RESUMO
Osteoarthritis (OA) is the most common musculoskeletal disorder causing a great disability and a reduction in the quality of life. In OA, articular chondrocytes (AC) and synovial fibroblasts (SF) release innate-derived immune mediators that initiate and perpetuate inflammation, inducing cartilage extracellular matrix (ECM) degradation. Given the lack of therapies for the treatment of OA, in this study, we explore biomarkers that enable the development of new therapeutical approaches. We analyze the set of secreted proteins in AC and SF co-cultures by stable isotope labeling with amino acids (SILAC). We describe, for the first time, 115 proteins detected in SF-AC co-cultures stimulated by fibronectin fragments (Fn-fs). We also study the role of the vasoactive intestinal peptide (VIP) in this secretome, providing new proteins involved in the main events of OA, confirmed by ELISA and multiplex analyses. VIP decreases proteins involved in the inflammatory process (CHI3L1, PTX3), complement activation (C1r, C3), and cartilage ECM degradation (DCN, CTSB and MMP2), key events in the initiation and progression of OA. Our results support the anti-inflammatory and anti-catabolic properties of VIP in rheumatic diseases and provide potential new targets for OA treatment.
Assuntos
Condrócitos/metabolismo , Fibroblastos/metabolismo , Osteoartrite/metabolismo , Proteoma , Proteômica , Membrana Sinovial/citologia , Peptídeo Intestinal Vasoativo/metabolismo , Biomarcadores , Condrócitos/efeitos dos fármacos , Técnicas de Cocultura , Citocinas/metabolismo , Suscetibilidade a Doenças , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Osteoartrite/etiologia , Osteoartrite/patologia , Proteômica/métodos , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
In osteoarthritis (OA), impairment of cartilage regeneration can be related to a defective chondrogenic differentiation of mesenchymal stromal cells (MSCs). Therefore, understanding the proteomic- and metabolomic-associated molecular events during the chondrogenesis of MSCs could provide alternative targets for therapeutic intervention. Here, a SILAC-based proteomic analysis identified 43 proteins related with metabolic pathways whose abundance was significantly altered during the chondrogenesis of OA human bone marrow MSCs (hBMSCs). Then, the level and distribution of metabolites was analyzed in these cells and healthy controls by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), leading to the recognition of characteristic metabolomic profiles at the early stages of differentiation. Finally, integrative pathway analysis showed that UDP-glucuronic acid synthesis and amino sugar metabolism were downregulated in OA hBMSCs during chondrogenesis compared with healthy cells. Alterations in these metabolic pathways may disturb the production of hyaluronic acid (HA) and other relevant cartilage extracellular matrix (ECM) components. This work provides a novel integrative insight into the molecular alterations of osteoarthritic MSCs and potential therapeutic targets for OA drug development through the enhancement of chondrogenesis.
Assuntos
Redes e Vias Metabólicas , Terapia de Alvo Molecular , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Estudos de Casos e Controles , Condrogênese , Humanos , Células-Tronco Mesenquimais/metabolismo , Metaboloma , Via de Pentose Fosfato , Uridina Difosfato Ácido Glucurônico/biossínteseRESUMO
Osteoarthritis (OA) is a pathology characterized by the loss of articular cartilage. In this study, we performed a peptidomic strategy to identify endogenous peptides (neopeptides) that are released from human osteoarthritic tissue, which may serve as disease markers. With this aim, secretomes of osteoarthritic and healthy articular cartilages obtained from knee and hip were analyzed by shotgun peptidomics. This discovery step led to the identification of 1175 different peptides, corresponding to 101 proteins, as products of the physiological or pathological turnover of cartilage extracellular matrix. Then, a targeted multiple reaction monitoring-mass spectrometry method was developed to quantify the panel of best marker candidates on a larger set of samples (n = 62). Statistical analyses were performed to evaluate the significance of the observed differences and the ability of the neopeptides to classify the tissue. Eight of them were differentially abundant in the media from wounded zones of OA cartilage compared with the healthy tissue (p < 0.05). Three neopeptides belonging to Clusterin and one from Cartilage Oligomeric Matrix Protein showed a disease-dependent decrease specifically in hip OA, whereas two from Prolargin (PRELP) and one from Cartilage Intermediate Layer Protein 1 were significantly increased in samples from knee OA. The release of one peptide from PRELP showed the best metrics for tissue classification (AUC = 0.834). The present study reveals specific neopeptides that are differentially released from knee or hip human osteoarthritic cartilage compared with healthy tissue. This evidences the intervention of characteristic pathogenic pathways in OA and provides a novel panel of peptidic candidates for biomarker development.
Assuntos
Biomarcadores/metabolismo , Cartilagem Articular/citologia , Osteoartrite do Quadril/metabolismo , Osteoartrite do Joelho/metabolismo , Peptídeos/metabolismo , Proteômica/métodos , Idoso , Idoso de 80 Anos ou mais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Estudos de Casos e Controles , Células Cultivadas , Cromatografia Líquida , Meios de Cultivo Condicionados/química , Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , Especificidade de Órgãos , Osteoartrite do Quadril/patologia , Osteoartrite do Joelho/patologia , Espectrometria de Massas em TandemRESUMO
Endometrial cancer (EC) is the most frequent gynecological cancer. Tumor dissemination affecting â¼20% of EC patients is characterized at the primary carcinoma by epithelial-to-mesenchymal transition (EMT) associated with myometrial infiltration. At distant sites, the interaction of circulating tumor cells (CTCs) with the microenvironment is crucial for metastatic colonization, with a participation of the extracellular vesicles (EVs). We comprehensively approached these primary and secondary sites to study the impact of tumor EVs on the metastatic efficiency of CTCs in EC. Tumor EVs in circulation reproduce the epithelial phenotype predominant in the primary carcinoma, whereas CTCs are characterized by an EMT phenotype. We modeled this EMT-related clinical scenario in the Hec1A endometrial cell line and characterized the epithelial-like EVs in circulation by SILAC proteome analysis. The identification of proteins involved in cell-cell and cell-matrix interaction and binding, together with in vitro evidence of an improved adhesion of CTC to a functionalized endothelium, suggests a contribution of the epithelial-like EVs in the homing of CTCs at metastatic sites. Accordingly, adhesion protein LGALS3BP was found to be significantly enriched in circulating EVs from a cohort of EC patients with a high risk of recurrence by targeted proteomics (multiple reaction monitoring), highlighting its potential in liquid biopsy in EC.
Assuntos
Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Neoplasias do Endométrio/genética , Proteoma/genética , Proteômica , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/sangue , Biomarcadores Tumorais/sangue , Neoplasias do Endométrio/sangue , Neoplasias do Endométrio/patologia , Transição Epitelial-Mesenquimal/genética , Vesículas Extracelulares/genética , Vesículas Extracelulares/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Marcação por Isótopo , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Microambiente Tumoral/genéticaRESUMO
Carcinoma, the most common type of cancer, arises from epithelial cells. The transition from adenoma to carcinoma is associated with the loss of E-cadherin and, in consequence, the disruption of cell-cell contacts. E-cadherin is a tumor suppressor, and it is down-regulated during epithelial-to-mesenchymal transition (EMT); indeed, its loss is a predictor of poor prognosis. Hakai is an E3 ubiquitin-ligase protein that mediates E-cadherin ubiquitination, endocytosis and finally degradation, leading the alterations of cell-cell contacts. Although E-cadherin is the most established substrate for Hakai activity, other regulated molecular targets for Hakai may be involved in cancer cell plasticity during tumor progression. In this work we employed an iTRAQ approach to explore novel molecular pathways involved in Hakai-driven EMT during tumor progression. Our results show that Hakai may have an important influence on cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. Moreover, a profound decreased expression in several proteasome subunits during Hakai-driven EMT was highlighted. Since proteasome inhibitors are becoming increasingly used in cancer treatment, our findings suggest that the E3 ubiquitin-ligase, such as Hakai, may be a better target than proteasome for using novel specific inhibitors in tumor subtypes that follow EMT.
Assuntos
Citoesqueleto/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteômica/métodos , Ubiquitina-Proteína Ligases/análise , Animais , Antineoplásicos/química , Caderinas/metabolismo , Adesão Celular , Cães , Transição Epitelial-Mesenquimal , Humanos , Células Madin Darby de Rim Canino , Terapia de Alvo Molecular/métodos , Complexo de Endopeptidases do Proteassoma/químicaRESUMO
The methods currently available for the diagnosis and monitoring of osteoarthritis (OA) are very limited and lack sensitivity. Being the most prevalent rheumatic disease, one of the most disabling pathologies worldwide and currently untreatable, there is a considerable interest pointed in the verification of specific biological markers for improving its diagnosis and disease progression studies. Considering the remarkable development of targeted proteomics methodologies in the frame of the Human Proteome Project, the aim of this work was to develop and apply a MRM-based method for the multiplexed analysis of a panel of 6 biomarker candidates for OA encoded by the Chromosome 16, and another 8 proteins identified in previous shotgun studies as related with this pathology, in specimens derived from the human joint and serum. The method, targeting 35 different peptides, was applied to samples from human articular chondrocytes, healthy and osteoarthritic cartilage, synovial fluid and serum. Subsequently, a verification analysis of the biomarker value of these proteins was performed by single point measurements on a set of 116 serum samples, leading to the identification of increased amounts of Haptoglobin and von Willebrand Factor in OA patients. Altogether, the present work provides a tool for the multiplexed monitoring of 14 biomarker candidates for OA, and verifies for the first time the increased amount of two of these circulating markers in patients diagnosed with this disease. SIGNIFICANCE: We have developed an MRM method for the identification and relative quantification of a panel of 14 protein biomarker candidates for osteoarthritis. This method has been applied to analyze human articular chondrocytes, articular cartilage, synovial fluid, and finally a collection of 116 serum samples from healthy controls and patients suffering different degrees of osteoarthritis, in order to verify the biomarker usefulness of the candidates. HPT and VWF were validated as increased in OA patients.
Assuntos
Espectrometria de Massas/métodos , Osteoartrite/diagnóstico , Biomarcadores/análise , Biomarcadores/sangue , Cartilagem Articular/química , Cartilagem Articular/patologia , Estudos de Casos e Controles , Linhagem Celular , Condrócitos/química , Cromossomos Humanos Par 16 , Progressão da Doença , Humanos , Peptídeos/análise , Manejo de Espécimes , Líquido Sinovial/químicaRESUMO
Mesenchymal stromal cells (MSCs) secrete a large variety of proteins and factors, which shape the secretome. These proteins participate in multiple cellular functions, including the promotion of regenerative processes in the damaged tissue. Secretomes derived from either undifferentiated MSCs or these cells undergoing osteogenic, chondrogenic, or adipogenic differentiation have been characterized using different liquid chromatography tandem mass spectrometry (LC-MS/MS)-based quantitative proteomic approaches. In this chapter, we describe the use of the Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) strategy for the identification and relative quantification of the mesenchymal stromal cell secretome, specifically during chondrogenesis.
Assuntos
Marcação por Isótopo/métodos , Células-Tronco Mesenquimais/citologia , Proteoma/metabolismo , Proteômica/métodos , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células Cultivadas , Condrogênese , Cromatografia Líquida , Meios de Cultivo Condicionados/isolamento & purificação , Humanos , Células-Tronco Mesenquimais/metabolismo , Espectrometria de Massas em TandemRESUMO
PURPOSE: Osteoarthritis (OA) is a degenerative joint pathology characterized by articular cartilage degradation that lacks from efficient therapy. Since previous epidemiological data show a high controversy regarding the role of smoking in OA, we aimed to evaluate the effects of nicotine (the most physiologically active compound of tobacco) on the joint. EXPERIMENTAL DESIGN: Secretome analyses, based on metabolic labeling followed by LC-MALDI-TOF/TOF analysis, were carried out using an in vitro model of articular inflammation (primary human articular chondrocytes treated with interleukin-1ß), and also on osteoarthritic cells. ELISA and Western blot assays were performed to verify some of the results. RESULTS: Nineteen proteins were altered by nicotine in the model of articular inflammation, including several cytokines and proteases. We confirmed the increased secretion by nicotine of matrix metalloproteinase 1 and two proposed markers of OA, fibronectin, and chitinase 3-like protein 1. Finally, four components of the extracellular matrix of cartilage were decreased by nicotine in OA chondrocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Our data contribute to a better understanding of the molecular mechanisms that are modulated by nicotine in cartilage cells, suggesting a negative effect of this drug on the joint.
Assuntos
Proteína 1 Semelhante à Quitinase-3/metabolismo , Condrócitos/efeitos dos fármacos , Fibronectinas/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Metabolismo/efeitos dos fármacos , Nicotina/farmacologia , Osteoartrite/metabolismo , Idoso , Idoso de 80 Anos ou mais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Estudos de Casos e Controles , Proteína 1 Semelhante à Quitinase-3/genética , Condrócitos/citologia , Condrócitos/metabolismo , Matriz Extracelular/química , Matriz Extracelular/efeitos dos fármacos , Feminino , Fibronectinas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/farmacologia , Masculino , Metaloproteinase 1 da Matriz/genética , Metabolismo/genética , Modelos Biológicos , Osteoartrite/genética , Osteoartrite/patologia , Cultura Primária de Células , Fumar/fisiopatologiaRESUMO
The Spanish Chromosome 16 consortium is integrated in the global initiative Human Proteome Project, which aims to develop an entire map of the proteins encoded following a gene-centric strategy (C-HPP) in order to make progress in the understanding of human biology in health and disease (B/D-HPP). Chromosome 16 contains many genes encoding proteins involved in the development of a broad range of diseases, which have a significant impact on the health care system. The Spanish HPP consortium has developed a B/D platform with five programs focused on selected medical areas: cancer, obesity, cardiovascular, infectious and rheumatic diseases. Each of these areas has a clinical leader associated to a proteomic investigator with the responsibility to get a comprehensive understanding of the proteins encoded by Chromosome 16 genes. Proteomics strategies have enabled great advances in the area of rheumatic diseases, particularly in osteoarthritis, with studies performed on joint cells, tissues and fluids. BIOLOGICAL SIGNIFICANCE: In this manuscript we describe how the Spanish HPP-16 consortium has developed a B/D platform with five programs focused on selected medical areas: cancer, obesity, cardiovascular, infectious and rheumatic diseases. Each of these areas has a clinical leader associated to a proteomic investigator with the responsibility to get a comprehensive understanding of the proteins encoded by Chromosome 16 genes. We show how the Proteomic strategy has enabled great advances in the area of rheumatic diseases, particularly in osteoarthritis, with studies performed on joint cells, tissues and fluids. This article is part of a Special Issue entitled: HUPO 2014.
Assuntos
Cromossomos Humanos Par 16/genética , Osteoartrite/genética , Osteoartrite/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , Feminino , Humanos , Masculino , EspanhaRESUMO
Osteoarthritis (OA) is the most common rheumatic pathology and is characterized primarily by articular cartilage degradation. Despite its high prevalence, there is no effective therapy to slow disease progression or regenerate the damaged tissue. Therefore, new diagnostic and monitoring tests for OA are urgently needed, which would also promote the development of alternative therapeutic strategies. In the present study, we have performed an iTRAQ-based quantitative proteomic analysis of secretomes from healthy human articular cartilage explants, comparing their protein profile to those from unwounded (early disease) and wounded (advanced disease) zones of osteoarthritic tissue. This strategy allowed us to identify a panel of 76 proteins that are distinctively released by the diseased tissue. Clustering analysis allowed the classification of proteins according to their different profile of release from cartilage. Among these proteins, the altered release of osteoprotegerin (decreased in OA) and periostin (increased in OA), both involved in bone remodelling processes, was verified in further analyses. Moreover, periostin was also increased in the synovial fluid of OA patients. Altogether, the present work provides a novel insight into the mechanisms of human cartilage degradation and a number of new cartilage-characteristic proteins with possible biomarker value for early diagnosis and prognosis of OA.
Assuntos
Cartilagem Articular/metabolismo , Osteoartrite/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Western Blotting , Cartilagem Articular/patologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Cromatografia Líquida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Osteoartrite/diagnóstico , Osteoartrite/genética , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Proteoma/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Líquido Sinovial/metabolismo , Espectrometria de Massas em Tandem , Técnicas de Cultura de TecidosRESUMO
Human mesenchymal stem cells (hMSCs) can be triggered to differentiate toward chondrocytes and thus harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we followed a double-stable isotope labeling by amino acids in cell culture (SILAC) strategy to evaluate the quantitative modulation of the secretome of stem cells isolated from bone marrow (hBMSCs) during the first steps of their chondrogenic differentiation. Analysis by LC-ESI-MS/MS led to the identification of 221 proteins with a reported extracellular localization. Most of them were characteristic of cartilage extracellular matrix, and 34 showed statistically significant quantitative alterations during chondrogenesis. These include, among others, cartilage markers such as Proteoglycan 4 or COMP, anticatabolic markers (TIMP1), reported markers of cartilage development (Versican), and a suggested marker of chondrogenesis, CRAC1. Altogether, our work demonstrates the usefulness of secretome analysis for understanding the mechanisms responsible for cartilage matrix formation, and it reports a panel of extracellular markers potentially useful for the evaluation of tissue development in cell therapy- or tissue engineering-based approaches for cartilage repair.
Assuntos
Diferenciação Celular , Condrócitos/citologia , Células-Tronco Mesenquimais/metabolismo , Western Blotting , Células Cultivadas , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Humanos , Células-Tronco Mesenquimais/citologia , Proteínas/química , Proteínas/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
INTRODUCTION: Chondroitin sulfate (CS) is a symptomatic slow-acting drug for osteoarthritis (OA) widely used in the clinic. The aim of this work is to find proteins whose secretion from cartilage cells under proinflammatory stimuli (IL-1ß) is regulated by CS, employing a novel quantitative proteomic approach. METHODS: Human articular chondrocytes released from three normal cartilages were grown in SILAC medium. When complete incorporation of the heavy isotope was achieved, chondrocytes were stimulated with IL-1ß 5 ng/ml with or without CS pretreatment (200 µg/ml). Forty-eight hours later, chondrocyte secretomes were analyzed by nano-scale liquid chromatography-mass spectrometry. Real-time PCR, western blot and immunohistochemistry analyses were employed to confirm some of the results. RESULTS: We could identify 75 different proteins in the secretome of human articular chondrocytes. Eighteen of these were modulated by CS with statistical significance (six increased and 12 decreased). In normal chondrocytes stimulated with IL-1ß, CS reduces inflammation directly by decreasing the presence of several complement components (CFAB, C1S, CO3, and C1R) and also indirectly by increasing proteins such as TNFα-induced protein (TSG6). TSG6 overexpression correlates with a decrease in pro-matrix metalloproteinase activation (observed in MMP1 and MMP3 levels). Finally, we observed a strong CS-dependent increase of an angiogenesis inhibitor, thrombospondin-1. CONCLUSION: We have generated a quantitative profile of chondrocyte extracellular protein changes driven by CS in the presence of IL-1ß. We have also provided novel evidences of its anti-angiogenic, anti-inflammatory, and anti-catabolic properties. Demonstration of the anti-angiogenic action of CS might provide a novel therapeutic approach for OA targeting.
Assuntos
Anabolizantes/metabolismo , Inibidores da Angiogênese/metabolismo , Anti-Inflamatórios/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Sulfatos de Condroitina/farmacologia , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Complemento C1r/metabolismo , Complemento C1s/metabolismo , Humanos , Interleucina-1beta/farmacologia , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Trombospondina 1/metabolismoRESUMO
Human mesenchymal stem cells (hMSCs), residing in bone marrow as well as in the synovial lining of joints, can be triggered to differentiate toward chondrocytes. Thus, hMSCs harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we applied for the first time the stable isotope labeling by amino acids in cell culture (SILAC) technique for the quantitative analysis of protein modulation during the chondrogenic differentiation process of hMSCs. First, we have standardized the metabolic labeling procedure on MSCs isolated from bone marrow (hBMSCs), and we have assessed the quality of chondrogenesis taking place in these conditions. Then, chondrogenic differentiation was induced on these labeled cells, and a quantitative proteomics approach has been followed to evaluate protein changes between two differentiation days. With this strategy, we could identify 622 different proteins by LC-MALDI-TOF/TOF analysis and find 65 proteins whose abundance was significantly modulated between day 2 and day 14 of chondrogenesis. Immunohistochemistry analyses were performed to verify the changes on a panel of six proteins that play different biological roles in the cell: fibronectin, gelsolin, vimentin, alpha-ATPase, mitochondrial superoxide dismutase, and cyclophilin A. All of these proteins were increased at day 14 compared to day 2 of chondrogenic induction, thus being markers of the enhanced extracellular matrix synthesis, cell adhesion, metabolism, and response to stress processes that take place in the early steps of chondrogenesis. Our strategy has allowed an additional insight into both specific protein function and the mechanisms of chondrogenesis and has provided a panel of protein markers of this differentiation process in hBMSCs.
Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular , Condrócitos/citologia , Células-Tronco Mesenquimais/metabolismo , Sequência de Bases , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Células Cultivadas , Cromatografia Líquida , Primers do DNA , Bases de Dados de Proteínas , Humanos , Células-Tronco Mesenquimais/citologia , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Umbilical cord stroma mesenchymal stem cells were differentiated toward chondrocyte-like cells using a new in vitro model that consists of the random formation of spheroids in a medium supplemented with fetal bovine serum on a nonadherent surface. The medium was changed after 2 days to one specific for the induction of chondrocyte differentiation. We assessed this model using reverse transcriptase-polymerase chain reaction, flow cytometry, immunohistochemistry, and secretome analyses. The purpose of this study was to determine which proteins were differentially expressed during chondrogenesis. Differential gel electrophoresis analysis was performed, followed by matrix-assisted laser desorption/ionization mass spectrometry protein identification. A total of 97 spots were modulated during the chondrogenesis process, 54 of these spots were identified as 39 different proteins and 15 were isoforms. Of the 39 different proteins identified 15 were down-regulated, 21 were up-regulated, and 3 were up- and down-regulated during the chondrogenesis process. Using Pathway Studio 7.0 software, our results showed that the major cell functions modulated during chondrogenesis were cellular differentiation, proliferation, and migration. Five proteins involved in cartilage extracellular matrix metabolism found during the differential gel electrophoresis study were confirmed using Western blot. The results indicate that our in vitro chondrogenesis model is an efficient and rapid technique for obtaining cells similar to chondrocytes that express proteins characteristic of the cartilage extracellular matrix. These chondrocyte-like cells could prove useful for future cell therapy treatment of cartilage pathologies.
Assuntos
Diferenciação Celular , Condrogênese/fisiologia , Células-Tronco Mesenquimais/metabolismo , Modelos Biológicos , Proteoma/análise , Células Estromais/metabolismo , Cordão Umbilical/metabolismo , Adulto , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Condrócitos , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Células-Tronco Mesenquimais/citologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Estromais/citologia , Cordão Umbilical/citologiaRESUMO
BACKGROUND: Hsp90ß is a member of the Hsp90 family of protein chaperones. This family plays essential roles in the folding, maturation and activity of many proteins that are involved in signal transduction and transcriptional regulation. The role of this protein in chondrocytes is not well understood, although its increase in osteoarthritic cells has been reported. The present study aimed to explore the role of Hsp90ß in key aspects of OA pathogenesis. METHODS: Human OA chondrocytes were isolated from cartilage obtained from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with proinflammatory cytokines (IL-1ß or TNF-α) and nitric oxide donors (NOC-12 or SNP). For Hsp90ß inhibition, two different chemical inhibitors (Geldanamycin and Novobiocin) were employed, or siRNA transfection procedures were carried out. Gene expression was determined by real-time PCR, apoptosis was quantified by flow cytometry and ELISA, and nitric oxide (NO) production was evaluated by the Griess method. Indirect immunofluorescence assays were performed to evaluate the presence of Hsp90ß in stimulated cells. RESULTS: Hsp90ß was found to be increased by proinflammatory cytokines. Inhibition of Hsp90ß by the chemicals Geldanamycin (GA) and Novobiocin (NB) caused a dose-dependent decrease of the NO production induced by IL-1ß in chondrocytes, up to basal levels. Immunofluorescence analyses demonstrate that the NO donors NOC-12 and SNP also increased Hsp90ß. Chemical inhibition or specific gene silencing of this chaperone reduced the DNA condensation and fragmentation, typical of death by apoptosis, that is induced by NO donors in chondrocytes. CONCLUSIONS: The present results show how Hsp90ß modulates NO production and NO-mediated cellular death in human OA chondrocytes.
Assuntos
Apoptose/efeitos dos fármacos , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Óxido Nítrico/metabolismo , Idoso , Benzoquinonas/farmacologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Citocinas/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP90/genética , Humanos , Lactamas Macrocíclicas/farmacologia , Masculino , Pessoa de Meia-Idade , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Compostos Nitrosos/farmacologia , Novobiocina/farmacologia , Osteoartrite do Joelho/tratamento farmacológico , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , RNA Interferente Pequeno/farmacologia , TransfecçãoRESUMO
Osteoarthritis (OA) is a degenerative disease characterized by the degradation of articular cartilage. This tissue is avascular, and it is characterized by the low oxygen tension and poor nutrient availability for its cells, the chondrocytes. Hypoxia conditions have been reported to stimulate chondrogenesis and synthesis of extracellular matrix components. Therefore, we aimed to analyze the effect of hypoxia on normal and osteoarthritic cartilage cell cultures by a proteomic approach based in Two-dimensional gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry protein identification. Twenty-eight proteins were found to be modulated by hypoxia in normal chondrocytes and 11 in OA cells when compared to their normoxia controls. In both cases, a hypoxia-dependent decrease in metabolism-related proteins was detected. We also identified 42 protein forms that were altered in OA chondrocytes under hypoxia when compared to normal cells. The upregulation of cyclophylin A (PPIA) and Tumor necrosis factor receptor associated protein 1 (TRAP1) was confirmed both in cultured chondrocytes and in cartilage tissue. Our work shows how hypoxia conditions induce diverse modifications in the proteomic profile of normal and OA human articular chondrocytes, which probably renders a different capacity of OA and normal cells to react under a hypoxic environment.
Assuntos
Condrócitos/metabolismo , Osteoartrite/metabolismo , Proteoma , Proteômica/métodos , Adulto , Sequência de Aminoácidos , Cartilagem Articular , Estudos de Casos e Controles , Hipóxia Celular/fisiologia , Células Cultivadas , Ciclofilina A/química , Ciclofilina A/metabolismo , Regulação para Baixo , Eletroforese em Gel Bidimensional , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Dados de Sequência Molecular , Osteoartrite/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteoma/química , Proteoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estatísticas não Paramétricas , Tripsina/metabolismo , Regulação para CimaRESUMO
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-alpha 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.