Chondrocyte Protein Research Articles

PTEROCARPUS ANGOLENSIS CRUDE EXTRACTS INDUCE THE EXPRESSION OF COLLAGEN TYPE II IN ARTICULAR CARTILAGE IN VITRO

Background: Pterocarpus angolensis (P. angolensis) is a large deciduous tree native to Sub-Saharan Africa. This tree has
 traditionally been used to treat malaria, gonorrhoea, inflammation and wounds. The use of P. angolensis for tissue
 engineering has not been explored. This study investigated the potential of the bark and root water extracts of P. angolensis
 to induce the expression of collagen type II protein in the articular chondrocytes.
 Materials and Methods: Surface and middle zone chondrocytes, as well as explants, were stimulated with crude root and
 bark plant extracts at different concentrations. After 96 hours of stimulation, microscopic observation, XCELLigence assay
 and collagen type II ELISA, histology and immunohistochemistry assays were done.
 Results and Conclusion: Results showed no significant difference in the cell index between the controls and chondrocytes
 that had been treated with the plant extracts at concentrations of 15 and 30 μg/ml. A significant increase in the expression of
 collagen type II protein by the chondrocytes was observed and found to be optimal at a concentration of 30 μg/ml. There was
 an increase in the production of proteoglycans. However, the plant extracts at a concentration of 50 μg/ml induced apoptosis
 in the middle zone chondrocytes. These results show the potential of P. angolensis extracts in chondrocyte tissue
 regeneration. This potential could be exploited in the treatment of osteoarthritis.

An in silico axisymmetric multistage model of linear biphasic mesenchymal stem cell and chondrocyte protein secreted-matrix interactions for the reconstruction of the dynamic mechanical environment of a time varying magnetic field expanded chondrocyte biphasic finite elemental model in articular cartilage for the evaluation of the ability of natural and synthetic scaffolds in providing an appropriate environment for growth and sustain

An in silico axisymmetric multistage model of linear biphasic mesenchymal stem cell and chondrocyte protein secreted-matrix interactions for the reconstruction of the dynamic mechanical environment of a time varying magnetic field expanded chondrocyte biphasic finite elemental model in articular cartilage for the evaluation of the ability of natural and synthetic scaffolds in providing an appropriate environment for growth and sustain

Platelets promote cartilage repair and chondrocyte proliferation via ADP in a rodent model of osteoarthritis

Osteoarthritis (OA) is the most common age-related degenerative joint disease and platelet-rich plasma (PRP) has been shown to be beneficial in OA. Therefore, in this study, we aimed to investigate the effects of platelets on chondrocytes and the underlying mechanisms. Anabolic and catabolic activity and the proliferation rate of chondrocytes were evaluated after co-culture with platelets. Chondrocyte gene expression was measured by real-time PCR. Chondrocyte protein expression and phosphorylation were measured by western blot. Chondrocytes treated with or without platelets were transplanted into a rat model of OA induced by intra-articular injection of monosodium iodoacetate and the repair of articular cartilage was evaluated macroscopically and histologically. Platelets significantly promoted the proliferation of chondrocytes, while mildly influencing anabolic and catabolic activity. Chondrocytes co-cultured with platelets showed significantly increased production of bone morphogenetic protein 7 (BMP7). The autocrine/paracrine effect of BMP7 was responsible for the increased proliferation of chondrocytes, via the ERK/CDK1/cyclin B1 signaling pathway. Transplantation of platelet-treated chondrocytes showed better cartilage repair in the OA model. Platelet-derived ADP was identified as the major mediator to promote the production of BMP7 and the proliferation of chondrocytes, through the ADP receptor P2Y1. Finally, direct injection of α,β-methyleneadenosine-5′-diphosphate into OA joints also enhanced cartilage repair. This study has identified that platelet-derived ADP, but not ATP, is the key mediator for platelet-promoted chondrocyte proliferation and cartilage repair in osteoarthritis. This finding may provide a key explanation for the therapeutic effect of platelets in OA and help shaping a strategy to improve OA therapy.

Increased expression and activation of cathepsin K in human osteoarthritic cartilage and synovial tissues.

Few studies have analyzed Cathepsin K (CatK) expression in human osteoarthritic tissues. We investigated CatK expression and activation in human articular cartilage using clinical specimens. Human osteoarthritic cartilage was obtained during surgery of total hip arthroplasty (n = 10), and control cartilage was from that of femoral head replacement for femoral neck fracture (n = 10). CatB, CatK, CatL, CatS, and Cystatin C (CysC) expressions were evaluated immunohistochemically and by real-time PCR. Intracellular CatK protein was quantified by ELISA. Intracellular CatK activity was also investigated. Osteoarthritis (OA) chondrocytes were strongly stained with CatK, particularly in the superficial layer and more damaged areas. CatB, CatL, CatS, and CysC were weakly stained. CatK mRNA expression was significantly higher in OA group compared to that in control group (p = 0.043), whereas those of CatB, CatL, CatS, and CysC did not differ significantly. Mean CatK concentration (4.83 pmol/g protein) in OA chondrocytes was higher than that (3.91 pmol/g protein) in control chondrocytes (p = 0.001). CatK was enzymatically more activated in OA chondrocytes as compared with control chondrocytes. This study, for the first time, revealed increased CatK expression and activation in human OA cartilage, suggesting possible crucial roles for it in the pathogenesis of osteoarthritic change in articular cartilage.

Convergence behaviour and Control in Non-Linear Biological Networks.

Control of genetic regulatory networks is challenging to define and quantify. Previous control centrality metrics, which aim to capture the ability of individual nodes to control the system, have been found to suffer from plausibility and applicability problems. Here we present a new approach to control centrality based on network convergence behaviour, implemented as an extension of our genetic regulatory network simulation framework Jimena ( http://stefan-karl.de/jimena). We distinguish three types of network control, and show how these mathematical concepts correspond to experimentally verified node functions and signalling pathways in immunity and cell differentiation: Total control centrality quantifies the impact of node mutations and identifies potential pharmacological targets such as genes involved in oncogenesis (e.g. zinc finger protein GLI2 or bone morphogenetic proteins in chondrocytes). Dynamic control centrality describes relaying functions as observed in signalling cascades (e.g. src kinase or Jak/Stat pathways). Value control centrality measures the direct influence of the value of the node on the network (e.g. Indian hedgehog as an essential regulator of proliferation in chondrocytes). Surveying random scale-free networks and biological networks, we find that control of the network resides in few high degree driver nodes and networks can be controlled best if they are sparsely connected.

Cysteine-mediated redox regulation of cell signaling in chondrocytes stimulated with fibronectin fragments

Objective Oxidative posttranslational modifications of intracellular proteins can potentially regulate signaling pathways relevant to cartilage destruction in arthritis. In this study, oxidation of cysteine residues to form sulfenic acid (S-sulfenylation) was examined in osteoarthritic (OA) chondrocytes and investigated in normal chondrocytes as a mechanism by which fragments of fibronectin (FN-f) stimulate chondrocyte catabolic signaling. Methods Chondrocytes isolated from OA and normal human articular cartilage were analyzed using analogs of dimedone that specifically and irreversibly react with protein S-sulfenylated cysteines. Global S-sulfenylation was measured in cell lysates with and without FN-f stimulation by immunoblotting and in fixed cells by confocal microscopy. S-sulfenylation in specific proteins was identified by mass spectroscopy and confirmed by immunoblotting. Src activity was measured in live cells using a fluorescence resonance energy transfer biosensor. Results Proteins in chondrocytes isolated from OA cartilage were found to have elevated basal levels of S-sulfenylation relative to those of chondrocytes from normal cartilage. Treatment of normal chondrocytes with FN-f induced increased levels of S-sulfenylation in multiple proteins, including the tyrosine kinase Src. FN-f treatment also increased the levels of Src activity. Pretreatment with dimedone to alter S-sulfenylation function or with Src kinase inhibitors inhibited FN-f–induced production of matrix metalloproteinase 13. Conclusion These results demonstrate for the first time the presence of oxidative posttranslational modification of proteins in human articular chondrocytes by S-sulfenylation. Due to the ability to regulate the activity of a number of cell signaling pathways, including catabolic mediators induced by fibronectin fragments, S-sulfenylation may contribute to cartilage destruction in OA and warrants further investigation.

The DOT1L protein and gene network in chondrocytes identifies H3K79 histone methylation as a key regulator of WNT and other growth factor cascades

Purpose: DOT1L is the only known H3K79 histone methyltransferase and has been linked to epigenetic regulation of the Wingless-like (Wnt) signaling pathway. Genome-wide association and functional studies identified the DOT1L gene to be associated with cartilage thickness and hip osteoarthritis (OA) and showed an interaction of DOT1L with canonical Wnt signaling. Here, we further investigated the biology of DOT1L in cartilage health and disease. Specifically, our objective was to define the transcriptional complexes and transcriptome associated with DOT1L in articular cartilage, and to link these DOT1L targets and interactors with the molecular characteristics of the articular chondrocyte during the loss of phenotype in a dedifferentiation setup. Finally, we aimed for a translational validation of our data in cartilage samples from OA patients. Methods: Human articular chondrocytes (hACs) stimulated or not with LiCl were used to carefully map the presence or absence of DOT1L protein complexes suggested earlier in leukemia cells, using a series of specific immunoprecipitation experiments. To elucidate the transcriptional network of Dot1l, we performed a microarray of hACs from 5 non-OA fracture patients treated with a specific DOT1L inhibitor (EPZ5676) or vehicle control. The differentially expressed genes were explored with the limma package. String-DB and Panther were used for gene network analyses. Based on the microarray analysis, selected genes were confirmed by quantitative PCR. To establish the role of DOT1L in the maintenance of hACs phenotype, hACs were cultured in monolayer in the presence or absence of EPZ5676, and RNA and protein were isolated at serial passages (P0 to P5). We analyzed by quantitative PCR the gene expression of known genes important for chondrocyte biology and/or genes that appeared in the microarray of DOT1L inhibition. Activation of the Wnt canonical and non-canonical signaling cascades was analyzed by Western blot. For translational validation we analyzed the differential gene expression profile of damaged as compared to intact cartilage areas within the same joint of patients with hip OA. Results: The presence of different DOT1L elongation complexes was confirmed in hACs. The CoIP experiments revealed that DOT1L interacts directly with ENL, AF10 and active beta-catenin (more strongly when cells were treated with LiCl to stabilize beta-catenin) and with AF4 and AF5 (only with cells were treated with LiCl). The formation of these complexes was disrupted when DOT1L was inhibited. In the microarray, 1937 genes significantly changed (p<0.05) in DOT1L inhibited samples compared to vehicle treated samples (1048 genes were down-regulated, 889 up-regulated). In EPZ5676 treated samples, chondrocyte differentiation-associated genes, such as ACAN, RGS5, GDF10 and LOXL2 were down-regulated; OA-related genes, such as MMP1, CCL7, CCL8 and GPNMB were up-regulated; and WNT target genes, ligands and antagonists such as LEF1, WNT5A and DKK1 were significantly up-regulated. In the de-differentiation experiment, in vitro expanded hACs showed progressive changes in gene expression. Genes involved in relevant pathways for cartilage biology such as the WNT, NOTCH and BMP pathways exhibited significant changes that were highly accentuated by DOT1L inhibition. We successfully validated these targets of interest in samples from patients with OA condition. Conclusions: Our transcriptomic, protein and gene interaction approach provides novel insights into the DOT1L molecular network and its putative role in osteoarthritis and cartilage. These data further support an important role for DOT1L in joint homeostasis as a key regulator of WNT signaling and other growth factor cascades in the joint.

Increased classical endoplasmic reticulum stress is sufficient to reduce chondrocyte proliferation rate in the growth plate and decrease bone growth.

Mutations in genes encoding cartilage oligomeric matrix protein and matrilin-3 cause a spectrum of chondrodysplasias called multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). The majority of these diseases feature classical endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) as a result of misfolding of the mutant protein. However, the importance and the pathological contribution of ER stress in the disease pathogenesis are unknown. The aim of this study was to investigate the generic role of ER stress and the UPR in the pathogenesis of these diseases. A transgenic mouse line (ColIITgcog) was generated using the collagen II promoter to drive expression of an ER stress-inducing protein (Tgcog) in chondrocytes. The skeletal and histological phenotypes of these ColIITgcog mice were characterised. The expression and intracellular retention of Tgcog induced ER stress and activated the UPR as characterised by increased BiP expression, phosphorylation of eIF2α and spliced Xbp1. ColIITgcog mice exhibited decreased long bone growth and decreased chondrocyte proliferation rate. However, there was no disruption of chondrocyte morphology or growth plate architecture and perturbations in apoptosis were not apparent. Our data demonstrate that the targeted induction of ER stress in chondrocytes was sufficient to reduce the rate of bone growth, a key clinical feature associated with MED and PSACH, in the absence of any growth plate dysplasia. This study establishes that classical ER stress is a pathogenic factor that contributes to the disease mechanism of MED and PSACH. However, not all the pathological features of MED and PSACH were recapitulated, suggesting that a combination of intra- and extra-cellular factors are likely to be responsible for the disease pathology as a whole.

The effects of static pressure on chondrogenic and osteogenic differentiation in condylar chondrocytes from temporomandibular joint

ObjectiveThe goal of the study was to investigate the production of collagen, type II, alpha 1 (COL2A1), SOX9, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), Indian hedgehog (Ihh), and periarticular cell-derived parathyroid hormone-related protein (PTHrP) in mandibular condylar chondrocytes under static pressure stimuli. MethodsMandibular condylar chondrocytes separated from rabbit temporomandibular joint (TMJ) were treated with a static pressure of 100kPa for 0, 1, 2, 3, and 4h by an in-house-designed pressure chamber. A CCK-8 kit was used to analyze the cell viability. The production of COL2A1, SOX9, ALP, Runx2, Ihh, and PTHrP was detected by Western blot or real-time polymerase chain reaction (PCR). Changes in cell morphology were observed by scanning electron microscopy. ResultsCompared with the control group (0h), the cytoplasmic processes of treated chondrocytes obviously increased and elongated, and the cell viability of pressurized chondrocytes were 91.13% (1h), 103.41% (2h), 103.47% (3h), and 104.94% (4h), respectively. The exposure of condylar chondrocytes to a static pressure of 100kPa for 3–4h resulted in a significant increase in COL2A1, SOX9, ALP, and Runx2. After a static pressure loading of 100kPa, the activation of Ihh and PTHrP was also observed. ConclusionsMandibular condylar chondrocytes adapt to alterations of the microenvironment. Ihh and PTHrP are sensitive to static pressure. Our findings suggest that static pressure accelerated the chondrogenic and osteogenic differentiation of condylar chondrocytes, which may influence the pathological progress of temporomandibular diseases.

Effects of tofacitinib on nucleic acid metabolism in human articular chondrocytes

Objective. In our previous screening of chondrocyte protein profiles, the amount of adenosine monophosphate deaminase (AMPD) 2 was found to be decreased by tofacitinib. Extending the study, here we confirmed the decrease of AMPD2 by tofacitinib and further investigated effects of tofacitinib on purine nucleotide metabolism.Methods. Human articular chondrocytes and a chondrosarcoma cell line: OUMS-27 were stimulated with tofacitinib. Then the levels of AMPD2 and its related enzymes were investigated by Western blot. The levels of AMP and adenosine were assessed by mass spectrometry.Results. We confirmed the significant decrease of AMPD2 by tofacitinib in chondrocytes (p = 0.025). The levels of adenosine kinase and 5’-nucleotidase were decreased in chondrocytes, although they did not meet statistical significance (p = 0.067 and p = 0.074, respectively). The results from OUMS-27 were similar to those from the chondrocytes. The cellular adenosine levels were significantly decreased by tofacitinib in OUMS-27 (p = 0.014). The cellular AMP levels were increased, although they did not meet statistical significance in OUMS-27 (p = 0.066).Conclusion. Our data indicate that tofacitinib increases the cellular levels of adenosine, which is known to have anti-inflammatory activity, through the downregulation of AMPD2. This would be a novel functional aspect of tofacitinib.

Current status of temporomandibular joint disorders and the therapeutic system derived from a series of biomechanical, histological, and biochemical studies

This article was designed to report the current status of temporomandibular joint disorders (TMDs) and the therapeutic system on the basis of a series of clinical, biomechanical, histological and biochemical studies in our research groups. In particular, we have focused on the association of degenerative changes of articular cartilage in the mandibular condyle and the resultant progressive condylar resorption with mechanical stimuli acting on the condyle during the stomatognathic function. In a clinical aspect, the nature and prevalence of TMDs, association of malocclusion with TMDs, association of condylar position with TMDs, association of craniofacial morphology with TMDs, and influences of TMDs, TMJ-osteoarthritis (TMJ-OA) in particular, were examined. In a biomechanical aspect, the nature of stress distribution in the TMJ from maximum clenching was analyzed with finite element method. In addition, the pattern of stress distribution was examined in association with varying vertical discrepancies of the craniofacial skeleton and friction between the articular disk and condyle. The results demonstrated an induction of large compressive stresses in the anterior and lateral areas on the condyle by the maximum clenching and the subsequent prominent increases in the same areas of the mandibular condyle as the vertical skeletal discrepancy became more prominent. Increase of friction at the articular surface was also indicated as a cause of larger stresses and the relevant disk displacement, which further induced an increase in stresses in the tissues posterior to the disks, indicating an important role of TMJ disks as a stress absorber. In a histological or biological aspect, increase in TMJ loading simulated by vertical skeletal discrepancy, which has already been revealed by the preceding finite element analysis or represented by excessive mouth opening, produced a decrease in the thickness of cartilage layers, an increase in the numbers of chondroblasts and osteoclasts and the subsequent degenerative changes in the condylar cartilage associated with the expression of bone resorption-related factors. In a biochemical or molecular and cellular aspect, excessive mechanical stimuli, irrespective of compressive or tensile stress, induced HA fragmentation, expression of proinflammatory cytokines, an imbalance between matrix metalloproteinases and the tissue inhibitors, all of which are assumed to induce lower resistance to external stimuli and degenerative changes leading to bone and cartilage resorption. Excessive mechanical stimuli also reduced the synthesis of superficial zone protein in chondrocytes, which exerts an important role in the protection of cartilage and bone layers from the degenerative changes. It is also revealed that various cytoskeletal changes induced by mechanical stimuli are transmitted through a stretch-activated or Ca2+channel. Finally, on the basis of the results from a series of studies, it is demonstrated that optimal intra-articular environment can be achieved by splint therapy, if indicated, followed by occlusal reconstruction with orthodontic approach in patients with myalgia of the masticatory muscles, and TMJ internal derangement or anterior disk displacement with or without reduction. It is thus shown that orthodontic treatment is available for the treatment of TMDs and the long-term stability after treatment.

CRISPR/Cas9 mediated generation of stable chondrocyte cell lines with targeted gene knockouts; analysis of an aggrecan knockout cell line

The Swarm rat chondrosarcoma (RCS) cell lines derived from a spontaneous neoplasm in a rat spine several decades ago have provided excellent models of chondrosarcoma tumor development. In addition the robust chondrocyte phenotype (expression of a large panel of genes identical to that seen in normal rat cartilage) and the ability to generate cell clones have facilitated their extensive use in the identification of chondrocyte proteins and genes. The clustered regularly interspersed short palindromic repeat (CRISPR) technology employing the RNA-guided nuclease Cas9 has rapidly dominated the genome engineering field as a unique and powerful gene editing tool. We have generated a stable RCS cell line (RCS Cas9) expressing the nuclease Cas9 that enables the editing of any target gene or non-coding RNA by simple transfection with a guide RNA. As proof of principle, stable cell lines with targeted ablation of aggrecan expression (Acan KO) were generated and characterized. The studies show that stable chondrocyte cell lines with targeted genome editing can be quickly generated from RCS Cas9 cells using this system. The Acan KO cell lines also provided a tool for characterizing the response of chondrocytes to aggrecan loss and the role of aggrecan in chondrosarcoma development. Loss of aggrecan expression while not affecting the chondrocyte phenotype resulted in a much firmer attachment of cells to their substrate in culture. Large changes in the expression of several genes were observed in response to the absence of the proteoglycan matrix, including those for several small leucine rich proteoglycans (SLRPs), transcription factors and membrane transporters. Acan KO cells failed to form a substantial chondrosarcoma when injected subcutaneously in nude mice consistent with previous suggestions that the glycosaminoglycan-rich matrix surrounding the chondrosarcoma protects it from destruction by the host immune system. The studies provide new understanding of aggrecan function and the RCS Cas9 cell line is expected to provide a very valuable tool for the study gene function in chondrocytes.

Down-regulation of adiponectin receptors in osteoarthritic chondrocytes.

The objective of this study was to investigate the expression of adiponectin receptors (AdipoR1, R2, and T-cadherin) in both normal subjects and patients with knee osteoarthritis (OA). We used immunofluorescence to assess expression of adiponectin receptors in the chondrocytes of normal subjects (n = 3) and OA patients (n = 3). We also studied mRNA expression of adiponectin receptors in both groups by real-time polymerase chain reaction (real-time PCR). Finally, we utilized Western blotting to confirm the presence of adiponectin receptors. As compared with osteoarthritic chondrocytes, normal chondrocytes showed stronger immunoreactivity for AdipoR1, AdipoR2, and T-cadherin. The expression levels of both AdipoR1 and AdipoR2 mRNA were significantly lower in the osteoarthritic chondrocytes compared with those in the normal chondrocytes, 19 ± 2 and 36 ± 3 % of normal chondrocytes, respectively (P < 0.001). T-cadherin mRNA expression levels of the osteoarthritic chondrocytes were also lower than those in the normal chondrocytes, but not statistical significant (P = 0.072). The expression levels of AdipoR1 and AdipoR2 protein were significantly higher in the normal chondrocytes compared with those in the osteoarthritic chondrocytes (P < 0.001, P < 0.01, respectively). T-cadherin protein expression level of the normal chondrocytes was also higher than those in the osteoarthritic chondrocytes, but the difference is not statistical significant (P = 0.114). Expression of adiponectin receptors protein in normal and osteoarthritic chondrocytes is consistent with its mRNA expression levels. In conclusion, we report for the first time down-regulation of adiponectin receptors (AdipoR1, R2, and T-cadherin) in osteoarthritic chondrocytes. Decreased adiponectin receptors in OA may reduce the tissue sensitivity to adiponectin, thus lost the protection from adiponectin in the progression of OA.

Proteomic analysis of hyaluronate‐regulated proteins involved in chondroprotective effect under oxidative stress (LB177)

Osteoarthritis (OA), the most common type of arthritis, is a degenerative joint disease. Oxidative stress is well known to play important roles in cartilage degradation and pathogenesis of OA. The intra‐articular injection of hyaluronic acid (IAHA) is accepted as an effective clinical therapy for OA, but we do not yet fully understand the mechanisms underlying the effects of HA on OA chondrocytes under oxidative stress. Here, we show for the first time that IAHA significantly reduces the synovial fluid levels of hydrogen peroxide (H2O2) and superoxide (O2−) in patients with knee OA. We also demonstrate that HA suppresses H2O2‐induced cell death in human OA chondrocytes. Proteomic approaches (2‐DE combined with mass spectrometry) allowed us to identify 13 protein spots corresponding to 12 non‐redundant proteins as HA‐regulated proteins in OA chondrocytes under oxidative stress. The expression levels of three putative HA‐regulated proteins (TALDO, ANXA1 and EF2) in control, H2O2‐, HA‐ and HA/H2O2‐treated OA chondrocytes were verified by Western blotting and the results indeed support the notion that HA acts in anti‐oxidation, anti‐apoptosis, and the promotion of cell survival. Our results collectively demonstrate the utility of proteomic approaches and provide new insights into the chondroprotective effects of HA on OA.Grant Funding Source: Supported by National Science Council, R.O.C.

The effects of low intensity pulsed ultrasound on expression of mechanochemical transduction pathway-related protein of integrin-focal adhesion kinase-mitogen-activated protein kinases in the chondrocytes of rabbits with knee osteoarthritis

Objective To observe any effect of low intensity pulsed ultrasound (LIPUS) on the expression of integrin-focal adhesion kinase (FAK)-mitogen-activated protein kinase (MAPK) mechanochemical transduction pathway-related proteins in the chondrocytes of rabbits with knee osteoarthritis (OA).Methods Of the 18 New Zealand white rabbits selected for the study,twelve received knee anterior cruciate ligament transection to model OA.The remaining 6 rabbits served as normal controls.At the 4th week after modeling the rabbits were sacrificed and chondrocytes were isolated and cultured in vitro.All the cultured cells were randomly divided into three groups:a normal control group (NC),an OA model group (OA) and an OA model plus LIPUS group (OA + LIPUS).When the cells had been cultured to the 2nd passage,the NC group and OA group cells had received no treatment.The OA + LIPUS group cells were exposed to 40 mW/cm2 of LIPUS for 20 min,once a day for 6 days.The expression of collagen protein type Ⅱ,aggrecan,MMP-13,integrin β1 p-FAK and p-p38,p-ERK1/2 and p-JNK Mapks were detected by Western blotting.Results Compared with the NC group,the expression of collagen type Ⅱ and aggrecan was significantly lower in the OA and OA + LIPUS groups,with more significantly lower expression of collagen type Ⅱ and aggrecan in the OA group than in the OA + LIPUS group.Compared with the NC group,the expression of MMP-13 was significantly higher in the OA and OA + LIPUS groups,with a significantly larger increase in the OA group.Compared with the NC group,the expression of integrin β1 and p-FAK was also significantly higher in the OA and OA + LIPUS groups,with a significantly larger increase in the OA + LIPUS group.Compared with the NC group,the expression of p-p38,p-ERK1/2 and p-JNK was also significantly higher in the OA group,but compared with the OA group,the expression of those kinases was,on average,significantly lower in the OA + LIPUS group.Conclusions LIPUS can inhibit the degradation of collagen type Ⅱ and aggrecan,and inhibit the expression of MMP-13,p-p38,p-ERK1/2 and p-JNK in OA chondrocytes,at least in vitro.At the same time,LIPUS can increase the expression of integrin β1 and p-FAK.The results show that LIPUS may activate an integrin-FAK-MAPK mechanochemical transduction pathway to induce changes in the extracellular matrix of chondrocytes. Key words: Ultrasound ; Chondrocytes; Integrin; Focal adhesion kinase ; Mitogen-activated protein kinases

The procatabolic cytokine interleukin-1 alfa induces a dysregulation of o-linked n-acetylglucosamine modified proteins in human osteoarthritic chondrocytes

Purpose: Nuclear and cytoplasmic protein glycosylation by the addition of O-linked N-Acetylglucosamine (O-GlcNAc) to serine and threonine residues is a widespread post-translational modification that has been described in degenerative and age-related diseases, such as Alzheimer´s and diabetes. This modification is catalyzed by the O-GlcNAc transferase (OGT) which uses UDP-GlcNAc as a donor, and O-GlcNAc can be removed by the O-GlcNAc-glucosaminidase (OGA). We have seen that O-GlcNAc glycosylation levels in the articular cartilage of patients with OA were increased as well as the expression of the different isoforms of OGT and OGA was dysregulated. The goal of this study was to assess if a procatabolic cytokine, IL-1α, can be responsible of the changes in O-GlcNAc system found in OA human cartilage. Methods: OA knee articular cartilage was obtained during replacement surgery. Twelve OA patients were included in this study (6 woman/6 men; mean age 71±3 years), with a Mankin score for knee specimens between 11 and 14. Human osteoarthritic chondrocytes (HOC) were isolated by protease digestion for primary culture. At confluence, cells were stimulated with different concentrations of IL-1α for different doses and periods of time after they were deprived of serum for 48 h. The level of O-GlcNAcylation, OGT and O-GlcNAcase expression was assessed by western-blot employing specific antibodies. Results: The amount of O-GlcNAcylated proteins was increased in HOC 24 h after stimulation with different doses of IL-1α (1, 10 and 100 ng/ml), showing the highest accumulation with 10 ng/ml. This concentration was chosen for stimulation of HOC during different periods of time (3, 6, and 24 h). The increase in O-GlcNAc proteins amount showed a peak of induction at 6 h. This accumulation occurs especially in proteins between 50 to 150 KDa therefore showing a similar O-GlcNAcylated proteins pattern to that observed in OA cartilage. We also observed an increased expression of the three OGT isoforms, nucleo-cytoplasmic (ncOGT), mitochondrial (mOGT) and small (sOGT) in HOC as early as 3 h, although the peak of induction was at 6 h. Three isoforms were increased in a similar grade, without prevalence of sOGT as found in OA cartilage. IL-1α also increased the presence of both OGA isoforms, long (OGA-L) and short (OGA-S) after 3 and 6 h of stimuli. A clear prevalence of long isoform was found along IL-1α stimulation whereas OGA-S had the highest prevalence in OA cartilage. Conclusions: To our knowledge, this is first evidence of procatabolic cytokines involvement in O-GlcNAc modifications. IL-1α can induce accumulation of O-GlcNAc proteins in human OA chondrocytes. Although both regulatory enzymes of this modification, OGT and OGA, are also increased with IL-1α stimulation, characteristic distribution of the different isoforms in OA cartilage was not reached. IL-1α is in part responsible of a dysregulation in the O-GlcNAc proteins modification during OA although other processes must be involved.

Brazilin blocks the induction of NFKB1/P50 in human chondrocytes and synoviocytes

Purpose: Brazilin is one major constituent of Caesalpinia sappan and is known to possess anti-bacterial, anti-oxidative, and anti-inflammatory activities. Osteoarthritis (OA) creates a disequilibrium between the catabolic and anabolic activities of chondrocytes in remodeling the cartilage extracellular matrix (ECM). Pro-inflammatory signaling contributes to the degradation of the cartilage matrix. Under OA conditions, synoviocytes produce a range of inflammatory mediators, including IL-1β. Upon binding to chondrocytes, these mediators activate specific signal transduction cascades, such as the nuclear factor-kappa-B (NF-κB) pathway, which acts as one central regulator of the catabolic processes that suppresses the synthesis of ECM components, and induces the expression of matrix metalloproteinases and enzymes of the ADAMTS family, leading to perpetuated cartilage breakdown. This study aimed to evaluate the effects of brazilin in osteoarthritic chondrocytes and synoviocytes with particular focus on the NF-κB pathway. Methods: Brazilin was isolated from Caesalpinia sappan extract (CSE) and identified using HPLC and NMR methods. Chondrocytes and synoviocytes were isolated from OA patients undergoing total knee replacement surgery. Cells were either left untreated for control samples, were treated with 10 ng/ml IL-1β, or were pre-treated with 10 μg/ml brazilin prior to the addition of 10 ng/ml IL-1β. Genes associated with NF-κB-mediated signal transduction were examined in primary human chondrocytes using an NF-κB signaling array and in silico pathway analysis. The induction and inhibition of NFKB1, its gene product p50, and the precursor p105 were investigated in primary human chondrocytes and synoviocytes using RT-qPCR and Western blotting. Results: Nine NF-κB pathway-related genes (BIRC3, CCL2, CSF1, CSF3, IL1B, IL8, NFKB1, NFKB2 and TNFAIP3) were found to be significantly up-regulated by IL-1β-stimulation and significantly down-regulated after pre-incubation with brazilin. Pathway analysis revealed NFKB1 as one major gene regulating the anti-inflammatory activities of brazilin. RT-qPCR assays confirmed that the up-regulation of NFKB1 mRNA in IL-1β-stimulated primary chondrocytes and synoviocytes was significantly reduced by pre-treatment with brazilin. Western blotting showed that IL-1β treatment increased p105 protein in chondrocytes with a peak after 1 h followed by a steady decline, while increasing constantly the amount of p50 over 24 h. Brazilin suppressed the IL-1β-mediated induction of NFKB1/p50 in chondrocytes and synoviocytes. Conclusions: The present study suggests that brazilin effectively blocks the induction of NFKB1/p50 in cytokine-stimulated primary human chondrocytes and synoviocytes, pointing towards a chondroprotective potential of brazilin, which may be beneficial for reducing cartilage breakdown in OA.

Network medicine analysis of chondrocyte proteins towards new treatments of osteoarthritis

Osteoarthritis (OA) is a progressive disorder with high incidence in the ageing human population that still has no treatment currently. This disorder induces the breakdown of articular cartilage, leading to the exposure and damage of bone surfaces. For a global understanding of OA development, the systematic integration of known OA-related proteins with protein-protein interaction (PPI) networks is required. In this work, the OA-related interactome was reconstructed using multiple data sources to have the most up-to-date information on OA-related proteins and their interactions. We then combined emergent concepts in network medicine to detect new unclassified OA-related proteins. The mapping of known OA-related proteins with PPI networks showed that these proteins are locally connected to each other and agglomerated in a large component. To expand this module, we applied a diffusion-based algorithm that probabilistically induces more searches in the vicinity of the seed OA-related proteins. As a result, the 10 topmost ranked proteins were connected to the OA disease module, supporting the local hypothesis. We computed structural modules and selected those that had the highest enrichment of OA-related proteins. The identified molecules show a link between structural topology and disease dysfunctionality. Interestingly, the protein Q6EEV6 was highlighted for OA association by both methods, reinforcing the potential involvement of this protein. These results suggest that similar disease-connected modules may exist in different human disorders, which could lead to systematic identification of genes or proteins that have a joint role in specific disease phenotypes.

Low‐dose γ‐radiation inhibits IL‐1β‐induced dedifferentiation and inflammation of articular chondrocytes via blockage of catenin signaling

Although low-dose radiation (LDR) regulates a wide range of biological processes, limited information is available on the effects of LDR on the chondrocyte phenotype. Here, we found that LDR, at doses of 0.5–2 centiGray (cGy), inhibited interleukin (IL)-1β-induced chondrocyte destruction without causing side effects, such as cell death and senescence. IL-1β treatment induced an increase in the expression of α-, β-, and γ-catenin proteins in chondrocytes via Akt signaling, thereby promoting dedifferentiation through catenin-dependent suppression of Sox-9 transcription factor expression and induction of inflammation through activation of the NF-κB pathway. Notably, LDR blocked cartilage disorders by inhibiting IL-1β-induced catenin signaling and subsequent catenin-dependent suppression of the Sox-9 pathway and activation of the NF-κB pathway, without directly altering catenin expression. LDR also inhibited chondrocyte destruction through the catenin pathway induced by epidermal growth factor, phorbol 12-myristate 13-acetate, and retinoic acid. Collectively, these results identify the molecular mechanisms by which LDR suppresses pathophysiological processes and establish LDR as a potentially valuable therapeutic tool for patients with cytokine- or soluble factors-mediated cartilage disorders.

Proteomic analysis of osteoarthritic chondrocyte reveals the hyaluronic acid-regulated proteins involved in chondroprotective effect under oxidative stress

Osteoarthritis (OA), the most common type of arthritis, is a degenerative joint disease. Oxidative stress is well known to play important roles in cartilage degradation and pathogenesis of OA. The intra-articular injection of hyaluronic acid (IAHA) is accepted as an effective clinical therapy for OA, but we do not yet fully understand the mechanisms underlying the effects of HA on OA chondrocytes under oxidative stress. Here, we show for the first time that IAHA significantly reduces the synovial fluid levels of hydrogen peroxide (H2O2) and superoxide (O2(-)) in patients with knee OA. We also demonstrate that HA suppresses H2O2-induced cell death in human OA chondrocytes. Proteomic approaches (2-DE combined with mass spectrometry) allowed us to identify 13 protein spots corresponding to 12 non-redundant proteins as HA-regulated proteins in OA chondrocytes under oxidative stress. The expression levels of three putative HA-regulated proteins (TALDO, ANXA1 and EF2) in control, H2O2-, HA- and HA/H2O2-treated OA chondrocytes were verified by Western blotting and the results indeed support the notion that HA acts in anti-oxidation, anti-apoptosis, and the promotion of cell survival. Our results collectively demonstrate the utility of proteomic approaches and provide new insights into the chondroprotective effects of HA on OA. In the present study, we show for the first time that IAHA reduces the levels of H2O2 and O2(-) in synovial fluids from OA patients. We used primary cultured human OA chondrocytes as a model, treated cells with H2O2 to partly mimic their physiological conditions under oxidative stress, and examined the protection effects of HA. The proteomic approach allowed us to identify candidate proteins regulated by H2O2 and/or HA in OA chondrocytes. We found that proteins functioning in stress responses, apoptosis and protein synthesis were consistently regulated by HA in chondrocytes under oxidative stress. These novel results contribute to our understanding of the molecular mechanisms underlying HA-mediated chondroprotection.