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Human Chondrocytes (human + chondrocyte)
Kinds of Human Chondrocytes Selected AbstractsDevelopment of selective tolerance to interleukin-1, by human chondrocytes in vitro,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2002Greta M. Lee Interleukin-1 induces release of NO and PGE2 and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1,. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1, (0.05,10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1, declined over the pretreatment period. In response to IL-1, (0.1 ng/ml), NO and PGE2 release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1, on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1,-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1, increased glucose consumption and lactate production throughout the treatment. IL-1, stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE2 and MMP-13 in response to IL-1, than OA cultures. Like the OA, IL-1,-induced NO and PGE2 release decreased over time. In conclusion, with prolonged exposure to IL-1,, human chondrocytes develop selective tolerance involving NO and PGE2 release but not MMP-13 production, metabolic activity, or matrix metabolism. © 2002 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] Development of selective tolerance to interleukin-1, by human chondrocytes in vitro,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2002Greta M. Lee Interleukin-1 induces release of NO and PGE2 and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1,. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1, (0.05,10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1, declined over the pretreatment period. In response to IL-1, (0.1 ng/ml), NO and PGE2 release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1, on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1,-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1, increased glucose consumption and lactate production throughout the treatment. IL-1, stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE2 and MMP-13 in response to IL-1, than OA cultures. Like the OA, IL-1,-induced NO and PGE2 release decreased over time. In conclusion, with prolonged exposure to IL-1,, human chondrocytes develop selective tolerance involving NO and PGE2 release but not MMP-13 production, metabolic activity, or matrix metabolism. © 2002 Wiley-Liss, Inc. [source] MMP-mediated collagen breakdown induced by activated protein C in equine cartilage is reduced by corticosteroidsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 3 2010Elaine R. Garvican Abstract The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin-1, (IL-1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities. Intraarticular corticosteroids are a commonly prescribed treatment for joint disease, however their role in disease modification remains unclear. APC synergized with IL-1 or tumor necrosis factor-, (TNF,), promoting significant collagen degradation from equine cartilage explants within 4 days, but did not augment glycoaminoglycan (GAG) release. APC activated pro-matrix metalloproteinases (MMP)-2 but not pro-MMP-9, as assessed by gelatin zymography. APC did not directly activate pro-MMP-13. Dexamethasone, triamcinolone, and methylprednisolone acetate (MPA) were evaluated at concentrations between 10, 5M and 10,10M. High concentrations significantly increased GAG release from IL-1+APC,treated explants. With the exception of MPA at 10,10M, all concentrations of corticosteroids caused significant decreases in IL-1+APC-driven hydroxyproline loss. Treatment with corticosteroids suppressed expression of MMP-1, -3, and -13 mRNA. The collagenolysis associated with IL-1+APC synergy, and the inhibition of this effect by corticosteroids may involve gelatinase activation and downregulation of MMP expression, respectively. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:370,378, 2010 [source] Expression of mutant cartilage oligomeric matrix protein in human chondrocytes induces the pseudoachondroplasia phenotypeJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2006Thomas M. Merritt Abstract Over 70 mutations in the cartilage oligomeric matrix protein (COMP), a large extracellular pentameric glycoprotein synthesized by chondrocytes, have been identified as causing two skeletal dysplasias: multiple epiphyseal dysplasia (MED/EDM1), and a dwarfing condition, pseudoachondroplasia (PSACH). These mutations induce misfolding of intracellular COMP, resulting in retention of the protein in the rough endoplasmic reticulum (rER) of chondrocytes. This accumulation of COMP in the rER creates the phenotypic enlarged rER cisternae in the cells, which is believed to compromise chondrocyte function and eventually cause cell death. To study the molecular mechanisms involved with the disease, we sought to develop an in vitro model that recapitulates the PSACH phenotype. Normal human chondrocytes were transfected with wildtype (wt-) COMP or with mutant COMP (D469del; mt-) recombinant adenoviruses and grown in a nonattachment redifferentiating culture system that provides an environment allowing formation of a differentiated chondrocyte nodule. Visualization of normal cells expressing COMP suggested the hallmarks of the PSACH phenotype. Mutant COMP expressed in normal cells was retained in enlarged rER cisternae, which also retained IX collagen (COL9) and matrilin-3 (MATN3). Although these proteins were secreted normally into the ECM of the wt-COMP nodules, reduced secretion of these proteins was observed in nodules composed of cells transfected with mt-COMP. The findings complement those found in chondrocytes from PSACH patient growth plates. This new model system allows for production of PSACH chondrocyte pathology in normal costochondral chondrocytes and can be used for future mechanistic and potential gene therapy studies. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [source] Discoidin domain receptor 2 mediates the collagen II-dependent release of interleukin-6 in primary human chondrocytes,THE JOURNAL OF PATHOLOGY, Issue 2 2009Andreas R Klatt Abstract We deciphered constituent parts of a signal transduction cascade that is initiated by collagen II and results in the release of various pro-inflammatory cytokines, including interleukin-6 (IL-6), in primary human chondrocytes. This cascade represents a feed-forward mechanism whereby cartilage matrix degradation is exacerbated by the mutually inducing effect of released collagen II fragments and pro-inflammatory cytokines. We previously proposed discoidin domain receptor 2 as a central mediator in this event. Since this cascade plays a prominent role in the pathogenesis of osteoarthritis, our study further investigates the hypothesis that discoidin domain receptor 2 is a candidate receptor for collagen II, and that transcription factor NF,B, lipid kinase PI3K, and the MAP kinases are constituent parts of this very signal transduction cascade. To accomplish this, we selectively knocked down the molecules of interest in primary human chondrocytes, induced the specified cascade by incubating primary human chondrocytes with collagen II, and observed the outcome, specifically the changes in interleukin-6 release. Knockdown was performed by siRNA-mediated gene silencing in the case of discoidin domain receptor 2 (DDR2) or by using specific inhibitors for the remainder of the molecules. Results indicated that discoidin domain receptor 2 mediates the collagen II-dependent release of interleukin-6 in primary human chondrocytes and that MAP kinases p38, JNK and ERK, as well as transcription factor NF,B, are integral components of intracellular collagen II signalling. Given the detrimental role of these molecules in osteoarthritis, our findings provide new targets for more specific therapeutics, which may have fewer side effects than those currently applied. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Matrix metalloproteinase 13 loss associated with impaired extracellular matrix remodeling disrupts chondrocyte differentiation by concerted effects on multiple regulatory factorsARTHRITIS & RHEUMATISM, Issue 8 2010Rosa Maria Borzí Objective To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. Methods MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and ,-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage. Results Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, ,-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular localization. SOX9 was largely excluded from the nuclei of chondrocytes with MMP-13,remodeled or ,degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. Conclusion MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors. [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] MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytesARTHRITIS & RHEUMATISM, Issue 5 2010Nahid Akhtar Objective Aberrant posttranscriptional regulation of matrix metalloproteinases (MMPs) by microRNA has emerged as an important factor in human diseases. The aim of this study was to determine whether the expression of MMP-13 in human osteoarthritis (OA) chondrocytes is regulated by microRNA. Methods Chondrocytes were stimulated with interleukin-1, (IL-1,) in vitro. Total RNA was prepared using TRIzol reagent. Polymerase chain reaction (PCR),based arrays were used to determine the expression profile of 352 human microRNA. Gene expression was quantified using TaqMan assays, and microRNA targets were identified using bioinformatics. Transfection with reporter construct and microRNA mimic was used to verify suppression of target messenger RNA (mRNA). Gene expression of argonaute and Dicer was determined by reverse transcription,PCR, and expression of protein was determined by immunoblotting. The role of activated MAP kinases (MAPKs) and NF-,B was evaluated using specific inhibitors. Results In IL-1,,stimulated OA chondrocytes, 42 microRNA were down-regulated, 2 microRNA were up-regulated, and the expression of 308 microRNA remained unchanged. In silico analysis identified a sequence in the 3,-untranslated region (3,-UTR) of MMP-13 mRNA complementary to the seed sequence of microRNA-27b (miR-27b). Increased expression of MMP-13 correlated with down-regulation of miR-27b. Overexpression of miR-27b suppressed the activity of a reporter construct containing the 3,-UTR of human MMP-13 mRNA and inhibited the IL-1,,induced expression of MMP-13 protein in chondrocytes. NF-,B and MAPK activation down-regulated the expression of miR-27b. Conclusion Our data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1,,induced activation of signal transduction pathways associated with the expression of MMP-13 down-regulated the expression of miR-27b. Thus, miR-27b may play a role in regulating the expression of MMP-13 in human chondrocytes. [source] SirT1 enhances survival of human osteoarthritic chondrocytes by repressing protein tyrosine phosphatase 1B and activating the insulin-like growth factor receptor pathwayARTHRITIS & RHEUMATISM, Issue 5 2010Viktoria Gagarina Objective The protein deacetylase SirT1 inhibits apoptosis in a variety of cell systems by distinct mechanisms, yet its role in chondrocyte death has not been explored. We undertook the present study to assess the role of SirT1 in the survival of osteoarthritic (OA) chondrocytes in humans. Methods SirT1, protein tyrosine phosphatase 1B (PTP1B), and PTP1B mutant expression plasmids as well as SirT1 small interfering RNA (siRNA) and PTP1B siRNA were transfected into primary human chondrocytes. Levels of apoptosis were determined using flow cytometry, and activation of components of the insulin-like growth factor receptor (IGFR)/Akt pathway was assessed using immunoblotting. OA and normal knee cartilage samples were subjected to immunohistochemical analysis. Results Expression of SirT1 in chondrocytes led to increased chondrocyte survival in either the presence or the absence of tumor necrosis factor ,/actinomycin D, while a reduction of SirT1 by siRNA led to increased chondrocyte apoptosis. Expression of SirT1 in chondrocytes led to activation of IGFR and the downstream kinases phosphatidylinositol 3-kinase, phosphoinosite-dependent protein kinase 1, mTOR, and Akt, which in turn phosphorylated MDM2, inhibited p53, and blocked apoptosis. Activation of IGFR occurs at least in part via SirT1-mediated repression of PTP1B. Expression of PTP1B in chondrocytes increased apoptosis and reduced IGFR phosphorylation, while down-regulation of PTP1B by siRNA significantly decreased apoptosis. Examination of cartilage from normal donors and OA patients revealed that PTP1B levels are elevated in OA cartilage in which SirT1 levels are decreased. Conclusion For the first time, it has been demonstrated that SirT1 is a mediator of human chondrocyte survival via down-regulation of PTP1B, a potent proapoptotic protein that is elevated in OA cartilage. [source] Proteomic analysis by two-dimensional electrophoresis to identify the normal human chondrocyte proteome stimulated by tumor necrosis factor , and interleukin-1,ARTHRITIS & RHEUMATISM, Issue 3 2010Berta Cillero-Pastor Objective To determine the intracellular proteome of normal human chondrocytes stimulated with interleukin-1, (IL-1,) and tumor necrosis factor , (TNF,) and to ascertain differences in the protein expression patterns of these 2 cytokines. Methods Normal human knee cartilage chondrocytes were incubated for 48 hours without stimulation or stimulated with IL-1, (5 ng/ml) or with TNF, (10 ng/ml). For each culture condition, protein extracts from 4 normal subjects were pooled and resolved using 2-dimensional electrophoresis. Protein spots were visualized with Sypro stain, and qualitative and quantitative analyses were performed using PDQuest software. Protein spots were then identified by mass spectrometry, using matrix-assisted laser desorption ionization,time-of-flight/time-of-flight technology. Results We identified 37 spots by mass spectrometry (MS) or by MS/MS, corresponding to 35 different proteins. In IL-1,,stimulated chondrocytes, IL-1, was found to modulate 22 proteins, as compared with unstimulated chondrocytes. All of these proteins except connective tissue growth factor (CCND2) were up-regulated. Proteins involved in cellular metabolism and energy (23%) that were up-regulated or induced by IL-1, included nicotinamide phosphoribosyltransferase, long-chain fatty acid,coenzyme A ligase 4, ,-aminolevulinic acid dehydratase, triosephosphate isomerase, and an isoform of glyceraldehyde-3-phosphate dehydrogenase. In TNF,-stimulated chondrocytes, TNF, was found to modulate 20 proteins, as compared with unstimulated chondrocytes. All of these except chitinase 3,like 1 (cartilage glycoprotein 39), proteasome activator complex subunit 2, and G3PDH, were up-regulated. Eighteen proteins were differently modulated by IL-1, and TNF,. Of these, 45% were related to metabolism. Conclusion IL-1, and TNF, induce different profiles of intracellular protein expression in healthy human chondrocytes. Most of the proteins that are differently regulated are proteins that are implicated in the generation of cellular energy and in glycolysis. [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] Calcification of articular cartilage in human osteoarthritisARTHRITIS & RHEUMATISM, Issue 9 2009M. Fuerst Objective Hypertrophic chondrocyte differentiation is a key step in endochondral ossification that produces basic calcium phosphates (BCPs). Although chondrocyte hypertrophy has been associated with osteoarthritis (OA), chondrocalcinosis has been considered an irregular event and linked mainly to calcium pyrophosphate dihydrate (CPPD) deposition. The aim of this study was to determine the prevalence and composition of calcium crystals in human OA and analyze their relationship to disease severity and markers of chondrocyte hypertrophy. Methods One hundred twenty patients with end-stage OA undergoing total knee replacement were prospectively evaluated. Cartilage calcification was studied by conventional x-ray radiography, digital-contact radiography (DCR), field-emission scanning electron microscopy (FE-SEM), and synovial fluid analysis. Cartilage calcification findings were correlated with scores of knee function as well as histologic changes and chondrocyte hypertrophy as analyzed in vitro. Results DCR revealed mineralization in all cartilage specimens. Its extent correlated significantly with the Hospital for Special Surgery knee score but not with age. FE-SEM analysis showed that BCPs, rather than CPPD, were the prominent minerals. On histologic analysis, it was observed that mineralization correlated with the expression of type X collagen, a marker of chondrocyte hypertrophy. Moreover, there was a strong correlation between the extent of mineralization in vivo and the ability of chondrocytes to produce BCPs in vitro. The induction of hypertrophy in healthy human chondrocytes resulted in a prominent mineralization of the extracellular matrix. Conclusion These results indicate that mineralization of articular cartilage by BCP is an indissociable process of OA and does not characterize a specific subset of the disease, which has important consequences in the development of therapeutic strategies for patients with OA. [source] SIRT1 regulation of apoptosis of human chondrocytesARTHRITIS & RHEUMATISM, Issue 9 2009Koji Takayama Objective SIRT1 is known to inhibit apoptosis and to promote survival of various types of cells. However, the roles of SIRT1 in apoptosis of human chondrocytes have never been reported. We undertook this study to investigate the relationship of SIRT1 to apoptosis of human chondrocytes, which is a characteristic feature of osteoarthritis (OA). Methods The expression of SIRT1 in human chondrocytes was examined by reverse transcription,polymerase chain reaction, immunoblotting, and immunohistology of human cartilage samples. The expression of SIRT1 under catabolic, mechanical, and nutritional stresses was investigated by immunoblotting. To examine the effect of SIRT1 on apoptosis, SIRT1 was inhibited by small interfering RNA (siRNA) and activated by resveratrol during nitric oxide (NO),induced apoptosis. TUNEL staining and immunoblotting of cleaved poly(ADP-ribose) polymerase (PARP) were performed to detect apoptosis. To examine the mechanisms of apoptosis, we used immunoblotting to determine the levels of cleaved caspases and mitochondria-related apoptotic signaling proteins, Bax and Bcl-2, in the mitochondrial fraction. Results SIRT1 expression was confirmed in human chondrocytes and human cartilage samples. All catabolic, mechanical, and nutritional stresses inhibited SIRT1 expression. SIRT1 inhibition by siRNA for SIRT1 increased the percentage of TUNEL-positive cells and increased the amounts of cleaved PARP and cleaved caspases 3 and 9 induced by NO. In contrast, treatment with resveratrol decreased the percentage of TUNEL-positive cells and decreased the amounts of cleaved PARP and cleaved caspases 3 and 9 induced by NO. Furthermore, in the mitochondrial fraction, SIRT1 inhibition by siRNA for SIRT1 increased the amount of Bax but reduced the amount of Bcl-2, while resveratrol reduced the amount of Bax but increased the amount of Bcl-2. Conclusion These results indicate that SIRT1 regulates apoptosis in human chondrocytes through the modulation of mitochondria-related apoptotic signals. Further research on SIRT1 might contribute to resolving the pathogenesis of OA. [source] HLA,B27,restricted antigen presentation by human chondrocytes to CD8+ T cells: Potential contribution to local immunopathologic processes in ankylosing spondylitisARTHRITIS & RHEUMATISM, Issue 6 2009Maren Kuhne Objective Analysis of the histopathologic features of hip arthritis in patients with ankylosing spondylitis (AS) has revealed accumulation of infiltrating mononuclear cells in the bone end plate and presence of hyaline articular cartilage that is not found in areas of total cartilage destruction. This study was undertaken to assess whether chondrocytes attract lymphocytes and whether cartilage chondrocytes from patients with AS have the potential to directly stimulate T cells in an HLA-restricted manner. Methods Human HLA,B27+ T cell lines, specific for the Epstein-Barr virus,derived peptide EBNA258,266, and autologous chondrocytes, serving as nonprofessional antigen-presenting cells (APCs), were available for use in a model system to study chondrocyte functions in femoral head joint cartilage of patients with AS. Peptide functionality of cytotoxic T cells was assessed by flow cytometry, and cellular interactions were detected by fluorescence confocal microscopy. Results When maintained in an alginate matrix, chondrocytes isolated from the femoral heads of patients with AS constitutively expressed type II collagen and CD80. When pulsed with the EBNA258,266 peptide, autologous chondrocytes functioned as APCs and, specifically, induced interferon-, production in CD8+ T cells. In mixed chondrocyte,T cell cultures, cell,cell contacts were dependent on the presence of the EBNA258,266 peptide. T cells adjacent to chondrocytes produced perforin and granzyme B; both molecules were found in focal aggregates, a prerequisite for antigen-specific lysis of target cells. Conclusion Antigen presentation through human chondrocytes allows the stimulation of peptide-specific CD8+ T cells. These results indicate that human chondrocytes can act as nonprofessional APCs, and suggest that there is an interferon-,,triggered autocrine loop of immune cell,mediated chondrocyte activation in the already inflamed environment. Thus, local HLA-dependent activation of peptide-specific cytotoxic CD8+ T cells by chondrocytes might contribute to inflammatory processes in the spondylarthritides. [source] Expression and modulation of ghrelin O -acyltransferase in cultured chondrocytesARTHRITIS & RHEUMATISM, Issue 6 2009Rodolfo Gómez Objective To use reverse transcription,polymerase chain reaction to detect ghrelin O -acyltransferase (GOAT) transcripts in both murine and human chondrocytes, to evaluate the effect of pharmacologic in vitro treatments with lipopolysaccharide (LPS), growth hormone, ghrelin, and dexamethasone on GOAT messenger RNA (mRNA) expression, and to study the GOAT mRNA profile during chondrocyte differentiation. Methods Murine and human GOAT and ghrelin mRNA levels were determined by the SYBR Green,based quantitative real-time polymerase chain reaction method. Results GOAT mRNA was expressed in murine cartilage explants as well as in the cultured murine chondrogenic ATDC-5 cell line. GOAT was also expressed in human immortalized chondrocyte cell lines and in human cultured primary chondrocytes. In addition, GOAT mRNA expression in differentiating ATDC-5 cells was lower at the early stage of differentiation (days 3,7), whereas GOAT mRNA levels increased progressively at the late stages. Finally, among the drugs and hormones tested, only LPS was able to strongly decrease GOAT mRNA expression. Conclusion These data indicate that chondrocytes are equipped with biochemical machinery for the synthesis of acylated ghrelin and suggest a novel role of the ghrelin axis in prehypertrophic and hypertrophic chondrocyte differentiation during endochondral ossification. [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] Increased expression of the Akt/PKB inhibitor TRB3 in osteoarthritic chondrocytes inhibits insulin-like growth factor 1,mediated cell survival and proteoglycan synthesisARTHRITIS & RHEUMATISM, Issue 2 2009John D. Cravero Objective The chondrocyte response to insulin-like growth factor 1 (IGF-1) is reduced with aging and in osteoarthritis (OA). IGF-1 signals through the phosphatidylinositol 3-kinase/Akt pathway. TRB3, a tribbles homolog, has been shown to inhibit IGF-1,mediated activation of Akt in HEK 293 cells. This study was undertaken to determine if TRB3 is expressed in chondrocytes, and whether the chondrocyte response to IGF-1 is reduced by TRB3. Methods Human articular cartilage was obtained from normal tissue donors and from patients with OA at the time of knee replacement surgery. TRB3 was assessed in the tissue samples by reverse transcription,polymerase chain reaction, immunoblotting, and immunohistochemistry. Overexpression of TRB3 was induced by transient transfection to determine the effects of TRB3 on cell survival and proteoglycan synthesis. Results TRB3 messenger RNA was detected in normal human chondrocytes. TRB3 protein levels were low in cells from normal cartilage but significantly increased in cells from OA cartilage. Incubation with 2 agents that induce endoplasmic reticulum stress, tunicamycin and thapsigargin, increased TRB3 levels in normal cells. Overexpression of TRB3 inhibited Akt phosphorylation and reduced chondrocyte survival and proteoglycan synthesis. Conclusion These results are the first to demonstrate that TRB3 is present in human chondrocytes, and that the level of TRB3 is increased in OA cartilage and in isolated OA chondrocytes. Because it is an inhibitor of Akt activation, elevated TRB3 production could play a role in the increased cell death and reduced response to IGF-1 observed in OA cartilage. [source] Inhibition of interleukin-1,,induced matrix metalloproteinases 1 and 13 production in human osteoarthritic chondrocytes by prostaglandin D2ARTHRITIS & RHEUMATISM, Issue 11 2008Nadia Zayed Objective To investigate the effects of prostaglandin D2 (PGD2) on interleukin-1, (IL-1,),induced matrix metalloproteinase 1 (MMP-1) and MMP-13 expression in human chondrocytes and the signaling pathways involved in these effects. Methods Chondrocytes were stimulated with IL-1 in the presence or absence of PGD2, and expression of MMP-1 and MMP-13 proteins was evaluated by enzyme-linked immunosorbent assay. Messenger RNA (mRNA) expression and promoter activity were analyzed by real-time reverse transcription,polymerase chain reaction and transient transfections, respectively. The role of the PGD2 receptors D prostanoid receptor 1 (DP1) and chemoattractant receptor,like molecule expressed on Th2 cells (CRTH2) was evaluated using specific agonists and antibody-blocking experiments. The contribution of the cAMP/protein kinase A (PKA) pathway was determined using cAMP-elevating agents and PKA inhibitors. Results PGD2 decreased in a dose-dependent manner IL-1,induced MMP-1 and MMP-13 protein and mRNA expression as well as their promoter activation. DP1 and CRTH2 were expressed and functional in chondrocytes. The effect of PGD2 was mimicked by BW245C, a selective agonist of DP1, but not by 13,14-dihydro-15-keto-PGD2, a selective agonist of CRTH2. Furthermore, treatment with an anti-DP1 antibody reversed the effect of PGD2, indicating that the inhibitory effect of PGD2 is mediated by DP1. The cAMP-elevating agents 8-Br-cAMP and forskolin suppressed IL-1,induced MMP-1 and MMP-13 expression, and the PKA inhibitors KT5720 and H89 reversed the inhibitory effect of PGD2, suggesting that the effect of PGD2 is mediated by the cAMP/PKA pathway. Conclusion PGD2 inhibits IL-1,induced production of MMP-1 and MMP-13 by chondrocytes through the DP1/cAMP/PKA signaling pathway. These data also suggest that modulation of PGD2 levels in the joint may have therapeutic potential in the prevention of cartilage degradation. [source] Cartilage-like gene expression in differentiated human stem cell spheroids: A comparison of bone marrow,derived and adipose tissue,derived stromal cellsARTHRITIS & RHEUMATISM, Issue 2 2003Anja Winter Objective To compare the chondrogenic potential of human bone marrow,derived mesenchymal stem cells (BMSC) and adipose tissue,derived stromal cells (ATSC), because the availability of an unlimited cell source replacing human chondrocytes could be strongly beneficial for cell therapy, tissue engineering, in vitro drug screening, and development of new therapeutic options to enhance the regenerative capacity of human cartilage. Methods Quantitative gene expression of common cartilage and cell interaction molecules was analyzed using complementary DNA array technology and reverse transcription,polymerase chain reaction during optimization of cell differentiation, in order to achieve a molecular phenotype similar to that of chondrocytes in cartilage. Results The multilineage potential of BMSC and ATSC was similar according to cell morphology and histology, but minor differences in marker gene expression occurred in diverse differentiation pathways. Although chondrogenic differentiation of BMSC and ATSC was indistinguishable in monolayer and remained partial, only BMSC responded (with improved chondrogenesis) to a shift to high-density 3-dimensional cell culture, and reached a gene expression profile highly homologous to that of osteoarthritic (OA) cartilage. Conclusion Hypertrophy of chondrocytes and high matrix-remodeling activity in differentiated BMSC spheroids and in OA cartilage may be the basis for the strong similarities in gene expression profiles between these samples. Differentiated stem cell spheroids represent an attractive tool for use in drug development and identification of drug targets in OA cartilage,like tissue outside the human body. However, optimization of differentiation protocols to achieve the phenotype of healthy chondrocytes is desired for cell therapy and tissue engineering approaches. [source] Tachykinin expression in cartilage and function in human articular chondrocyte mechanotransductionARTHRITIS & RHEUMATISM, Issue 1 2003S. J. Millward-Sadler Objective To assess whether substance P and the corresponding neurokinin 1 (NK1) receptor are expressed in human articular cartilage, and whether these molecules have a role in chondrocyte mechanotransduction. Methods Transgenic studies, immunohistochemistry, Western blotting, and reverse transcriptase,polymerase chain reaction were used to assess the expression of the preprotachykinin (PPT) gene, substance P, and NK1 in developing mice, in adult human articular cartilage, and in human chondrocytes in culture. Chondrocytes obtained from PPT knockout mice and human articular chondrocytes were mechanically stimulated in the presence or absence of inhibitors of substance P signaling, and cell membrane potentials or relative levels of aggrecan messenger RNA (mRNA) were measured. Results Replacing a region of the PPT gene transcriptional site that contains a dominant repressor of the proximal promoter activity with the constitutive minimal promoter of the human ,-globin promoter allowed expression of a marker gene in areas of chondrogenesis during mouse development and in adult chondrocytes grown in culture. Adult human articular chondrocytes expressed endogenous PPT mRNA, substance P, and the corresponding NK1 receptor in vivo and in vitro. Blockade of substance P signaling by a chemical antagonist to the NK1 receptor inhibited chondrocyte responses to mechanical stimulation. Conclusion Substance P is expressed in human articular cartilage and is involved in chondrocyte mechanotransduction via the NK1 receptor in an autocrine and paracrine manner. This suggests that substance P and the NK1 receptor have roles in the maintenance of articular cartilage structure and function that were previously unrecognized. [source] Role of novel protein kinase C isoforms in Lyme arthritisCELLULAR MICROBIOLOGY, Issue 8 2007Ok S. Shin Summary Inflammation caused by Borrelia burgdorferi infection occurs as a result of induction of pro-inflammatory cytokines from activation of multiple signalling pathways. It has previously been shown that mitogen-activated protein kinase (MAPK) and Janus kinase/signal transducer and activator of transcription signalling pathways are activated by B. burgdorferi in cultured human chondrocytes. Protein kinase C (PKC) signalling pathways are potential candidates that may control these downstream signalling pathways. Here we show that B. burgdorferi infection leads to phosphorylation and activation of novel PKC isoforms (PKC ,, ,, , and ,) in a time-dependent manner. A specific inhibitor of novel PKC isoforms blocked the induction of pro-inflammatory molecules in response to B. burgdorferi infection as did transient transfection of novel PKC dominant-negative plasmids into chondrocytes. B. burgdorferi -induced p38 MAPK phosphorylation was also significantly inhibited by an inhibitor of novel PKC isoforms, suggesting that PKC activation occurs upstream of p38 activation. In vivo, administration of an inhibitor of classical and novel PKC isoforms to C3H/HeN mice infected with B. burgdorferi resulted in significantly reduced ankle inflammation and swelling. In conclusion, these data suggest that novel PKC isoforms are specifically activated by B. burgdorferi infection and this can contribute to the regulation of inflammation in vitro and in vivo. [source] | |||||||||||||