression. In the early process of OA, articular chondrocytes increase type II collagen and aggrecan synthesis in an attempt to repair cartilage lesions.2 However, as OA progresses to an advanced stage, chondrocytes begin to express heterogenous phenotypes representatively, type X collagen, 3−6 in conjunction with decreases in type II collagen and aggrecan expression, which reduces the capability of the OA cartilage to repair lesions naturally. 7,8 Chondrocyte phenotype alternations serve as an important cellular event in OA progression, however,

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Proteomic analysis of human osteoarthritic chondrocytes reveals protein changes in stress and glycolysis

Cited by 111 publications

References 32 publications

Advanced Osteoarthritis in Humans Is Associated With Altered Collagen VI Expression and Upregulation of ER-stress Markers Grp78 and Bag-1

Advanced Osteoarthritis in Humans Is Associated With Altered Collagen VI Expression and Upregulation of ER-stress Markers Grp78 and Bag-1

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

REST corepressor (CoREST) repression induces phenotypic gene regulation in advanced osteoarthritic chondrocytes

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