Mitochondrial Dysregulation of Osteoarthritic Human Articular Chondrocytes Analyzed by Proteomics

Abstract

Mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including osteoarthritis (OA). Mitochondrial proteins are attractive targets for the study of metabolism of the chondrocyte, the unique cell type present in mature cartilage, and its role in tissue degradation. Using a proteomics approach based on two-dimensional DIGE and MALDI-TOF/TOF mass spectrometric identification of mitochondria- enriched protein fractions from human articular chondrocytes, we analyzed mitochondrial protein changes that are characteristic of OA chondrocytes. A total of 73 protein forms were unambiguously identified as significantly altered in OA; 23 of them have been previously described as mitochondrial. An extensive statistical and cluster analysis of the data revealed a mitochondrial protein profile characteristic for OA. This pattern includes alterations in energy production, maintenance of mitochondrial membrane integrity, and free radical detoxification. Real time PCR, Western blot, and immunohistofluorescence assays confirmed a significant decrease of the major mitochondrial antioxidant protein manganese-superoxide dismutase (SOD2) in the superficial layer of OA cartilage. As possible outputs for this antioxidant deficiency, we found an increase of intracellular reactive oxygen species generation in OA chondrocytes and also verified an OA-dependent increase in the mitochondrial tumor necrosis factor-α receptor-associated protein 1 (TRAP1), a chaperone with a reported reactive oxygen species antagonist role. Our results describe the differences between the mitochondrial protein profiles of normal and OA chondrocytes, demonstrating that mitochondrial dysregulation occurs in cartilage cells during OA and highlighting redox imbalance as a key factor in OA pathogenesis. Mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including osteoarthritis (OA). Mitochondrial proteins are attractive targets for the study of metabolism of the chondrocyte, the unique cell type present in mature cartilage, and its role in tissue degradation. Using a proteomics approach based on two-dimensional DIGE and MALDI-TOF/TOF mass spectrometric identification of mitochondria- enriched protein fractions from human articular chondrocytes, we analyzed mitochondrial protein changes that are characteristic of OA chondrocytes. A total of 73 protein forms were unambiguously identified as significantly altered in OA; 23 of them have been previously described as mitochondrial. An extensive statistical and cluster analysis of the data revealed a mitochondrial protein profile characteristic for OA. This pattern includes alterations in energy production, maintenance of mitochondrial membrane integrity, and free radical detoxification. Real time PCR, Western blot, and immunohistofluorescence assays confirmed a significant decrease of the major mitochondrial antioxidant protein manganese-superoxide dismutase (SOD2) in the superficial layer of OA cartilage. As possible outputs for this antioxidant deficiency, we found an increase of intracellular reactive oxygen species generation in OA chondrocytes and also verified an OA-dependent increase in the mitochondrial tumor necrosis factor-α receptor-associated protein 1 (TRAP1), a chaperone with a reported reactive oxygen species antagonist role. Our results describe the differences between the mitochondrial protein profiles of normal and OA chondrocytes, demonstrating that mitochondrial dysregulation occurs in cartilage cells during OA and highlighting redox imbalance as a key factor in OA pathogenesis. The mitochondrion is one of the most complex and important organelles found in eukaryotic cells and carries out a wide variety of biochemical processes. Mitochondria are critical subcellular organelles responsible for energy production through the coupling of respiration to the generation of ATP. Mitochondria consist of four components: an outer membrane, an intermembrane space, an inner membrane, and a matrix. These components all function in concert to convert pyruvate and fatty acids to acetyl CoA, which is metabolized by the citric acid cycle to produce NADH. High energy electrons from NADH are then passed to oxygen by means of the respiratory chain in the inner membrane, producing ATP by a chemiosmotic process. Transcription and translation take place in mitochondria, which also actively import proteins and metabolites from the cytosol, influence programmed cell death, and respond to cellular signals such as oxidative stress (1Green D.R. Reed J.C. Mitochondria and apoptosis.Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). In addition to their central role in energy metabolism, mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including Parkinson disease, diabetes mellitus, Alzheimer disease, and OA 1The abbreviations used are: OA, osteoarthritis; 2-D, two-dimensional; 2-DE, two-dimensional gel electrophoresis; BVA, biological variation analysis; Cy, cyanine; DCF, dichlorofluorescein; DIA, differential in-gel analysis; MRC, mitochondrial respiratory chain; PMF, peptide mass fingerprinting; ROS, reactive oxygen species; SOD2, manganese-superoxide dismutase; TNFα, tumor necrosis factor-α; TRAP1, TNFα receptor-associated protein 1; N, normal; PCA, principal component analysis; HC, hierarchical clustering; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBGD, porphobilinogen deaminase; FACS, fluorescence-activated cell sorting; IMMT, inner membrane protein mitofilin; SOD, superoxide dismutase. 1The abbreviations used are: OA, osteoarthritis; 2-D, two-dimensional; 2-DE, two-dimensional gel electrophoresis; BVA, biological variation analysis; Cy, cyanine; DCF, dichlorofluorescein; DIA, differential in-gel analysis; MRC, mitochondrial respiratory chain; PMF, peptide mass fingerprinting; ROS, reactive oxygen species; SOD2, manganese-superoxide dismutase; TNFα, tumor necrosis factor-α; TRAP1, TNFα receptor-associated protein 1; N, normal; PCA, principal component analysis; HC, hierarchical clustering; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBGD, porphobilinogen deaminase; FACS, fluorescence-activated cell sorting; IMMT, inner membrane protein mitofilin; SOD, superoxide dismutase. (1Green D.R. Reed J.C. Mitochondria and apoptosis.Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 2Finkel T. Holbrook N.J. Oxidants, oxidative stress and the biology of ageing.Nature. 2000; 408: 239-247Crossref PubMed Scopus (7120) Google Scholar, 3Blanco F.J. Lopez-Armada M.J. Maneiro E. Mitochondrial dysfunction in osteoarthritis.Mitochondrion. 2004; 4: 715-728Crossref PubMed Scopus (132) Google Scholar). OA, the most common age-related cartilage and joint pathology (4Heinegard D. Bayliss M. Lorenzo P. Biochemistry and metabolism of normal and osteoarthritic cartilage.in: Brandt K.D. Doherty M. Lohmander L.S. Osteoarthritis. Oxford University Press, New York1998: 74-84Google Scholar), is a slowly progressive degenerative disease characterized by degradation of the matrix and cell death, which result in a gradual loss of articular cartilage integrity (5Pritzker K. Pathology of osteoarthritis.in: Brandt K.D. Doherty M. Lohmander L.S. Osteoarthritis. Oxford University Press, New York1998: 50-61Google Scholar, 6Kim H.A. Blanco F.J. Cell death and apoptosis in osteoarthritic cartilage.Curr. Drug Targets. 2007; 8: 333-345Crossref PubMed Scopus (171) Google Scholar). The only cell type present in mature cartilage is the chondrocyte, which is responsible for repairing cartilage tissue damaged by OA. Recently the role of mitochondrial dysfunction in OA has been the subject of renewed interest. Some studies have shown that mitochondrial dysfunction mediates several pathways implicated in cartilage degradation (7Blanco M. Mitochondria and role in osteoarthritis.in: M. and A Press, Scholar). These oxidative of and and matrix cartilage matrix and death mitochondrial respiratory chain in OA chondrocytes in and with normal chondrocytes that a in mitochondrial membrane and in ATP the as a the number of mitochondria is in OA chondrocytes as by a significant increase in mitochondrial mass and in E. Lopez-Armada M.J. P. Blanco F.J. Mitochondrial respiratory is altered in osteoarthritic human articular PubMed Scopus Google Scholar). mitochondrial as a factor in degenerative cartilage disease K. Mitochondrial oxidative is a of on matrix and 2000; PubMed Scopus Google Scholar, M. M. M. mitochondrial respiration and of articular PubMed Scopus Google Scholar, oxidative stress with with intracellular PubMed Scopus Google Scholar). that mitochondrial proteins an attractive for study of the metabolism of chondrocytes and the role in cartilage degradation. studies mitochondrial proteins in chondrocytes proteins the mitochondrial The of proteomics has the analysis of changes in many proteomics studies two-dimensional gel to and this one of the key in proteomics to of proteins to their and mass and the and of protein gel to and Using this proteomics we have described the of normal and OA human chondrocytes as as their differential protein profile Lopez-Armada M.J. Blanco F.J. of human normal articular a for the study of osteoarthritis and PubMed Scopus Google Scholar, Lopez-Armada M.J. Blanco F.J. analysis of human osteoarthritic chondrocytes protein changes in stress and 8: PubMed Scopus Google Scholar). the of changes in in the as with mass analysis of complex protein PubMed Scopus Google Scholar, K. P. M. protein in 2004; PubMed Scopus Google Scholar). such as variation and of gel and of DIGE M. gel a gel for changes in protein PubMed Scopus Google an to and the of changes in protein with statistical E. M. and of two-dimensional differential gel proteomics PubMed Scopus Google Scholar). In this are with and only and also that in the gel with an The of a of changes and variation and for 2004; PubMed Scopus Google Scholar, E. 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E. P. M. to Cell PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). of identified proteins were reported intracellular of proteins are based on of the proteins as associated to this have a mitochondrial Some of the identified proteins were characterized previously in an analysis on and OA Lopez-Armada M.J. Blanco F.J. analysis of human osteoarthritic chondrocytes protein changes in stress and 8: PubMed Scopus Google Scholar). the reported increase in OA chondrocytes of an protein type and decrease of a protein intracellular protein were confirmed by the present the analysis on the proteins previously characterized as mitochondrial to that are in OA chondrocytes used to biological and of the altered mitochondrial which are shown in of proteins are involved in metabolism and energy production in metabolism as components of These data the mitochondrial dysregulation of energy production that place in OA that described by from the of of and of E. Lopez-Armada M.J. P. Blanco F.J. Mitochondrial respiratory is altered in osteoarthritic human articular PubMed Scopus Google Scholar). Using proteomics approach we found a decrease in a of complex a which a role in this complex The of of complex and also Mitochondrial membrane is for mitochondrial The inner membrane protein has been reported to M.J. The mitochondrial inner membrane protein PubMed Scopus Google to mitochondrial In proteomics we found in OA, a These data an OA-dependent of that protein involved in shown in this study to altered in OA. is a that identified as a for P. T. to PubMed Scopus Google Scholar, Lorenzo P. E. a mitochondrial is in 2000; PubMed Scopus Google Scholar). a role for as a major of the mitochondrial inner membrane its in E. P. of the mitochondrial inner membrane and integrity, to and PubMed Scopus Google Scholar). An in mitochondria described for D. of during apoptosis in the and of and mitochondrial PubMed Scopus Google Scholar), reported to from the of M. T. D. from mitochondrial PubMed Scopus Google Scholar). the role of in mitochondrial has been T. J.C. T. cell and apoptosis by in PubMed Scopus Google Scholar, mitochondrial through with the PubMed Scopus Google Scholar). data a for altered mitochondrial function in OA chondrocytes in which dysregulation of from in and to apoptosis in OA chondrocytes. we on mitochondrial proteins role is to oxidative Mitochondrial dysregulation and oxidative stress have been reported to an role in OA and K. D. of osteoarthritis by progressive ATP in 2004; PubMed Scopus Google Scholar, P. The role of reactive oxygen species in and degradation of PubMed Scopus Google Scholar). cartilage changes during the have been highlighting a possible between aging, and cartilage degradation in OA Blanco T. K. The of oxidative stress in articular cartilage and 2007; Scopus Google Scholar). The of ROS, for cartilage is by and which by oxidative free of the in OA this and is on the of chondrocytes T. and reactive oxygen species in cartilage PubMed Scopus Google Scholar). of the of a major intracellular antioxidant SOD2, in mitochondria a key to OA In an study K. T. K. T. K. of oxidative stress in cartilage and of oxidative stress and of PubMed Google Scholar), in the of OA cartilage. These that the oxidative stress and dysfunction of chondrocytes, to the and of OA. only found to in OA cartilage E. K. M. D. superoxide dismutase and in PubMed Scopus Google and also in OA are in osteoarthritic to with cartilage and PubMed Scopus Google Scholar). the of mitochondrial OA chondrocytes a that of ROS, which intracellular metabolism, is an factor in OA D. P. oxygen species and superoxide role in joint 2007; PubMed Scopus Google Scholar). These data are also by of generation in OA chondrocytes. a based on of osteoarthritic a of in OA tissue of osteoarthritic altered and protein 2007; PubMed Scopus Google Scholar). the of time of this an in the antioxidant of OA is with results on cartilage tissue and data a of redox in the cartilage, and of the OA joint that to a decrease of antioxidant proteins such as is this time that an antioxidant is for of OA and of in OA cartilage to Drug Targets. 2007; 8: PubMed Scopus Google Scholar), results to the of and as antioxidant in OA. is a of the of that identified by its to the type 1 tumor necrosis factor D. of a protein with to that the type 1 tumor necrosis factor PubMed Scopus Google Scholar). is to and proteins are by such as is reported to in the mitochondria that tumor necrosis factor receptor-associated protein 1 is a mitochondrial protein which also Cell 2000; PubMed Scopus Google and to have from its P. The protein is a mitochondrial protein with 2000; PubMed Scopus Google Scholar). studies have a role for in cells from apoptosis by of T. M. K. T. K. of tumor necrosis factor receptor-associated protein 1 in apoptosis by 2004; PubMed Scopus Google Scholar). describe this protein production, the cells from oxidative apoptosis study in a normal human cell that tumor necrosis factor receptor-associated protein 1 production of reactive oxygen 2007; PubMed Scopus Google Scholar, protein reactive oxygen species generation and cells from 2007; PubMed Scopus Google Scholar, E. M. necrosis protein 1 cells from oxidative stress and 2007; PubMed Scopus Google Scholar). As of production and apoptosis are characteristic of OA H.A. Blanco F.J. Cell death and apoptosis in osteoarthritic cartilage.Curr. Drug Targets. 2007; 8: 333-345Crossref PubMed Scopus (171) Google Scholar). we differences of in OA and protein confirmed the increase of in OA chondrocytes, assays its in mitochondria from OA cartilage the reported antagonist role of TRAP1, we that the of in OA tissue a by mitochondria to out a oxidative stress the pathways that are altered in OA in the production of mitochondrial membrane and apoptosis have been the of mitochondrial dysregulation in cartilage cells during the of the The decrease of mitochondrial we found is a to maintenance of a normal intracellular redox in chondrocytes. studies on all protein are to the study of OA pathogenesis. The of studies to and targets that of for OA. are to for in and to for to the Pathology and to and from the of the A for cartilage with