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Human Articular Chondrocytes (human + articular_chondrocyte)
Kinds of Human Articular Chondrocytes Selected AbstractsHuman articular chondrocytes suppress in vitro proliferation of anti-CD3 activated peripheral blood mononuclear cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2006Chiara Bocelli-Tyndall Objective: To investigate whether mature human articular chondrocytes (AC) exhibit an antiproliferative effect on activated peripheral blood mononuclear cells (PBMC) and to compare this effect with other cells of mesenchymal origin. Methods: AC from healthy cadaveric cartilage were grown for different passages, in the absence (control) or presence of factors enhancing cell de-differentiation (transforming growth factor (TGF),1, fibroblast growth factor (FGF)-2, and platelet derived growth factor (PDGF)bb-TFP medium). Cell ability to suppress PBMC proliferation driven by anti-CD3 antibody was measured by tritiated thymidine uptake following incubation for 48 h at different PBMC:AC ratios and expressed as percent of residual proliferation (RP). AC antiproliferative effect was compared to that of control dermal fibroblasts (DF) and bone marrow stromal cells (BMSC). Results: AC exhibited a cell number-dependent antiproliferative effect. The strongest effect (up to 2% RP) was measured using the least expanded AC cultures. The use of TFP medium for AC expansion resulted in a significantly lower antiproliferative effect, in the range of that induced by BMSC (up to 18% RP). Also DF induced a marked antiproliferative effect (up to 11% RP). Conclusion: We report for the first time that human AC have a marked antiproliferative effect on anti-CD3 stimulated PBMC, which is reduced upon culture in medium-inducing extensive cell de-differentiation. These results reflect the immunosuppressive properties observed for other different mesenchymal cell types and raise the question of a potential common physiological role in local tissue protection. J. Cell. Physiol. 209: 732,734, 2006. © 2006 Wiley-Liss, Inc. [source] Human articular chondrocytes secrete parathyroid hormone,related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesisARTHRITIS & RHEUMATISM, Issue 9 2010J. Fischer Objective The use of bone marrow,derived mesenchymal stem cells (MSCs) has shown promise in cell-based cartilage regeneration. A yet-unsolved problem, however, is the unwanted up-regulation of markers of hypertrophy, such as alkaline phosphatase (AP) and type X collagen, during in vitro chondrogenesis and the formation of unstable calcifying cartilage at heterotopic sites. In contrast, articular chondrocytes produce stable, nonmineralizing cartilage. The aim of this study was to address whether coculture of MSCs with human articular chondrocytes (HACs) can suppress the undesired hypertrophy in differentiating MSCs. Methods MSCs were differentiated in chondrogenic medium that had or had not been conditioned by parallel culture with HAC pellets, or MSCs were mixed in the same pellet with the HACs (1:1 or 1:2 ratio) and cultured for 6 weeks. Following in vitro differentiation, the pellets were transplanted into SCID mice. Results The gene expression ratio of COL10A1 to COL2A1 and of Indian hedgehog (IHH) to COL2A1 was significantly reduced by differentiation in HAC-conditioned medium, and less type X collagen protein was deposited relative to type II collagen. AP activity was significantly lower (P < 0.05) in the cells that had been differentiated in conditioned medium, and transplants showed significantly reduced calcification in vivo. In mixed HAC/MSC pellets, suppression of AP was dose-dependent, and in vivo calcification was fully inhibited. Chondrocytes secreted parathyroid hormone,related protein (PTHrP) throughout the culture period, whereas PTHrP was down-regulated in favor of IHH up-regulation in control MSCs after 2,3 weeks of chondrogenesis. The main inhibitory effects seen with HAC-conditioned medium were reproducible by PTHrP supplementation of unconditioned medium. Conclusion HAC-derived soluble factors and direct coculture are potent means of improving chondrogenesis and suppressing the hypertrophic development of MSCs. PTHrP is an important candidate soluble factor involved in this effect. [source] Resistin induces expression of proinflammatory cytokines and chemokines in human articular chondrocytes via transcription and messenger RNA stabilizationARTHRITIS & RHEUMATISM, Issue 7 2010Zhiqi Zhang Objective To elucidate the effects of resistin on human articular chondrocytes and to generate a picture of their regulation at the transcriptional and posttranscriptional levels. Methods Human articular chondrocytes were cultured with resistin. Changes in gene expression were analyzed at various doses and times. Cells were also treated with the transcription inhibitor actinomycin D after resistin treatment or with the NF-,B inhibitor IKK-NBD before resistin treatment. Gene expression was tested by quantitative real-time polymerase chain reaction. Computational analysis for transcription factor binding motifs was performed on the promoter regions of differentially expressed genes. TC-28 chondrocytes were transfected with CCL3 and CCL4 promoter constructs, pNF-,B reporter, and NF-,B and CCAAT/enhancer binding protein , (C/EBP,) expression vectors with or without resistin. Results Resistin-treated human articular chondrocytes increased the expression of cytokines and chemokines. Levels of messenger RNA (mRNA) for matrix metalloproteinase 1 (MMP-1), MMP-13, and ADAMTS-4 also increased, while type II collagen ,1 (COL2A1) and aggrecan were down-regulated. The cytokine and chemokine genes could be categorized into 3 groups according to the pattern of mRNA expression over a 24-hour time course. One pattern suggested rapid regulation by mRNA stability. The second and third patterns were consistent with transcriptional regulation. Computational analysis suggested the transcription factors NF-,B and C/EBP, were involved in the resistin-induced up-regulation. This prediction was confirmed by the cotransfection of NF-,B and C/EBP, and the IKK-NBD inhibition. Conclusion Resistin has diverse effects on gene expression in human chondrocytes, affecting chemokines, cytokines, and matrix genes. Messenger RNA stabilization and transcriptional up-regulation are involved in resistin-induced gene expression in human chondrocytes. [source] The melanocortin system in articular chondrocytes: Melanocortin receptors, pro-opiomelanocortin, precursor proteases, and a regulatory effect of ,-melanocyte,stimulating hormone on proinflammatory cytokines and extracellular matrix componentsARTHRITIS & RHEUMATISM, Issue 10 2009Susanne Grässel Objective The pro-opiomelanocortin (POMC),derived neuropeptide ,-melanocyte,stimulating hormone (,-MSH) mediates its effects via melanocortin (MC) receptors. This study was carried out to investigate the expression patterns of the MC system and the effects of ,-MSH in human articular chondrocytes. Methods Articular chondrocytes established from human osteoarthritic joint cartilage were analyzed by reverse transcription,polymerase chain reaction (RT-PCR) and Western blotting for the expression of MC receptors, POMC, and prohormone convertases (PCs). MC-1 receptor (MC-1R) expression in articular cartilage was further studied by immunohistochemistry. Ca2+ and cAMP assays were used to monitor ,-MSH signaling, while studies of ,-MSH function were performed in cultures with chondrocyte micromass pellets stimulated with ,-MSH. Expression of cytokines and extracellular matrix (ECM) components was determined by real-time RT-PCR, Western immunoblotting, and enzyme-linked immunosorbent assays. Results MC-1R expression was detected in articular chondrocytes in vitro and in articular cartilage in situ. In addition, expression of transcripts for MC-2R, MC-5R, POMC, and PCs was detected in articular chondrocytes. Stimulation with ,-MSH increased the levels of intracellular cAMP, but not Ca2+, in chondrocytes. Both messenger RNA and protein expression of various proinflammatory cytokines, collagens, matrix metalloproteinases (MMPs), and SOX9 was modulated by ,-MSH. Conclusion Human articular chondrocytes are target cells for ,-MSH. The effects of ,-MSH on expression of cytokines and MMPs suggest that this neuropeptide plays a role in inflammatory and degenerative processes in cartilage. It is conceivable that inflammatory reactions can be mitigated by the induction of endogenous MCs or administration of ,-MSH to the affected joints. The induction pattern of regulatory and structural ECM components such as collagens as well as SOX9 and anabolic and catabolic cytokines points to a function of ,-MSH as a trophic factor in skeletal development during endochondral ossification rather than as a factor in homeostasis of permanent cartilage. [source] Nitrite-mediated protection against hypochlorous acid,induced chondrocyte toxicity: A novel cytoprotective role of nitric oxide in the inflamed joint?ARTHRITIS & RHEUMATISM, Issue 11 2003Matthew Whiteman Objective To examine the potential consequences of overproduction of nitric oxide (NO) and nitrite (NO) in the inflamed rheumatoid joint. Methods Human articular chondrocytes in culture were exposed to HOCl (hypochlorous acid, a physiologic oxidant formed in increased amounts at sites of chronic inflammation), and assays of cell viability, intracellular ATP and glutathione (GSH), and lactate dehydrogenase (LDH) were performed. HOCl-induced lipid peroxidation and activation of the MAP kinases ERK-1/2, JNK-1/2, and p38 were also measured. The modulatory effects of NO-derived nitrite (NO) and nitrate (NO) on HOCl-mediated chondrocyte toxicity were investigated. Results Exposure of human articular chondrocytes to HOCl resulted in a concentration- and time-dependent loss of viability, decrease in ATP and GSH levels, LDH leakage, and cell death. HOCl induced significant lipid peroxidation as well as activation of the MAP kinases ERK-1/2 and p38 but not JNK-1/2. However, the presence of NO but not NO substantially decreased HOCl-dependent cellular toxicity even when NO was added at low (,M) concentrations. In sharp contrast, NO (1 mM) did not inhibit superoxide-, hydroxyl radical,, H2O2 -, or peroxynitrite-mediated cytotoxicity. Furthermore, culture media from cells treated with interleukin-1, (to generate NO and NO) offered significantly more protection against HOCl-mediated cytotoxicity than culture media from untreated cells. Conclusion These data suggest that NO accumulation at chronically inflamed sites where both HOCl and NO are overproduced may be cytoprotective against damage induced by HOCl. Accumulation of NO could represent a novel cytoprotective role of NO in inflamed joints. A mechanism for this is suggested. [source] Human articular chondrocytes suppress in vitro proliferation of anti-CD3 activated peripheral blood mononuclear cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2006Chiara Bocelli-Tyndall Objective: To investigate whether mature human articular chondrocytes (AC) exhibit an antiproliferative effect on activated peripheral blood mononuclear cells (PBMC) and to compare this effect with other cells of mesenchymal origin. Methods: AC from healthy cadaveric cartilage were grown for different passages, in the absence (control) or presence of factors enhancing cell de-differentiation (transforming growth factor (TGF),1, fibroblast growth factor (FGF)-2, and platelet derived growth factor (PDGF)bb-TFP medium). Cell ability to suppress PBMC proliferation driven by anti-CD3 antibody was measured by tritiated thymidine uptake following incubation for 48 h at different PBMC:AC ratios and expressed as percent of residual proliferation (RP). AC antiproliferative effect was compared to that of control dermal fibroblasts (DF) and bone marrow stromal cells (BMSC). Results: AC exhibited a cell number-dependent antiproliferative effect. The strongest effect (up to 2% RP) was measured using the least expanded AC cultures. The use of TFP medium for AC expansion resulted in a significantly lower antiproliferative effect, in the range of that induced by BMSC (up to 18% RP). Also DF induced a marked antiproliferative effect (up to 11% RP). Conclusion: We report for the first time that human AC have a marked antiproliferative effect on anti-CD3 stimulated PBMC, which is reduced upon culture in medium-inducing extensive cell de-differentiation. These results reflect the immunosuppressive properties observed for other different mesenchymal cell types and raise the question of a potential common physiological role in local tissue protection. J. Cell. Physiol. 209: 732,734, 2006. © 2006 Wiley-Liss, Inc. [source] Experimental and mathematical study of the influence of growth factors on the growth kinetics of adult human articular chondrocytes,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005Andrea Barbero This study aimed at determining how kinetic parameters of adult human articular chondrocytes (AHAC) growth are modulated by the growth factor combination TGF,1, FGF-2, and PDGF BB (TFP), recently shown to stimulate AHAC proliferation. AHAC, isolated from cartilage biopsies of three individuals, were cultured in medium without (CTR) or with TFP. For growth curves, AHAC were seeded at 1,000 cells/cm2 and cultured for 12 days, with cell numbers measured fluorimetrically in the same wells every 12 h. For microcolony tests, AHAC were seeded at 2.5 cells/cm2 and cultured for 6 days, with cell numbers determined for each microcolony by phase contrast microscopy every 8 h. A mathematical model combining delay and logistic equations was developed to capture the growth kinetic parameters and to enable the description of the complete growth process of the cell culture. As compared to CTR medium, the presence of TFP increased the number of cells/well starting from the fifth day of culture, and a four-fold larger cell number was reached at confluency. For single microcolonies, TFP reduced the time for the first cell division by 26.6%, the time for subsequent cell divisions (generation time) by 16.8%, and the percentage of quiescent cells (Qc) by 42.5%. The mathematical model fitted well the experimental data of the growth kinetic. Finally, using both microcolony tests and the mathematical model, we determined that prolonged cell expansion induces an enrichment of AHAC with shorter first division time, but not of those with shorter generation time. © 2005 Wiley-Liss, Inc. [source] Immunophenotypic analysis of human articular chondrocytes: Changes in surface markers associated with cell expansion in monolayer cultureJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005Jose Diaz-Romero Cartilage tissue engineering relies on in vitro expansion of primary chondrocytes. Monolayer is the chosen culture model for chondrocyte expansion because in this system the proliferative capacity of chondrocytes is substantially higher compared to non-adherent systems. However, human articular chondrocytes (HACs) cultured as monolayers undergo changes in phenotype and gene expression known as "dedifferentiation." To gain a better understanding of the cellular mechanisms involved in the dedifferentiation process, our research focused on the characterization of the surface molecule phenotype of HACs in monolayer culture. Adult HACs were isolated by enzymatic digestion of cartilage samples obtained post-mortem. HACs cultured in monolayer for different time periods were analyzed by flow cytometry for the expression of cell surface markers with a panel of 52 antibodies. Our results show that HACs express surface molecules belonging to different categories: integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51/61, CD54, CD106, CD166, CD58, CD44), tetraspanins (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ectoenzymes (CD10, CD26), and other surface molecules (CD90, CD99). Moreover, differential expression of certain markers in monolayer culture was identified. Up-regulation of markers on HACs regarded as distinctive for mesenchymal stem cells (CD10, CD90, CD105, CD166) during monolayer culture suggested that dedifferentiation leads to reversion to a primitive phenotype. This study contributes to the definition of HAC phenotype, and provides new potential markers to characterize chondrocyte differentiation stage in the context of tissue engineering applications. © 2004 Wiley-Liss, Inc. [source] Control of human articular chondrocyte differentiation by reduced oxygen tensionJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Christopher L. Murphy Cell number is often a limiting factor in studies of chondrocyte physiology, particularly for human investigations. Chondrocytes can be readily proliferated in monolayer culture, however, differentiated phenotype is soon lost. We therefore endeavored to restore normal phenotype to human chondrocytes after serial passage in monolayer culture by manipulating cell morphology and oxygen tension towards the in vivo state. Third passage cells were encapsulated in alginate and exposed to either 20% or more physiologic 5% oxygen tensions. To assess cell phenotype, gene expression was measured using TaqMan real-time PCR. Encapsulated, primary chondrocytes cultured in 20% oxygen were used as a positive reference. Passaged human chondrocytes were fibroblastic in appearance and had lost normal phenotype as evidenced by a decrease in expression of collagen II, aggrecan, and sox9 genes of 66, 6, and 14 fold, respectively; with concomitant high expression of type I collagen (22 fold increase). A partial regaining of the differentiated phenotype was observed by encapsulation in 20% oxygen; however, even after 4 weeks, collagen II gene expression was not fully restored. Collagen II and aggrecan expression were increased, on average, 3 fold, in 5% oxygen tension compared to 20% cultures. Furthermore, matrix glycosaminoglycan (GAG) levels were significantly increased in reduced oxygen. In fact, after 4 weeks in 5% oxygen, encapsulated third passage cells had collagen II expression fully regained and aggrecan and sox9 levels actually exceeding primary cell levels in 20% oxygen. Our results show that the phenotype of serially passaged human articular chondrocytes is more fully restored by combining encapsulation with culture in more physiological levels of oxygen. Sox9, an essential transcription factor for chondrocyte differentiation is strongly implicated in this process since its expression was upregulated almost 27 fold. These findings have implications for the optimal conditions for the in vitro culture of chondrocytes. © 2004 Wiley-Liss, Inc. [source] Quantitative analysis of gene expression in human articular chondrocytes assigned for autologous implantationJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2008Ariana Barli Abstract Autologous chondrocyte implantation (ACI) relies on the implantation of in vitro expanded cells. The aim was to study the dedifferentiation of human articular chondrocytes under different cultivating conditions [days 0,10 in the primary culture (P0); passages in a monolayer from P0 to P3; monolayer vs. alginate and monolayer vs. alginate/agarose hydrogels] using real-time PCR analysis. The relative gene expressions for collagen type I and II, aggrecan and versican were quantified and the corresponding differentiation indexes (Col2/Col1, Agr/Ver) were calculated. The values of both differentiation indexes decreased exponentially with time in the P0 monolayer culture, and continued with a significant decrease over the subsequent monolayer passages. On the contrary, the chondrocytes seeded in either of the hydrogels significantly increased the indexes compared to their parallel monolayer cultures. These results indicate that alginate and alginate/agarose hydrogels offer an appropriate environment for human articular chondrocytes to redifferentiate after being expanded in vitro. Therefore the three-dimensional (3D) hydrogel chondrocyte cultures present not only surgical, but also biological advantage over the classic suspension,periosteum chondrocyte implantation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:847,853, 2008 [source] Quantification of expression levels of cellular differentiation markers does not support a general shift in the cellular phenotype of osteoarthritic chondrocytesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2003Pia Margarethe Gebhard Abstract Many studies have shown increased anabolic activity in osteoarthritic cartilage and have suggested changes in the cellular phenotypes of articular chondrocytes. Most of these studies relied on non-quantitative technologies, which did not allow the estimation of the relative importance of the different differentiation phenomena. In the present study, we developed and used quantitative PCR assays for collagen types I, II(total), IIA, III, and X as marker genes indicating cellular synthetic activity (collagen type II) as well as differentiation pattern of chondrocytes (collagen types I, IIA, III, and X) and quantified these genes in normal, early degenerative, and late stage osteoarthritic cartilage in parallel. At first sight, our results confirmed previously published data showing hardly any expression of collagen genes in normal and significantly enhanced expression in osteoarthritic cartilage. This included collagen types II, III, and IIA, but also collagen types I(,1) and X. However, if one considers the ratios of the various markers of chondrocytic differentiation in comparison to collagen type II, the main synthetic product of differentiated chondrocytes, no shift in the cellular phenotype was detectable. In fact, expression ratios remained constant or were even decreased in osteoarthritic cartilage. Our results confirm that normal adult human articular chondrocytes display hardly any expression activity of the collagen types investigated, whereas osteoarthritic chondrocytes show very increased synthetic activity. The largely unchanged ratios of collagen subtypes investigated indicate that no general shift in the cellular phenotype does occur in osteoarthritic cartilage as suggested by previous investigations. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage regeneration when subcutaneously implanted in nude miceJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 7 2009J. T. Oliveira Abstract Gellan gum is a polysaccharide that has been recently proposed by our group for cartilage tissue-engineering applications. It is commonly used in the food and pharmaceutical industry and has the ability to form stable gels without the use of harsh reagents. Gellan gum can function as a minimally invasive injectable system, gelling inside the body in situ under physiological conditions and efficiently adapting to the defect site. In this work, gellan gum hydrogels were combined with human articular chondrocytes (hACs) and were subcutaneously implanted in nude mice for 4 weeks. The implants were collected for histological (haematoxylin and eosin and Alcian blue staining), biochemical [dimethylmethylene blue (GAG) assay], molecular (real-time PCR analyses for collagen types I, II and X, aggrecan) and immunological analyses (immunolocalization of collagen types I and II). The results showed a homogeneous cell distribution and the typical round-shaped morphology of the chondrocytes within the matrix upon implantation. Proteoglycans synthesis was detected by Alcian blue staining and a statistically significant increase of proteoglycans content was measured with the GAG assay quantified from 1 to 4 weeks of implantation. Real-time PCR analyses showed a statistically significant upregulation of collagen type II and aggrecan levels in the same periods. The immunological assays suggest deposition of collagen type II along with some collagen type I. The overall data shows that gellan gum hydrogels adequately support the growth and ECM deposition of human articular chondrocytes when implanted subcutaneously in nude mice. Copyright © 2009 John Wiley & Sons, Ltd. [source] Effect of cell seeding concentration on the quality of tissue engineered constructs loaded with adult human articular chondrocytesJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 1 2008Sebastian Concaro Abstract Many aspects of the process of in vitro differentiation of chondrocytes in three-dimensional (3D) scaffolds need to be further investigated. Chitosan scaffolds were produced by freeze-drying 3% w/v 90% DDA chitosan gels. The effect of the cell seeding concentration was evaluated by culturing human adult chondrocytes in chitosan scaffolds After the first passage, cells were seeded into chitosan scaffolds with a diameter of 8 mm. The final cell seeding concentration per cm3 of chitosan scaffold was: Group A, 3 × 106; Group B, 6 × 106; Group C, 12 × 106; and Group D, 25 × 106 cells. After 14 and 28 days in 3D culture, the constructs were assesed for collagen, glucosaminoglycans and DNA content. The mechanical properties of the constructs were determined using a dynamic oscillatory shear test. The histological aspect of the constructs was evaluated using the Bern score. The collagen and GAG concentration increased, varying the cell seeding concentration. There was a significant increase in proteoglycan and hydroxyproline production between groups C and D. The sulphated GAG content increased significantly in the group D as compared to the other groups. The mechanical properties of the different constructs increased over time, from 9.6 G,/kPa at 14 days of 3D culture to 14.6 G,/kPa at 28 days under the same culture conditions. In this study we were able to determine that concentrations of 12,25 million cells/cm2 are needed to increase the matrix production and mechanical properties of human adult chondrocytes under static conditions. Copyright © 2008 John Wiley & Sons, Ltd. [source] Human articular chondrocytes secrete parathyroid hormone,related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesisARTHRITIS & RHEUMATISM, Issue 9 2010J. Fischer Objective The use of bone marrow,derived mesenchymal stem cells (MSCs) has shown promise in cell-based cartilage regeneration. A yet-unsolved problem, however, is the unwanted up-regulation of markers of hypertrophy, such as alkaline phosphatase (AP) and type X collagen, during in vitro chondrogenesis and the formation of unstable calcifying cartilage at heterotopic sites. In contrast, articular chondrocytes produce stable, nonmineralizing cartilage. The aim of this study was to address whether coculture of MSCs with human articular chondrocytes (HACs) can suppress the undesired hypertrophy in differentiating MSCs. Methods MSCs were differentiated in chondrogenic medium that had or had not been conditioned by parallel culture with HAC pellets, or MSCs were mixed in the same pellet with the HACs (1:1 or 1:2 ratio) and cultured for 6 weeks. Following in vitro differentiation, the pellets were transplanted into SCID mice. Results The gene expression ratio of COL10A1 to COL2A1 and of Indian hedgehog (IHH) to COL2A1 was significantly reduced by differentiation in HAC-conditioned medium, and less type X collagen protein was deposited relative to type II collagen. AP activity was significantly lower (P < 0.05) in the cells that had been differentiated in conditioned medium, and transplants showed significantly reduced calcification in vivo. In mixed HAC/MSC pellets, suppression of AP was dose-dependent, and in vivo calcification was fully inhibited. Chondrocytes secreted parathyroid hormone,related protein (PTHrP) throughout the culture period, whereas PTHrP was down-regulated in favor of IHH up-regulation in control MSCs after 2,3 weeks of chondrogenesis. The main inhibitory effects seen with HAC-conditioned medium were reproducible by PTHrP supplementation of unconditioned medium. Conclusion HAC-derived soluble factors and direct coculture are potent means of improving chondrogenesis and suppressing the hypertrophic development of MSCs. PTHrP is an important candidate soluble factor involved in this effect. [source] Resistin induces expression of proinflammatory cytokines and chemokines in human articular chondrocytes via transcription and messenger RNA stabilizationARTHRITIS & RHEUMATISM, Issue 7 2010Zhiqi Zhang Objective To elucidate the effects of resistin on human articular chondrocytes and to generate a picture of their regulation at the transcriptional and posttranscriptional levels. Methods Human articular chondrocytes were cultured with resistin. Changes in gene expression were analyzed at various doses and times. Cells were also treated with the transcription inhibitor actinomycin D after resistin treatment or with the NF-,B inhibitor IKK-NBD before resistin treatment. Gene expression was tested by quantitative real-time polymerase chain reaction. Computational analysis for transcription factor binding motifs was performed on the promoter regions of differentially expressed genes. TC-28 chondrocytes were transfected with CCL3 and CCL4 promoter constructs, pNF-,B reporter, and NF-,B and CCAAT/enhancer binding protein , (C/EBP,) expression vectors with or without resistin. Results Resistin-treated human articular chondrocytes increased the expression of cytokines and chemokines. Levels of messenger RNA (mRNA) for matrix metalloproteinase 1 (MMP-1), MMP-13, and ADAMTS-4 also increased, while type II collagen ,1 (COL2A1) and aggrecan were down-regulated. The cytokine and chemokine genes could be categorized into 3 groups according to the pattern of mRNA expression over a 24-hour time course. One pattern suggested rapid regulation by mRNA stability. The second and third patterns were consistent with transcriptional regulation. Computational analysis suggested the transcription factors NF-,B and C/EBP, were involved in the resistin-induced up-regulation. This prediction was confirmed by the cotransfection of NF-,B and C/EBP, and the IKK-NBD inhibition. Conclusion Resistin has diverse effects on gene expression in human chondrocytes, affecting chemokines, cytokines, and matrix genes. Messenger RNA stabilization and transcriptional up-regulation are involved in resistin-induced gene expression in human chondrocytes. [source] Trefoil factor 3 is induced during degenerative and inflammatory joint disease, activates matrix metalloproteinases, and enhances apoptosis of articular cartilage chondrocytesARTHRITIS & RHEUMATISM, Issue 3 2010Sophie Rösler Objective Trefoil factor 3 (TFF3, also known as intestinal trefoil factor) is a member of a family of protease-resistant peptides containing a highly conserved motif with 6 cysteine residues. Recent studies have shown that TFF3 is expressed in injured cornea, where it plays a role in corneal wound healing, but not in healthy cornea. Since cartilage and cornea have similar matrix properties, we undertook the present study to investigate whether TFF3 could induce anabolic functions in diseased articular cartilage. Methods We used reverse transcriptase,polymerase chain reaction, Western blot analysis, and immunohistochemistry to measure the expression of TFF3 in healthy articular cartilage, osteoarthritis (OA),affected articular cartilage, and septic arthritis,affected articular cartilage and to assess the effects of cytokines, bacterial products, and bacterial supernatants on TFF3 production. The effects of TFF3 on matrix metalloproteinase (MMP) production were measured by enzyme-linked immunosorbent assay, and effects on chondrocyte apoptosis were studied by caspase assay and annexin V assay. Results Trefoil factors were not expressed in healthy human articular cartilage, but expression of TFF3 was highly up-regulated in the cartilage of patients with OA. These findings were confirmed in animal models of OA and septic arthritis, as well as in tumor necrosis factor ,, and interleukin-1,,treated primary human articular chondrocytes, revealing induction of Tff3/TFF3 under inflammatory conditions. Application of the recombinant TFF3 protein to cultured chondrocytes resulted in increased production of cartilage-degrading MMPs and increased chondrocyte apoptosis. Conclusion In this study using articular cartilage as a model, we demonstrated that TFF3 supports catabolic functions in diseased articular cartilage. These findings widen our knowledge of the functional spectrum of TFF peptides and demonstrate that TFF3 is a multifunctional trefoil factor with the ability to link inflammation with tissue remodeling processes in articular cartilage. Moreover, our data suggest that TFF3 is a factor in the pathogenesis of OA and septic arthritis. [source] DNA demethylation at specific CpG sites in the IL1B promoter in response to inflammatory cytokines in human articular chondrocytesARTHRITIS & RHEUMATISM, Issue 11 2009Ko Hashimoto Objective To determine whether changes in the DNA methylation status in the promoter region of the gene encoding interleukin-1, (IL-1,) account for expression of IL1B messenger RNA (mRNA) after long-term treatment of human articular chondrocytes with inflammatory cytokines. Methods IL-1,, tumor necrosis factor , (TNF,) plus oncostatin M (OSM), or 5-azadeoxycytidine (5-aza-dC) was added twice weekly for 4,5 weeks to primary cultures of normal human articular chondrocytes derived from the femoral head cartilage of patients with a fracture of the femoral neck. Expression of MMP13, IL1B, TNFA, and DNMT1 was determined by SYBR Green,based quantitative reverse transcription,polymerase chain reaction (RT-PCR) analysis of genomic DNA and total RNA extracted from the same sample before and after culture. Bisulfite modification was used to identify which CpG sites in the IL1B promoter showed differential methylation between IL1B -expressing and IL1B -nonexpressing cells. The percentages of cells that were methylated at that critical CpG site (,299 bp) were quantified by a method that depended on methylation-sensitive restriction enzymes and real-time RT-PCR. Secretion of IL-1, into the culture media was assessed by enzyme-linked immunosorbent assay. Results Healthy chondrocytes did not express IL1B mRNA, but the levels were increased 5-fold by treatment with 5-aza-dC and were increased 100,1,000-fold by treatment with TNF,/OSM. The percentage CpG methylation was decreased by 5-aza-dC treatment but was reduced considerably more by IL-1, and was almost abolished by TNF,/OSM. The mRNA was translated into protein in cytokine-treated chondrocytes. Conclusion These novel findings indicate that inflammatory cytokines can change the DNA methylation status at key CpG sites, resulting in long-term induction of IL1B in human articular chondrocytes. [source] The melanocortin system in articular chondrocytes: Melanocortin receptors, pro-opiomelanocortin, precursor proteases, and a regulatory effect of ,-melanocyte,stimulating hormone on proinflammatory cytokines and extracellular matrix componentsARTHRITIS & RHEUMATISM, Issue 10 2009Susanne Grässel Objective The pro-opiomelanocortin (POMC),derived neuropeptide ,-melanocyte,stimulating hormone (,-MSH) mediates its effects via melanocortin (MC) receptors. This study was carried out to investigate the expression patterns of the MC system and the effects of ,-MSH in human articular chondrocytes. Methods Articular chondrocytes established from human osteoarthritic joint cartilage were analyzed by reverse transcription,polymerase chain reaction (RT-PCR) and Western blotting for the expression of MC receptors, POMC, and prohormone convertases (PCs). MC-1 receptor (MC-1R) expression in articular cartilage was further studied by immunohistochemistry. Ca2+ and cAMP assays were used to monitor ,-MSH signaling, while studies of ,-MSH function were performed in cultures with chondrocyte micromass pellets stimulated with ,-MSH. Expression of cytokines and extracellular matrix (ECM) components was determined by real-time RT-PCR, Western immunoblotting, and enzyme-linked immunosorbent assays. Results MC-1R expression was detected in articular chondrocytes in vitro and in articular cartilage in situ. In addition, expression of transcripts for MC-2R, MC-5R, POMC, and PCs was detected in articular chondrocytes. Stimulation with ,-MSH increased the levels of intracellular cAMP, but not Ca2+, in chondrocytes. Both messenger RNA and protein expression of various proinflammatory cytokines, collagens, matrix metalloproteinases (MMPs), and SOX9 was modulated by ,-MSH. Conclusion Human articular chondrocytes are target cells for ,-MSH. The effects of ,-MSH on expression of cytokines and MMPs suggest that this neuropeptide plays a role in inflammatory and degenerative processes in cartilage. It is conceivable that inflammatory reactions can be mitigated by the induction of endogenous MCs or administration of ,-MSH to the affected joints. The induction pattern of regulatory and structural ECM components such as collagens as well as SOX9 and anabolic and catabolic cytokines points to a function of ,-MSH as a trophic factor in skeletal development during endochondral ossification rather than as a factor in homeostasis of permanent cartilage. [source] Parathyroid hormone 1,34 inhibits terminal differentiation of human articular chondrocytes and osteoarthritis progression in ratsARTHRITIS & RHEUMATISM, Issue 10 2009Je-Ken Chang Objective Parathyroid hormone 1,34 (PTH[1,34]), a parathyroid hormone analog, shares the same receptor, PTH receptor 1, with parathyroid hormone,related peptide (PTHrP). This study was undertaken to address the hypothesis that PTH(1,34) inhibits terminal differentiation of articular chondrocytes and in turn suppresses the progression of osteoarthritis (OA). Methods We studied the effect of PTH(1,34) on human articular chondrocytes with azacytidine (azaC),induced terminal differentiation in vitro and on papain-induced OA in the knee joints of rats. In the in vitro study, we measured the levels of messenger RNA for SOX9, aggrecan, type II collagen, type X collagen, alkaline phosphatase (AP), Indian hedgehog (IHH), Bcl-2, and Bax by real-time polymerase chain reaction, levels of glycosaminoglycan (GAG) by dimethylmethylene blue assay, and rate of apoptosis by TUNEL staining. In the in vivo study, we evaluated the histologic changes in GAG, type II collagen, type X collagen, and chondrocyte apoptosis in the articular cartilage of rat knees. Results AzaC induced terminal differentiation of human chondrocytes, including down-regulation of aggrecan, type II collagen, and GAG and up-regulation of type X collagen, alkaline phosphatase, and IHH. Apoptosis was reversed by 3,10 days of treatment with 10 nM PTH(1,34). SOX9 expression was not changed by either azaC or PTH(1,34) treatment. Bcl-2 and Bax were up-regulated on day 10 and day 14, respectively, after azaC induction of terminal differentiation, but PTH(1,34) treatment did not reverse this effect. Furthermore, PTH(1,34) treatment reversed papain-induced OA changes (decreasing GAG and type II collagen, and increasing type X collagen and chondrocyte apoptosis) in the knee joints of rats. Conclusion Our findings indicate that PTH(1,34) inhibits the terminal differentiation of human articular chondrocytes in vitro and inhibits progression of OA in rats in vivo, and may be used to treat OA. [source] MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responsesARTHRITIS & RHEUMATISM, Issue 9 2009Shigeru Miyaki Objective MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). Methods To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1, (IL-1,) on miR-140 expression. Double-stranded miR-140 (ds,miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. Results Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1, suppressed miR-140 expression. Transfection of chondrocytes with ds,miR-140 down-regulated IL-1,,induced ADAMTS5 expression and rescued the IL-1,,dependent repression of AGGRECAN gene expression. Conclusion This study shows that miR-140 has a chondrocyte differentiation,related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1, may contribute to the abnormal gene expression pattern characteristic of OA. [source] Changes in surface markers of human mesenchymal stem cells during the chondrogenic differentiation and dedifferentiation processes in vitroARTHRITIS & RHEUMATISM, Issue 8 2009Hyun Jung Lee Objective To investigate surface markers showing specific changes during the chondrogenic differentiation and dedifferentiation of human mesenchymal stem cells (MSCs). Methods Human MSCs from adult bone marrow were subjected to chondrogenic differentiation in 3-dimensional (3-D) alginate culture with or without transforming growth factor ,3 (TGF,3) for 2 weeks, followed by dedifferentiation in monolayer for 1 week. Surface antigens were selected from those previously reported to show changes in expression during dedifferentiation of human articular chondrocytes (HACs). Results Flow cytometry was used to identify 3 groups of surface antigens with differential expression patterns that were quite different from those previously reported on HACs. Two groups of antigens were expressed at high levels on human MSCs. The expression of the first group of antigens (CD44, CD58, CD81, CD90, CD105, and CD166) was decreased reversibly by the 3-D alginate culture and irreversibly in the presence of TGF,3, except for CD81, which showed reversible changes regardless of TGF,3. The expression of the second group of antigens (CD49c, CD49e, and CD151) was decreased during chondrogenic differentiation only in the presence of TGF,3. During all experimental stages, the expression of the third group of antigens (CD14, CD26, CD49f, CD54, CD106, CD119, and CD140a) was maintained at low levels (expressed on <30% of cells), although with some fluctuations. Conclusion We speculate that the second group of surface antigens could be negative markers for chondrogenic differentiation of human MSCs. [source] Regulation of plasminogen activator inhibitor 1 expression in human osteoarthritic chondrocytes by fluid shear stress: Role of protein kinase C,ARTHRITIS & RHEUMATISM, Issue 8 2009Chih-Chang Yeh Objective To test a fluid flow system for the investigation of the influence of shear stress on expression of plasminogen activator inhibitor 1 (PAI-1) in human osteoarthritic (OA) articular chondrocytes (from lesional and nonlesional sites) and human SW-1353 chondrocytes. Methods Human SW-1353 chondrocytes and OA and normal human articular chondrocytes were cultured on type II collagen,coated glass plates under static conditions or placed in a flow chamber to form a closed fluid-circulation system for exposure to different levels of shear stress (2,20 dyn/cm2). Real-time polymerase chain reaction was used to analyze PAI-1 gene expression, and protein kinase C (PKC) inhibitors and small interfering RNA were used to investigate the mechanism of shear stress,induced signal transduction in SW-1353 and OA (lesional and nonlesional) articular chondrocytes. Results There was a significant reduction in PAI-1 expression in OA chondrocytes obtained from lesional sites compared with those obtained from nonlesional sites. In SW-1353 chondrocytes subjected to 2 hours of shear flow, moderate shear stresses (5 and 10 dyn/cm2) generated significant PAI-1 expression, which was regulated through PKC, phosphorylation and Sp-1 activation. These levels of shear stress also increased PAI-1 expression in articular chondrocytes from nonlesional sites and from normal healthy cartilage through the activation of PKC, and Sp-1 signal transduction, but no effect of these levels of fluid shear stress was observed on OA chondrocytes from lesional sites. Conclusion OA chondrocytes from lesional sites and those from nonlesional sites of human cartilage have differential responses to shear stress with regard to PAI-1 gene expression, and therefore diverse functional consequences can be observed. [source] Expression of MicroRNA-146a in osteoarthritis cartilageARTHRITIS & RHEUMATISM, Issue 4 2009Keiichiro Yamasaki Objective A role of microRNA, which are ,22-nucleotide noncoding RNAs, has recently been recognized in human diseases. The objective of this study was to identify the expression pattern of microRNA-146a (miR-146a) in cartilage from patients with osteoarthritis (OA). Methods The expression of miR-146a in cartilage from 15 patients with OA was analyzed by quantitative reverse transcription,polymerase chain reaction (RT-PCR) and by in situ hybridization. Induction of the expression of miR-146a by cultures of normal human articular chondrocytes following stimulation with interleukin-1, (IL-1,) was examined by quantitative RT-PCR. Results All cartilage samples were divided into 3 groups according to a modification of the Mankin score (grade I = mild OA scored 0,5, grade II = moderate OA scored 6,10, and grade III = severe OA scored 11,14). In grade I OA cartilage samples, the expression of miR-146a and COL2A1 was significantly higher than that in the other groups (P < 0.05). In grades II and III OA cartilage, the expression of miR-146a and COL2A1 was decreased, whereas the expression of matrix metalloproteinase 13 (MMP-13) was elevated in grade II OA cartilage. These data showed that miR-146a is expressed intensely in cartilage with a low Mankin grade and that miR-146a expression decreases in parallel with the level of MMP-13 expression. Tissue section in situ hybridization of primary miR-146a (pri-miR-146a) revealed that pri-miR-146a was expressed in chondrocytes residing in all tissue layers, especially in the superficial layer, where it was intensely expressed. The expression of miR-146 was markedly elevated by IL-1, stimulation in human chondrocytes in vitro. Conclusion This study shows that miR-146 is intensely expressed in low-grade OA cartilage and that its expression is induced by stimulation of IL-1,. Thus, miR-146 might play a role in OA cartilage pathogenesis. [source] CCAAT/ENHANCER binding protein , mediates expression of matrix metalloproteinase 13 in human articular chondrocytes in inflammatory arthritisARTHRITIS & RHEUMATISM, Issue 3 2009Mitsumasa Hayashida Objective To determine the function of CCAAT/enhancer binding protein , (C/EBP,) in the expression of matrix metalloproteinase 13 (MMP-13) in chondrocytes in inflammatory arthritis. Methods Cartilage obtained from patients with rheumatoid arthritis and osteoarthritis was immunostained for expression of C/EBP, or MMP-13. Interleukin-1,, or tumor necrosis factor , (TNF,),stimulated chondrocytes were subjected to Western blotting and real-time reverse transcriptase,polymerase chain reaction (RT-PCR). MMP-13 promoter assays were conducted, and the C/EBP, response element was characterized by deletion and mutation analysis. C-28/I2 cells were treated with TNF, and subjected to chromatin immunoprecipitation (ChIP) assays. Finally, C/EBP,,liver-enriched activator protein (LAP) was overexpressed in C-28/I2 cells or cartilage tissues, and MMP-13 expression was analyzed. Results C/EBP, and MMP-13 expression was colocalized in chondrocytes in arthritic cartilage. MMP-13 promoter activity was stimulated by C/EBP, overexpression in a dose-dependent manner. Luciferase assays revealed that a ,981-bp promoter had the greatest activity, while deletion to ,936 bp strongly diminished promoter activity. Luciferase activity was repressed to basal levels by mutations in potential C/EBP binding sites. The stimulatory effects of C/EBP, overexpression were diminished by mutation. ChIP assays revealed that TNF, treatment enhanced the binding of C/EBP, to the MMP-13 promoter. When C/EBP,-LAP was overexpressed in C-28/I2 cells, endogenous MMP-13 expression was stimulated up to 32-fold as detected by real-time RT-PCR. Furthermore, following adenoviral overexpression of C/EBP,-LAP in organ culture of articular cartilage, stimulation of MMP-13 was also detected by immunohistochemistry. Conclusion C/EBP, directly binds to the MMP-13 promoter region and stimulates the expression of MMP-13 in chondrocytes in inflammatory arthritis. [source] Interleukin-7 stimulates secretion of S100A4 by activating the JAK/STAT signaling pathway in human articular chondrocytesARTHRITIS & RHEUMATISM, Issue 3 2009Raghunatha R. Yammani Objective S100A4 has been shown to be increased in osteoarthritic (OA) cartilage and to stimulate chondrocytes to produce matrix metalloproteinase 13 (MMP-13) through activation of the receptor for advanced glycation end products (RAGE). The aim of this study was to examine the mechanism of S100A4 secretion by chondrocytes. Methods Human articular chondrocytes isolated from ankle cartilage were stimulated with 10 ng/ml of interleukin-1, (IL-1,), IL-6, IL-7, or IL-8. Cells were pretreated with either a JAK-3 inhibitor, brefeldin A, or cycloheximide. Immunoblotting with phospho-specific antibodies was used to determine the activation of signaling proteins. Secretion of S100A4 was measured in conditioned media by immunoblotting, and MMP-13 was measured by enzyme-linked immunosorbent assay. Results Chondrocyte secretion of S100A4 was observed after treatment with IL-6 or IL-8 but was much greater in cultures treated with equal amounts of IL-7 and was not observed after treatment with IL-1,. IL-7 activated the JAK/STAT pathway, with increased phosphorylation of JAK-3 and STAT-3, leading to increased production of S100A4 and MMP-13. Overexpression of a dominant-negative RAGE construct inhibited the IL-7,mediated production of MMP-13. Pretreatment of chondrocytes with a JAK-3 inhibitor or with cycloheximide blocked the IL-7,mediated secretion of S100A4, but pretreatment with brefeldin A did not. Conclusion IL-7 stimulates chondrocyte secretion of S100A4 via activation of JAK/STAT signaling, and then S100A4 acts in an autocrine manner to stimulate MMP-13 production via RAGE. Since both IL-7 and S100A4 are up-regulated in OA cartilage and can stimulate MMP-13 production by chondrocytes, this signaling pathway could contribute to cartilage destruction during the development of OA. [source] Prostaglandin E2 and its cognate EP receptors control human adult articular cartilage homeostasis and are linked to the pathophysiology of osteoarthritisARTHRITIS & RHEUMATISM, Issue 2 2009Xin Li Objective To elucidate the pathophysiologic links between prostaglandin E2 (PGE2) and osteoarthritis (OA) by characterizing the catabolic effects of PGE2 and its unique receptors in human adult articular chondrocytes. Methods Human adult articular chondrocytes were cultured in monolayer or alginate beads with and without PGE2 and/or agonists of EP receptors, antagonists of EP receptors, and cytokines. Cell survival, proliferation, and total proteoglycan synthesis and accumulation were measured in alginate beads. Chondrocyte-related gene expression and phosphatidylinositol 3-kinase/Akt signaling were assessed by real-time reverse transcription,polymerase chain reaction and Western blotting, respectively, using a monolayer cell culture model. Results Stimulation of human articular chondrocytes with PGE2 through the EP2 receptor suppressed proteoglycan accumulation and synthesis, suppressed aggrecan gene expression, did not appreciably affect expression of matrix-degrading enzymes, and decreased the type II collagen:type I collagen ratio. EP2 and EP4 receptors were expressed at higher levels in knee cartilage than in ankle cartilage and in a grade-dependent manner. PGE2 titration combined with interleukin-1 (IL-1) synergistically accelerated expression of pain-associated molecules such as inducible nitric oxide synthase and IL-6. Finally, stimulation with exogenous PGE2 or an EP2 receptor,specific agonist inhibited activation of Akt that was induced by insulin-like growth factor 1. Conclusion PGE2 exerts an antianabolic effect on human adult articular cartilage in vitro, and EP2 and EP4 receptor antagonists may represent effective therapeutic agents for the treatment of OA. [source] Effect of interleukin-1, on osteogenic protein 1,induced signaling in adult human articular chondrocytesARTHRITIS & RHEUMATISM, Issue 1 2009Amel M. Elshaier Objective Two major receptor-activated Smad (R-Smad) signaling pathways, bone morphogenetic protein (BMP) and MAPK, were examined in a model of interleukin-1, (IL-1,),induced cartilage degeneration to investigate the effect of IL-1, on osteogenic protein 1 (OP-1) signaling in adult human articular chondrocytes. Methods Chondrocytes from the ankles of 26 normal human donors were cultured in high-density monolayers in serum-free medium. The effect of IL-1, on BMP receptors was studied by reverse transcription,polymerase chain reaction and flow cytometry. Phosphorylation of R-Smads was tested in cells treated with IL-1, (10 ng/ml), OP-1 (100 ng/ml), or the combination of IL-1, and OP-1. Cell lysates were analyzed by Western blotting with polyclonal antibodies against 2 R-Smad phosphorylation sites (BMP- and MAPK-mediated) or with total, nonphosphorylated R-Smad as a control. To identify which MAPKs play a role in IL-1, activation of the linker region, chondrocytes were preincubated with specific MAPK inhibitors (PD98059 for MAP/ERK, SP600125 for JNK, and SB203580 for p38). Results IL-1, reduced the number of activin receptor,like kinase 2 (ALK-2) and ALK-3 receptors, inhibited expression of Smad1 and Smad6, delayed and prematurely terminated the onset of OP-1,mediated R-Smad phosphorylation, and affected nuclear translocation of R-Smad/Smad4 complexes. The alternative phosphorylation of R-Smad in the linker region via the MAPK pathway (primarily p38 and JNK) was observed to be a possible mechanism through which IL-1, offsets OP-1 signaling and the response to OP-1. Conversely, OP-1 was found to directly inhibit phosphorylation of p38. Conclusion These findings describe new mechanisms of the crosstalk between OP-1 and IL-1, in chondrocytes. The study also identifies potential targets for therapeutic interventions in the treatment of cartilage-degenerative processes. [source] A novel tumor necrosis factor ,,responsive CCAAT/enhancer binding protein site regulates expression of the cartilage-derived retinoic acid,sensitive protein gene in cartilageARTHRITIS & RHEUMATISM, Issue 5 2008Toshihiro Imamura Objective Inflammatory processes in rheumatoid arthritis are primarily regulated by the cytokines tumor necrosis factor , (TNF,) and interleukin-1, (IL-1,). Previous studies in our laboratory have shown that IL-1, represses expression of the cartilage characteristic genes, cartilage-derived retinoic acid,sensitive protein (cd - rap) and type II collagen (COL2A1); this mechanism of repression involves activation of a CCAAT/enhancer binding protein (c/EBP) site within promoter regions. The aim of this study was to investigate novel TNF,-mediated mechanisms that regulate the expression of cd - rap. Methods Rat chondrosarcoma cells were transiently transfected with complementary DNA constructs encoding cd - rap, in the presence of TNF,. The expression of c/EBP,, SOX9, and p300 in rat chondrosarcoma cells and primary human articular chondrocytes after treatment with TNF, was examined by reverse transcription,polymerase chain reaction and Western blotting. The effect of TNF, on endogenous binding of c/EBP, or SOX9 to the cd - rap promoter was examined by chromatin immunoprecipitation assays. Results We identified a new c/EBP binding site in the cd - rap promoter (from position ,1059 bp to position ,1046 bp). Binding of c/EBP to this site was regulated by TNF, but not IL-1,, resulting in down-regulation of cd - rap expression. This effect was reversed by mutational inactivation of the c/EBP motif. In addition, the activation potential of SOX9 and CREB binding protein/p300 on the cd - rap promoter was enhanced after mutation of the new c/EBP binding site, indicating that blockage of this site would increase transcription. Conclusion TNF, regulates the expression and/or DNA-binding potential of key positive-acting and negative-acting transcription factors that control the expression of the cartilage matrix gene, cd - rap. [source] Nitrite-mediated protection against hypochlorous acid,induced chondrocyte toxicity: A novel cytoprotective role of nitric oxide in the inflamed joint?ARTHRITIS & RHEUMATISM, Issue 11 2003Matthew Whiteman Objective To examine the potential consequences of overproduction of nitric oxide (NO) and nitrite (NO) in the inflamed rheumatoid joint. Methods Human articular chondrocytes in culture were exposed to HOCl (hypochlorous acid, a physiologic oxidant formed in increased amounts at sites of chronic inflammation), and assays of cell viability, intracellular ATP and glutathione (GSH), and lactate dehydrogenase (LDH) were performed. HOCl-induced lipid peroxidation and activation of the MAP kinases ERK-1/2, JNK-1/2, and p38 were also measured. The modulatory effects of NO-derived nitrite (NO) and nitrate (NO) on HOCl-mediated chondrocyte toxicity were investigated. Results Exposure of human articular chondrocytes to HOCl resulted in a concentration- and time-dependent loss of viability, decrease in ATP and GSH levels, LDH leakage, and cell death. HOCl induced significant lipid peroxidation as well as activation of the MAP kinases ERK-1/2 and p38 but not JNK-1/2. However, the presence of NO but not NO substantially decreased HOCl-dependent cellular toxicity even when NO was added at low (,M) concentrations. In sharp contrast, NO (1 mM) did not inhibit superoxide-, hydroxyl radical,, H2O2 -, or peroxynitrite-mediated cytotoxicity. Furthermore, culture media from cells treated with interleukin-1, (to generate NO and NO) offered significantly more protection against HOCl-mediated cytotoxicity than culture media from untreated cells. Conclusion These data suggest that NO accumulation at chronically inflamed sites where both HOCl and NO are overproduced may be cytoprotective against damage induced by HOCl. Accumulation of NO could represent a novel cytoprotective role of NO in inflamed joints. A mechanism for this is suggested. [source] Plasticity of clonal populations of dedifferentiated adult human articular chondrocytesARTHRITIS & RHEUMATISM, Issue 5 2003Andrea Barbero Objective To investigate whether adult human articular chondrocytes (AHACs), dedifferentiated by monolayer expansion, can differentiate toward diverse mesenchymal lineages and, if so, whether this ability is regulated by growth factors during monolayer expansion. Methods AHACs were expanded as multiclonal or clonal populations in medium without (control) or with factors enhancing cell dedifferentiation (transforming growth factor ,1, fibroblast growth factor 2, and platelet-derived growth factor type BB [TFP]). Cells were then cultured under conditions promoting chondrogenic, osteogenic, or adipogenic differentiation, and the acquired phenotypes were assessed histologically, biochemically, and by real-time reverse transcriptase,polymerase chain reaction. Results Multiclonal populations of both control- and TFP-expanded AHACs differentiated toward the chondrogenic, osteogenic, and adipogenic lineages. Compared with control-expanded AHACs, TFP-expanded cells displayed enhanced chondrogenic differentiation capacity (2.4-fold higher glycosaminoglycan/DNA content and 2,500-fold higher up-regulation of type II collagen) and osteogenic differentiation capacity (9.4-fold higher increase in alkaline phosphatase activity and 12.4-fold higher up-regulation of bone sialoprotein), but reduced formation of adipocytes (5.2-fold lower oil red O,positive cells/area). Clonal populations of AHACs could be efficiently expanded in TFP, but not in control medium. Most TFP-expanded clones were able to redifferentiate only into chondrocytes (7 of 20) or were unable to differentiate (6 of 20). However, some clones (2 of 20) differentiated toward all of the lineages investigated, thus displaying characteristics of mesenchymal progenitor cells. Conclusion Dedifferentiated AHACs exhibit differentiation plasticity, which is modulated by growth factors used during monolayer expansion and is highly heterogeneous across different clones. Clonal culture of AHACs in the presence of regulatory molecules could lead to the identification of AHAC subpopulations with enhanced cartilage repair capacity. [source] | |||||||||||||