CCN4 induces IL-6 production through αvβ5 receptor,PI3K,Akt, and NF-κB singling pathway in human synovial fibroblasts

Introduction

Osteoarthritis (OA) is the most common degenerative joint disease that is involved in the degradation of articular cartilage. The exact etiology of OA is not completely understood. CCN4 is related to up-regulation in the cartilage of patients with osteoarthritis. Previous studies have shown that CCN4 might be associated with the pathogenesis of OA, but the exact signaling pathways in CCN4-mediated IL-6 expression in synovial fibroblasts (SF) are largely unknown. Therefore, we explored the intracellular signaling pathway involved in CCN4-induced IL-6 production in human synovial fibroblast cells.

Methods

CCN4-induced IL-6 production was assessed with quantitative real-time qPCR and ELISA. The mechanisms of action of CCN4 in different signaling pathways were studied by using Western blotting. Neutralizing antibodies of integrin were used to block the integrin signaling pathway. Luciferase assays were used to study IL-6 and NF-κB promoter activity. Immunocytochemistry was used to examine the translocation activity of p65.

Results

Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCN4 and the expression was higher than in normal SFs. OASF stimulation with CCN4 induced concentration- and time-dependent increases in IL-6 production. Pretreatment of OASFs with αvβ5 but not α5β1 and αvβ3 integrin antibodies reduced CCN4-induced IL-6 production. CCN4-mediated IL-6 production was attenuated by PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and Wortmannin), Akt inhibitor (Akti), and NF-κB inhibitor (PDTC and TPCK). Stimulation of cells with CCN4 also increased PI3K, Akt, and NF-κB activation.

Conclusions

Our results suggest that CCN4 activates αvβ5 integrin, PI3K, Akt, and NF-κB pathways, leading to up-regulation of IL-6 production. According to our results, CCN4 may be an appropriate target for drug intervention in OA in the future.     

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

Objective

Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.

Methods

For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.

Results

In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage.

Conclusion

Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.     

Genes Involved in the Osteoarthritis Process Identified through Genome Wide Expression Analysis in Articular Cartilage; the RAAK Study

Objective

Identify gene expression profiles associated with OA processes in articular cartilage and determine pathways changing during the disease process.

Methods

Genome wide gene expression was determined in paired samples of OA affected and preserved cartilage of the same joint using microarray analysis for 33 patients of the RAAK study. Results were replicated in independent samples by RT-qPCR and immunohistochemistry. Profiles were analyzed with the online analysis tools DAVID and STRING to identify enrichment for specific pathways and protein-protein interactions.

Results

Among the 1717 genes that were significantly differently expressed between OA affected and preserved cartilage we found significant enrichment for genes involved in skeletal development (e.g. TNFRSF11B and FRZB). Also several inflammatory genes such as CD55, PTGES and TNFAIP6, previously identified in within-joint analyses as well as in analyses comparing preserved cartilage from OA affected joints versus healthy cartilage were among the top genes. Of note was the high up-regulation of NGF in OA cartilage. RT-qPCR confirmed differential expression for 18 out of 19 genes with expression changes of 2-fold or higher, and immunohistochemistry of selected genes showed a concordant change in protein expression. Most of these changes associated with OA severity (Mankin score) but were independent of joint-site or sex.

Conclusion

We provide further insights into the ongoing OA pathophysiological processes in cartilage, in particular into differences in macroscopically intact cartilage compared to OA affected cartilage, which seem relatively consistent and independent of sex or joint. We advocate that development of treatment could benefit by focusing on these similarities in gene expression changes and/or pathways.     

Lubricin is expressed in chondrocytes derived from osteoarthritic cartilage encapsulated in poly (ethylene glycol) diacrylate scaffold

Osteoarthritis (OA) is characterized by degenerative changes within joints that involved quantitative and/or qualitative alterations of cartilage and synovial fluid lubricin, a mucinous glycoprotein secreted by synovial fibroblasts and chondrocytes. Modern therapeutic methods, including tissue-engineering techniques, have been used to treat mechanical damage of the articular cartilage but to date there is no specific and effective treatment. This study aimed at investigating lubricin immunohistochemical expression in cartilage explant from normal and OA patients and in cartilage constructions formed by Poly (ethylene glycol) (PEG) based hydrogels (PEG-DA) encapsulated OA chondrocytes. The expression levels of lubricin were studied by immunohistochemistry: i) in tissue explanted from OA and normal human cartilage; ii) in chondrocytes encapsulated in hydrogel PEGDA from OA and normal human cartilage. Moreover, immunocytochemical and western blot analysis were performed in monolayer cells from OA and normal cartilage. The results showed an increased expression of lubricin in explanted tissue and in monolayer cells from normal cartilage, and a decreased expression of lubricin in OA cartilage. The chondrocytes from OA cartilage after 5 weeks of culture in hydrogels (PEGDA) showed an increased expression of lubricin compared with the control cartilage. The present study demonstrated that OA chondrocytes encapsulated in PEGDA, grown in the scaffold and were able to restore lubricin biosynthesis. Thus our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repairing cartilage lesions in patients with OA to reduce at least the progression of the disease.     

Lubricin: a novel potential biotherapeutic approaches for the treatment of osteoarthritis

Osteoarthritis (OA) is a multi-factor disorder of sinovial joints, which characterized by escalated degeneration and loss of articular cartilage. Treatment of OA is a critical unmet need in medicine for regeneration of damaged articular cartilage in elderly. On the other hand, lubricin, a glycoprotein specifically synthesized by chondrocytes located at the surface of articular cartilage, has been shown to provide boundary lubrication of congruent articular surfaces under conditions of high contact pressure and near zero sliding speed. Lubrication of these surfaces is critical to normal joint function, while different gene expressions of lubricin had been found in the synovium of rheumatoid arthritis (RA) and OA. Moreover, mutations or lacking of lubricin gene have been shown to link to the joint disease such as camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP), synovial hyperplasia and failure of joint function, suggesting an important role of lubricin in the pathogenesis of these joint disease. Recent studies demonstrate that administration with recombinant lubricin in the joint cavity would be effective in the prevention of cartilage degeneration in animal OA models. Therefore, a treatment with lubricin which would protect cartilage in vivo would be desirable. This article reviews recent findings with regard to the possible role of lubricin in the progression of OA, and further discusses lubricin as a novel potential biotherapeutic approaches for the treatment of OA.     

MicroRNA-488 regulates zinc transporter SLC39A8/ZIP8 during pathogenesis of osteoarthritis

Background

Even though osteoarthritis (OA) is the most common musculoskeletal dysfunction, there are no effective pharmacological treatments to treat OA due to lack of understanding in OA pathology. To better understand the mechanism in OA pathogenesis and investigate its effective target, we analyzed miRNA profiles during OA pathogenesis and verify the role and its functional targets of miR-488.

Results

Human articular chondrocytes were obtained from cartilage of OA patients undergoing knee replacement surgery and biopsy samples of normal cartilage and the expression profile of miRNA was analyzed. From expression profile, most potent miR was selected and its target and functional role in OA pathogenesis were investigated using target validation system and OA animal model system. Among miRNAs tested, miR-488 was significantly decreased in OA chondrocytes Furthermore, we found that exposure of IL-1β was also suppressed whereas exposure of TGF-β3 induced the induction of miR-488 in human articular chondrocytes isolated from biopsy samples of normal cartilages. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation.

Conclusions

miR-488 acts as a positive role for chondrocyte differentiation/cartilage development by inhibiting MMP-13 activity through targeting ZIP-8.     

NOV/CCN3 induces cartilage protection by inhibiting PI3K/AKT/mTOR pathway

Osteoarthritis (OA), an age‐related degenerative joint disease, is pathologically characterized by articular cartilage degeneration and synovial inflammation. Nephroblastoma overexpressed (NOV or CCN3), a matricellular protein, is a primary member of the CCN family (Cyr61, Ctgf, NOV) of proteins and is involved in various inflammatory disorders. Previous studies reported that CCN3 might play a therapeutic role in OA. However, the underlying mechanism remains unclear. In this study, we confirmed the expression of CCN3 was decreased in human and rat OA articular cartilage. Recombinant CCN3 ameliorated the IL‐1β‐induced matrix catabolism, as demonstrated by MMP1, MMP3, MMP13, ADAMTS5 and iNOS expression, in vitro. In addition, the degradation of cartilage matrix such as collagen 2 and aggrecan could be reversed by CCN3. Furthermore, we found CCN3 promoted autophagy as Atg5, Beclin1 and LC3‐II expression were increased. High‐mobility group box 1 was negatively correlated with CCN3 in IL‐1β‐induced osteoarthritis responses, and HMGB1 is involved in the protective effect of CCN3 in OA. Moreover, CCN3 overexpression decreased the expression of HMGB1 and reversed the IL‐1β induced MMPs production. Additionally, recombinant CCN3 or CCN3 overexpression attenuated the activation of PI3K/AKT/mTOR pathway induced by IL‐1β. Our study presents new mechanisms of CCN3 in osteoarthritis and indicates that CCN3 can serve as a novel potential therapeutic target for osteoarthritis.     

Articular Joint Lubricants during Osteoarthritis and Rheumatoid Arthritis Display Altered Levels and Molecular Species

Background

Hyaluronic acid (HA), lubricin, and phospholipid species (PLs) contribute independently or together to the boundary lubrication of articular joints that is provided by synovial fluid (SF). Our study is the first reporting quantitative data about the molecular weight (MW) forms of HA, lubricin, and PLs in SF from cohorts of healthy donors, patients with early (eOA)- or late (lOA)-stage osteoarthritis (OA), and patients with active rheumatoid arthritis (RA).

Methods

We used human SF from unaffected controls, eOA, lOA, and RA. HA and lubricin levels were measured by enzyme-linked immunosorbent assay. PLs was quantified by electrospray ionization tandem mass spectrometry. Fatty acids (FAs) were analyzed by gas chromatography, coupled with mass spectrometry. The MW distribution of HA was determined by agarose gel electrophoresis.

Results

Compared with control SF, the concentrations of HA and lubricin were lower in OA and RA SF, whereas those of PLs were higher in OA and RA SF. Moreover, the MW distribution of HA shifted toward the lower ranges in OA and RA SF. We noted distinct alterations between cohorts in the relative distribution of PLs and the degree of FA saturation and chain lengths of FAs.

Conclusions

The levels, composition, and MW distribution of all currently known lubricants in SF—HA, lubricin, PLs—vary with joint disease and stage of OA. Our study is the first delivering a comprehensive view about all joint lubricants during health and widespread joint diseases. Thus, we provide the framework to develop new optimal compounded lubricants to reduce joint destruction.     

If good things come from above,do bad things come from below?

Factors in the synovial fluid that maintain healthy articular cartilage, such as hyaluronic acid and lubricin, come from above. Is it possible that factors which lead to the destruction of cartilage come from below in the subchondral bone? The recent acquisition of tools to probe early events in osteoarthritis is shedding new light on possible contributions from this compartment on the initiation and progression of the disease. Tanamas and co-workers now provide evidence that bone marrow lesions in the subchondral bone are predictive, both of loss of cartilage and of formation of subchondral cysts. These data provoke questions about the nature and role of bone marrow lesions.Finding the factors that initiate, or the mechanisms that lead to progression of, osteoarthritis (OA) has proven frustrating and largely unproductive. Identification of risk factors for the condition - such as prior trauma to the joint, elevated body weight and female sex - may have helped with management of OA but has done little to progress understanding of the underlying factors that drive it. OA research has been more difficult than research for some other diseases of the skeleton, for several important reasons. Early OA, at the level of symptoms, can be episodic, making it difficult to identify the disease and to follow it longitudinally. Since the main early symptom is pain, clinical trials of new therapies have been problematic. Animal experiments have been bedevilled by a lack of models that accurately replicate the human disease. And perhaps, as argued by a minority of workers in the field, disease initiators have been sought in the wrong place; that is, cartilage versus bone.The recent study of Tanamas and colleagues highlights the way in which new-generation imaging holds the promise of shedding new light on this old problem [1]. In particular, high-resolution magnetic resonance imaging (MRI) can now deliver objective, measurable information about all structures of the joint, including the amount and quality of articular cartilage, and is also a powerful tool to investigate the subchondral bone. The holy grail of clinical investigation, namely longitudinal study with quantitative endpoints, is now accessible for OA. What Tanamas and colleagues'' study shows is important because it adds to emerging evidence that processes in the subchondral bone relate strongly to changes in the volumetric amount of articular cartilage. Specifically, bone marrow lesions (BMLs), the mysterious MRI-bright regions in the subchondral bone that occur more commonly in OA, were shown to be predictive of loss of cartilage and of formation of subchondral cysts. In turn, cysts were more likely than BMLs to occur in association with loss of cartilage.These data pose the intriguing question of whether BMLs encode key clues to the aetiology of OA. Longitudinal studies have shown that the presence of BMLs constitutes a potent risk factor for structural deterioration in knee OA [2]. BML enlargement has been strongly associated with increased cartilage loss, and Tanamas and colleagues'' data further suggest that their conversion into cysts is even more predictive of cartilage loss. Significantly, a reduction in the extent of BMLs on MRI has been shown to associate with a decrease in cartilage degradation [3]. Since the origin of BMLs is not known, its investigation needs to be prioritised as an important research topic. Current informed guesses are that BMLs comprise regions of oedema, perhaps secondary to episodes of local ischaemia. Although it is not possible to biopsy BMLs in patients with early OA, several studies have sought to correlate the MRI findings with histology in more severe disease. Regions of BMLs in end-stage OA patients at knee replacement were more likely to exhibit oedema, bone necrosis and trabecular abnormalities than were control sites [4].If BMLs are secondary to local ischaemia in the subchondral bone, there are several possible consequences. Firstly, the supply of nutrients and oxygen from regions of ischaemic subchondral bone, to the overlying articular cartilage, might be reduced. Cartilage nutrition has been considered to derive from the synovial fluid. The work of Imhof and colleagues, however, suggested that more than 50% of the glucose, oxygen and water requirements of cartilage are provided by perfusion from the subchondral vessels [5]. They described the dense subchondral vasculature in close proximity to the cartilage, and the micro-channels that penetrate the subchondral mineralisation zone and permit communication between the bone and the cartilage. More recent work indicates that small molecules can diffuse, in healthy joints, bidirectionally from the synovial compartment into the cartilage and underlying bone and from the subchondral bone into the overlying cartilage [6]. Inspection of the osteochondral junction of long bones reveals that osteocytes and osteocyte canaliculi, which are also probable conduits of nutrients, are intimately associated with the articular cartilage. Experimental interruption of contact between articular cartilage and subchondral bone results in degeneration of the cartilage, and osteoblasts from OA subchondral bone conferred catabolic changes in articular chondrocytes [7].Secondly, osteocyte death in bone is becoming recognised as a signalling event for osteoclastic removal of the nonviable bone and its replacement in a remodelling episode [8]. Although subchondral bone is constantly being remodelled, concentration of this activity in a particular region of the bone could alter its mechanical integrity and its ability to properly support the overlying cartilage.Tanamas and colleagues conclude that cysts (and BMLs) may provide therapeutic targets for the treatment of knee OA [1]. Certainly, the recent acquisition of tools to probe early events in subchondral bone in OA should deliver rapid advances in our understanding of the natural history of this condition.     

Intensity-dependent effect of treadmill running on lubricin metabolism of rat articular cartilage

Introduction

We aimed to understand the changes in cartilage lubricin expression and immunolocalisation in responsed to treadmill running with different intensities in a rat model.

Methods

A total of 24 male Wistar rats were randomly assigned into groups of control (CON), low-intensity running (LIR), moderate-intensity running (MIR), and high-intensity running (HIR). Rats in LIR, MIR, and HIR groups were trained for 8 weeks on the treadmill with low, moderate, and high intensity, respectively. After sacrifice, femoral condyles were collected to take histological observation for cartilage characteristics, and immunohistochemistry for lubricin. In addition, cartilage samples were obtained to assess PRG4 and TGF-β mRNA expression by quantitative RT-PCR.

Results

Histological examination showed osteoarthritic changes in rats after eight weeks of high intensity running. In comparison to CON group, significantly lower Mankin score was found in LIR and MIR groups, whereas, HIR group had significantly higher Mankin score than either CON, LIR, or MIR group. On the other hand, both LIR and MIR groups have significantly higher lubricin content than CON group, whereas, significantly lower lubricin content was found in HIR group compared with CON, LIR or MIR group. A significant inverse correlation was detected between the lubricin content and Mankin score. In addition, considerably higher mRNA gene expression of PRG4 and TGF-β was found in LIR and MIR groups, compared with those in CON and HIR groups.

Conclusions

There is a marked intensity-specific effect of running on the immunolocalisation and gene expression of lubricin in cartilage, which is inversely correlated with Mankin score. Our findings provide evidences that mechanical factors are key determinants of lubricin metabolism in vivo.     

Bevacizumab,an anti-vascular endothelial growth factor antibody,inhibits osteoarthritis

Introduction

Angiogenesis is an important factor in the development of osteoarthritis (OA). We investigated the efficacy of bevacizumab, an antibody against vascular endothelial growth factor and an inhibitor of angiogenesis, in the treatment of OA using a rabbit model of anterior cruciate ligament transection.

Methods

First, we evaluated the response of gene expression and histology of the normal joint to bevacizumab treatment. Next, in a rabbit model of OA induced by anterior cruciate ligament transection, we used macroscopic and histological evaluations and real-time polymerase chain reaction (PCR) to examine the responses to intravenous (systemic) administration of bevacizumab (OAB IV group). We also investigated the efficacy of intra-articular (local) administration of bevacizumab in OA-induced rabbits (OAB IA group).

Results

Histologically, bevacizumab had no negative effect in normal joints. Bevacizumab did not increase the expression of genes for catabolic factors in the synovium, subchondral bone, or articular cartilage, but it increased the expression of collagen type 2 in the articular cartilage. Macroscopically and histologically, the OAB IV group exhibited a reduction in articular cartilage degeneration and less osteophyte formation and synovitis compared with the control group (no bevacizumab; OA group). Real-time PCR showed significantly lower expression of catabolic factors in the synovium in the OAB IV group compared with the OA group. In articular cartilage, expression levels of aggrecan, collagen type 2, and chondromodulin-1 were higher in the OAB IV group than in the OA group. Histological evaluation and assessment of pain behaviour showed a superior effect in the OAB IA group compared with the OAB IV group 12 weeks after administration of bevacizumab, even though the total dosage given to the OAB IA group was half that received by the OAB IV group.

Conclusions

Considering the dosage and potential adverse effects of bevacizumab, the local administration of bevacizumab is a more advantageous approach than systemic administration. Our results suggest that intra-articular bevacizumab may offer a new therapeutic approach for patients with post-traumatic OA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0427-y) contains supplementary material, which is available to authorized users.     

The interaction of canonical bone morphogenetic protein- and Wnt-signaling pathways may play an important role in regulating cartilage degradation in osteoarthritis

Bone morphogenetic proteins (BMPs) and Wnts are important signaling protein families with key roles in embryologic, patterning, development, and tissue remodeling in growth. BMP and Wnt-β-catenin are highly evolutionarily conserved pathways that, though often regulating similar cellular events, are independent signaling mechanisms that can have complementary or antagonistic effects depending on various factors, including cell type and developmental stage. Although BMP and Wnt-β-catenin have the ability to act entirely independently, there is a developing body of evidence for specific extra- and intra-cellular molecular interactions and crosstalk that occur between BMP and Wnt-β-catenin signaling and that again this may be cell type-specific. In the previous issue of Arthritis Research & Therapy, Papathanasiou and colleagues provide novel insights into the role and direct interaction of BMP2 and canonical Wnt-β-catenin signaling in regulating chondrocyte hypertrophy and matrix metalloproteinase/a disintegrin like and metalloproteinase with thrombospondin type I motif (MMP/ADAMTS) synthesis in osteoarthritis.In the previous issue of Arthritis Research & Therapy, Papathanasiou and colleagues [1] provide novel insights into the role and direct interaction of bone morphogenetic protein 2 (BMP2) and canonical Wnt-β-catenin signaling in regulating chondrocyte hypertrophy and matrix metalloproteinase (MMP)/aggrecanolytic ADAMTS (a disintegrin like and metalloproteinase with thrombospondin type I motif) synthesis in osteoarthritis (OA). OA is the most common cause of joint pain and disability, and with increasing age and obesity of the population, the already major socioeconomic importance will continue to increase. Currently, in most Western cultures, OA afflicts more than 10% of the entire population and over a third of those over 65; an estimated 25 to 30 million people in the US suffer from this disease. The central pathological feature of OA is often considered to be the progressive destruction of articular cartilage that normally provides the load-bearing surface in the joint. Much has been learned in recent years about the mechanisms that drive cartilage matrix breakdown and loss in OA, and chondrocyte-derived metalloproteinases, particularly the ADAMTS and collagenolytic MMPs, have a key role. It is evident that a phenotypic shift in the mature articular chondrocyte to a cell type that displays many characteristics typical of hypertrophic cells in the lower zones of the growth plate is a typical feature of OA and is associated with the progressive cartilage breakdown observed (reviewed in [2]). Less clearly understood are the specific signaling pathways involved in regulating the chondrocyte phenotype, how they interact, and whether this changes in health and in diseases such as OA.BMPs and Wnts are important signaling protein families with key roles in embryologic, patterning, development, and tissue remodeling in growth. BMP and Wnt-β-catenin are highly evolutionarily conserved pathways that, though often regulating similar cellular events, are independent signaling mechanisms that can have complementary or antagonistic effects depending on various factors, including cell type and developmental stage (reviewed in [3]). Although BMP and Wnt-β-catenin have the ability to act entirely independently, there is a developing body of evidence for specific extra-and intra-cellular molecular interactions and crosstalk that occur between BMP and Wnt-β-catenin signaling and that again may be cell type-specific [3]. In addition to having a key role in development, BMPs and Wnts are emerging as critical regulators of bone and cartilage homeostasis in the adult and, importantly, in the onset and progression of musculoskeletal diseases.BMPs are multi-functional growth factors that belong to the transforming growth factor-β super family. Evidence suggests that BMP signaling is mediated primarily through the canonical BMP-Smad pathway in chondrocytes. BMPs bind the type II receptor and phosphorylate type I serine or threonine receptors, which subsequently phosphorylate Smad1, Smad5, and Smad8. BMPs are known to induce human mesenchymal stem cells to differentiate into chondrocytes, and BMP2 is a crucial local factor for chondrocyte proliferation and maturation during endochondral ossification [4,5]. In their report, Papathanasiou and colleagues show not only that human end-stage OA chondrocytes produce BMP2 and BMP4 but also, importantly, that BMP2, but not BMP4, can drive expression of low-density lipoprotein receptor 5 (LRP5). LRP5 is one of the most important co-receptors in the canonical Wnt-β-catenin signaling pathway; binding of Wnt ligands to the frizzled/LRP co-receptor complex leads to β-catenin stabilization, nuclear translocation, and activation of target genes.There is a large body of evidence demonstrating the central role for Wnt signaling in regulating adult bone turnover; increased β-catenin activity inducing bone production and inhibition of soluble antagonists is an emerging therapeutic approach for osteoporotic and inflammatory bone loss [6,7]. In cartilage, Wnt-β-catenin signaling plays a dual role; activity is essential for chondrocyte proliferation and maintenance of their phenotypic characteristics [8], but excessive activity increases chondrocyte hypertrophy and expression of cartilage degrades metalloproteinases [9]. The effect may be cell type- specific, and Wnt-β-catenin activation is essential for maintenance of the superficial zone chondrocyte phenotype and proteoglycan 4 (lubricin) expression [8]. Inhibition of β-catenin rapidly leads to downregulation of lubricin and increased collagen × expression in superficial zone chondrocytes. In chondrocytes from human end-stage OA cartilage, activation of canonical Wnt-β-catenin signaling by Wnt-2B and Wnt-16 can drive MMP and aggrecanase production [9]. Understanding the mechanisms that regulate Wnt signaling in chondrocytes in OA may provide keys to controlling cartilage degradation.One of the most important findings by Papathanasiou and colleagues is the demonstration of a new and unique function of BMP2 in chondrocytes in acting as a regulator of canonical Wnt-β-catenin signaling. Treatment of both normal and OA primary human chondrocytes with BMP2 for 12 hours enhanced total β-catenin expression while diminishing the degradation of β-catenin (phospho-β-catenin). This was accompanied by significant increases in mRNA for key cartilage-degrading enzymes MMP-13 and ADAMTS-5 in concert with a shift toward a hypertrophic chondrocyte phenotype as measured by increased collagen × expression. This effect was absent in LRP5 small interfering RNA (siRNA) pretreated chondrocytes and did not occur with BMP4, suggesting the unique function of BMP2 in specifically upregulating LRP5 and augmenting Wnt-β-catenin signaling. The BMP2-driven increase in LRP5 mRNA was mediated through Smad1/5/8 binding to the LRP5 promoter.The paper by Papathanasiou and colleagues adds to the accumulating evidence that increased or perhaps excessive activation of canonical Wnt-β-catenin signaling in chondrocytes is detrimental and contributes to OA cartilage degradation. Therapeutic approaches to block or suppress canonical Wnt-β-catenin signaling may protect cartilage damage in end-stage OA. There are many naturally occurring Wnt-β-catenin signaling antagonists, including dickkopf 1 (DKK1), secreted frizzled-related proteins (sFRPs), and sclerostin (SOST). Evidence suggests that circulating DKK1 levels negatively correlate with biomarkers of cartilage breakdown in patients with OA [10]; sFRP3 knockout mice have augmented cartilage proteoglycan loss in a collagenase-induced instability model of arthritis [11], and co-treatment of SOST with pro-inflammatory cytokines can attenuate cartilage matrix breakdown [12]. The role of SOST is interesting in light of the interaction between BMP2 and Wnt signaling pathways reported by Papathanasiou and colleagues. It appears that SOST can also function as a BMP antagonist in osteoblast and osteocytes by binding intra-cellularly to BMP7 and targeting the growth factor for proteosomal degradation [13]. This provides yet another mechanism by which BMP and Wnt signaling pathways may directly interact; it will be interesting to see whether this effect of SOST on BMP7 (and possibly other BMPs) also occurs in chondrocytes, particularly in OA, where chondrocyte SOST expression is increased [12].The BMP and Wnt signaling pathways are critical in regulating chondrocytes and maintaining the health and integrity of cartilage matrix. In other cell types/organs such as those in bone, it is the combinatorial integration and complex crosstalk between these two pathways that are emerging as significant regulators of development and tissue homeostasis [3]. The findings by Papathanasiou and colleagues suggest that similar signaling pathway interactions may be important in chondrocytes and could play a role in the development and progression of OA. A better appreciation of chondrocyte regulatory mechanisms may provide new avenues for development of therapeutic approaches for the treatment of OA.     

Mass spectrometry assays of plasma biomarkers to predict radiographic progression of knee osteoarthritis

Introduction

Biomarkers to identify osteoarthritis (OA) patients at risk for disease progression are needed. As part of a proteomic analysis of knee synovial fluid from normal and OA patients, differentially expressed proteins were identified that could represent potential biomarkers for OA. This study aimed to use mass spectrometry assays to identify representative peptides from several proteins in synovial fluid and peripheral blood, and assess their levels as biomarkers of OA progression.

Methods

Multiplexed high throughput selected reaction monitoring (SRM) assays were developed to measure tryptic peptides representative of 23 proteins in matched serum and synovial fluid samples from late OA subjects at the time of joint replacement. Subsequently plasma samples from the baseline visit of 173 subjects in an observational OA cohort were tested by SRM for peptides from nine of these proteins: afamin, clusterin, cartilage oligomeric matrix protein, hepatocyte growth factor, kallistatin, insulin-like growth factor binding protein, acid labile subunit, lubricin, lumican, and pigment epithelium-derived factor. Linear regression was used to determine the association between the peptide biomarker level at baseline and change in joint space width (ΔJSW) from baseline to 30 months, adjusting for age and sex.

Results

In the matched cohort, 17 proteins could be identified in synovial fluid and 16 proteins were detected in serum. For the progression cohort, the average age was 62 and average ΔJSW over 30 months was 0.68 mm. A high correlation between different peptides from individual proteins was observed, indicating our assays correctly measured their target proteins. Peptides representative of clusterin, lumican and lubricin showed statistically significant associations with joint space narrowing after adjustment for age and sex. Partial R² values showed clusterin FMETVAEK and lubricin LVEVNPK peptide biomarkers explains about 2 to 3% of the variability of ΔJSW, similar to that explained by age. A biomarker score combining normalized data for both lubricin and clusterin peptides increased the model R² to 0.079.

Conclusions

Our results suggest that when combined, levels of peptides representative of clusterin and lubricin in plasma are as predictive of OA progression as age. Replication of these findings in other prospective OA cohorts is planned.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0456-6) contains supplementary material, which is available to authorized users.     

The O-glycomap of Lubricin,a Novel Mucin Responsible for Joint Lubrication,Identified by Site-specific Glycopeptide Analysis

The lubricative, heavily glycosylated mucin-like synovial glycoprotein lubricin has previously been observed to contain glycosylation changes related to rheumatoid and osteoarthritis. Thus, a site-specific investigation of the glycosylation of lubricin was undertaken, in order to further understand the pathological mechanisms involved in these diseases. Lubricin contains an serine/threonine/proline (STP)-rich domain composed of imperfect tandem repeats (EPAPTTPK), the target for O-glycosylation. In this study, using a liquid chromatography–tandem mass spectrometry approach, employing both collision-induced and electron-transfer dissociation fragmentation methods, we identified 185 O-glycopeptides within the STP-rich domain of human synovial lubricin. This showed that adjacent threonine residues within the central STP-rich region could be simultaneously and/or individually glycosylated. In addition to core 1 structures responsible for biolubrication, core 2 O-glycopeptides were also identified, indicating that lubricin glycosylation may have other roles. Investigation of the expression of polypeptide N-acetylgalactosaminyltransferase genes was carried out using cultured primary fibroblast-like synoviocytes, a cell type that expresses lubricin in vivo. This analysis showed high mRNA expression levels of the less understood polypeptide N-acetylgalactosaminyltransferase 15 and 5 in addition to the ubiquitously expressed polypeptide N-acetylgalactosaminyltransferase 1 and 2 genes. This suggests that there is a unique combination of transferase genes important for the O-glycosylation of lubricin. The site-specific glycopeptide analysis covered 82% of the protein sequence and showed that lubricin glycosylation displays both micro- and macroheterogeneity. The density of glycosylation was shown to be high: 168 sites of O-glycosylation, predominately sialylated, were identified. These glycosylation sites were focused in the central STP-rich region, giving the domain a negative charge. The more positively charged lysine and arginine residues in the N and C termini suggest that synovial lubricin exists as an amphoteric molecule. The identification of these unique properties of lubricin may provide insight into the important low-friction lubricating functions of lubricin during natural joint movement.Human diarthrodial joints are surrounded by synovial fluid (SF),¹ a dense extracellular matrix fluid composed of proteins, glycoproteins, hyaluronic acid, proteoglycans, and phospholipids (1). During movement, the cartilage surfaces of the articulating joints slide over each other with an extremely low coefficient of friction that ranges from 0.0005 to 0.04 (2) and handle pressures up to ∼200 atm (3). In a healthy state, the joint surface and SF constitute a system of reduced friction that results in lifelong lubrication and wear resistance, primarily due to biolubricating molecules such as hyaluronic acid and lubricin (4). Human synovial lubricin is encoded by the proteoglycan 4 (Prg4) gene (5, 6) and is synthesized by fibroblast-like synoviocytes (FLSs) and superficial zone chondrocytes. Its 1404-amino-acid sequence contains a central mucin-like domain consisting of 59 imperfectly repeated sequences of EPAPTTPK. The O-glycosylation (in particular core 1 and sialylated core 1) of lubricin is suggested to be responsible for its lubricating properties (7), as the removal of these residues results in the loss of boundary lubrication. The molecule has also been suggested to play a key role in protecting the cartilage surface from excessive adsorption of proteins and cells (8).Arthritis results in the loss of this joint surface, leading to severe pain and a restricted range of motion. The two most common arthritic diseases, osteoarthritis (OA) and rheumatoid arthritis (RA), have different mechanisms of degradation. RA is an autoimmune systemic high inflammatory disease that increases the friction between articulating cartilage surfaces, resulting in degradation of the joint (9), whereas OA is a result of mechanical stress (10). Degeneration of the cartilage can be detected from proteoglycan fragments in the SF (11, 12). Because of the limited efficacy of available treatments, particularly for OA, understanding the biological factors related to arthritis is essential.The joints of arthritis patients, both RA and OA, have shown a down-regulation of expression and changes in glycosylation of lubricin (13). Studies using OA animal models suggest that there is a relationship between pathogenesis and the down-regulation of lubricin (9, 14, 15). This decrease in lubricin expression exacerbates the disease by accelerating the joint destruction, suggesting that certain characteristics of lubricin may be indicators of disease progression in RA and OA. Given the critical nature of lubricin glycosylation, we initiated a site-specific glycopeptide characterization of the lubricin mucin-like domain using liquid chromatography–tandem mass spectrometry with both collision-induced and electron-transfer dissociation fragmentation methods (LC-CID/ETD-MS²) after tryptic digestion of both intact and partly de-glycosylated lubricin.Collision-induced dissociation–tandem mass spectrometry (CID-MSⁿ) of O-linked (and N-linked) glycopeptides is capable of generating sequence information both for the attached glycan (in MS²) and for the de-glycosylated peptide (in MS³), but it lacks the site-specific information of the modified amino acids (16). This is due to extensive glycosidic bond cleavage of the precursor ion in MS² producing B/C and Y/Z ions (Domon and Costello carbohydrate fragmentation nomenclature (17)). In addition, the identification of the modified amino acids is even more difficult for peptides containing several Ser/Thr residues because of the lack of a consensus sequence for mucin-type O-glycosylation. Electron-capture dissociation and ETD are fragmentation techniques used for the site-specific characterization of protein post-translational modifications including phosphorylation (18) and glycosylation (19). Both techniques induce cleavage of the N-Cα bonds of the peptide backbone, producing c- and z-type fragment ions, while leaving the post-translational modification unaffected.In order to understand the biosynthesis of O-linked glycoproteins, one needs to link site localization of glycosylation to the expression of enzymes responsible for GalNAc-type (or mucin-type) O-glycosylation. This is necessary because the prediction of the site of GalNAc-type O-glycosylation is difficult. One reason for this is the large, redundant UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase (ppGalNAc T) gene family containing 20 gene-encoded isoenzymes, all possessing unique and/or overlapping substrate specificities (20, 21). These ppGalNAc Ts transfer GalNAc from the sugar nucleotide donor UDP-GalNAc to the hydroxyl groups of Ser and Thr residues in the proteins traversing the Golgi/endoplasmic reticulum. Altered protein O-glycosylation, suggested to be due to changes in the expression of distinct ppGalNAc Ts, has been reported in various disease states, including ulcerative colitis and cancer (21, 22). Thus, the connection of site-specific O-glycosylation with the responsible ppGalNAc Ts is important for understanding the functions of lubricin, as site-specific O-glycosylation has been shown to regulate the functions of proteins (23, 24) and may be involved in the pathological transformation of the joint in arthritis diseases.Although the type of glycosylation present on lubricin has been investigated previously, the site-specific glycopeptide characterization, including the analysis of the glycan types at these locations, was investigated for the first time in this study. In order to understand the nature of glycoproteins, it is essential to not only define the protein component or the glycan characteristics, but also understand how these two essential components interact. The macro- (different site occupation) and micro-heterogeneity (different glycan structure at each site) provided a heterogeneous mixture of lubricin O-linked glycopeptides that might help to explain the extraordinary properties of lubricin and how it can function as a lubricating agent in a demanding environment.     

Deletion of Dual Specificity Phosphatase 1 Does Not Predispose Mice to Increased Spontaneous Osteoarthritis

Background

Osteoarthritis (OA) is a degenerative joint disease with poorly understood etiology and pathobiology. Mitogen activated protein kinases (MAPKs) including ERK and p38 play important roles in the mediation of downstream pathways involved in cartilage degenerative processes. Dual specificity phosphatase 1 (DUSP1) dephosphorylates the threonine/serine and tyrosine sites on ERK and p38, causing deactivation of downstream signalling. In this study we examined the role of DUSP1 in spontaneous OA development at 21 months of age using a genetically modified mouse model deficient in Dusp1 (DUSP1 knockout mouse).

Results

Utilizing histochemical stains of paraffin embedded knee joint sections in DUSP1 knockout and wild type female and male mice, we showed similar structural progression of cartilage degeneration associated with OA at 21 months of age. A semi-quantitative cartilage degeneration scoring system also demonstrated similar scores in the various aspects of the knee joint articular cartilage in DUSP1 knockout and control mice. Examination of overall articular cartilage thickness in the knee joint demonstrated similar results between DUSP1 knockout and wild type mice. Immunostaining for cartilage neoepitopes DIPEN, TEGE and C1,2C was similar in the cartilage lesion sites and chondrocyte pericellular matrix of both experimental groups. Likewise, immunostaining for phosphoERK and MMP13 showed similar intensity and localization between groups. SOX9 immunostaining demonstrated a decreased number of positive cells in DUSP1 knockout mice, with correspondingly decreased staining intensity. Analysis of animal walking patterns (gait) did not show a discernable difference between groups.

Conclusion

Loss of DUSP1 does not cause changes in cartilage degeneration and gait in a mouse model of spontaneous OA at 21 months of age. Altered staining was observed in SOX9 immunostaining which may prove promising for future studies examining the role of DUSPs in cartilage and OA, as well as models of post-traumatic OA.     

Increased type II collagen cleavage by cathepsin K and collagenase activities with aging and osteoarthritis in human articular cartilage

Introduction

The intra-helical cleavage of type II collagen by proteases, including collagenases and cathepsin K, is increased with aging and osteoarthritis (OA) in cartilage as determined by immunochemical assays. The distinct sites of collagen cleavage generated by collagenases and cathepsin K in healthy and OA human femoral condylar cartilages were identified and compared.

Methods

Fixed frozen cartilage sections were examined immunohistochemically, using antibodies that react with the collagenase-generated cleavage neoepitopes, C2C and C1,2C, and the primary cleavage neoepitope (C2K) generated in type II collagen by the action of cathepsin K and possibly by other proteases, but not by any collagenases studied to date.

Results

In most cases, the staining patterns for collagen cleavage were similar for all three epitopes: weak to moderate mainly pericellular staining in non-OA cartilage from younger individuals and stronger, more widespread staining in aging and OA cartilages that often extended from the superficial to the mid/deep zone of the tissue. In very degenerate OA specimens, with significant disruption of the articular surface, staining was distributed throughout most of the cartilage matrix.

Conclusions

Cleavage of collagen by proteases usually arises pericellularly around chondrocytes at and near the articular surface, subsequently becoming more intense and extending progressively deeper into the cartilage with aging and OA. The close correspondence between the distributions of these products suggests that both collagenases and cathepsin K, and other proteases that may generate this distinct cathepsin K cleavage site, are usually active in the same sites in the degradation of type II collagen.     

The proton-activated G protein-coupled receptor GPR4 regulates the development of osteoarthritis via modulating CXCL12/CXCR7 signaling

Inflammatory diseases decrease the extracellular environmental pH. However, whether proton-activated G protein-coupled receptors (GPCRs) can regulate the development of osteoarthritis (OA) is largely unknown. In this study, we report that proton-activated GPR4 is essential for OA development. We found a marked increase in expression of the proton-activated GPR4 in human and mouse OA cartilage. Lentivirus-mediated overexpression of GPR4 in mouse joints accelerated the development of OA, including promotion of articular cartilage damage, synovial hyperplasia, and osteophyte formation, while Gpr4 knockout effectively attenuated the development of posttraumatic and aging-associated OA in mice. We also found that inhibition of GPR4 with the antagonist NE52-QQ57 ameliorated OA progression in mice, promoted extracellular matrix (ECM) production, and protected cartilage from degradation in human articular cartilage explants. Moreover, GPR4 overexpression upregulated matrix-degrading enzymes’ expression and inflammation factors under pro-inflammatory and slightly acidic conditions. Mechanistically, GPR4 suppressed chondrocyte differentiation and upregulated cartilage homeostasis through NF-κB/MAPK signaling activation by regulating CXCR7/CXCL12 expression. Together, our results take the lead to illustrate that proton-activated GPCR acts as a key regulator for OA pathogenesis in vivo, and support that GPR4 could be a promising therapeutic target for OA treatment.Subject terms: Cartilage development, Osteoarthritis     

Effect of eldecalcitol on articular cartilage through the regulation of transcription factor Erg in a murine model of knee osteoarthritis

Clinical studies have reported an association between low blood levels of 25-hydroxyvitamin D and the progression of osteoarthritis (OA), but the mechanism and effects of vitamin D signaling on articular chondrocytes and cartilage remains unclear. The purpose of this study was to investigate the effects of vitamin D on articular cartilage degeneration using eldecalcitol (ED-71), which is an active vitamin D₃ analog. Eight-week old male C57BL/6NCrSlc mice were subjected to experimental surgery to induce OA and local treatments with 10 μL ED-71 (0.5 μg/mL) were administered weekly. Four and 12 weeks after surgery, joints were evaluated using histological scoring systems. In addition, gene expression was analyzed in chondrocytes that were isolated from wildtype neonatal mice, cultured, and treated with ED-71 (10^?8 M). Joints treated with ED-71 demonstrated slowed progression of OA at 4 weeks after surgery, but few effects were observed at 12 weeks after surgery. Ets-related gene (Erg) expression was upregulated in OA articular cartilage, and further increased by ED-71 treatment. In primary chondrocytes cultured with ED-71, the gene expression of Erg and lubricin/proteoglycan 4 significantly increased, as compared to that of cells cultured without ED-71. Local treatment with ED-71 reduced degenerative changes to the articular cartilage during the early phase of experimental OA. Regulation of Erg by ED-71 in articular cartilage could confer resistance to early osteoarthritic changes.     

Targeting bone alleviates osteoarthritis in osteopenic mice and modulates cartilage catabolism

Objective

Subchondral bone modifications occur early in the development of osteoarthritis (OA). The level of bone resorption might impact cartilage remodeling. We therefore assessed the in vivo and in vitro effects of targeting bone resorption in OA and cartilage metabolism.

Methods

OA was induced by meniscectomy (MNX) in ovariectomized osteopenic mice (OP) treated with estradiol (E2), pamidronate (PAM), or phosphate buffered saline (PBS) for 6 weeks. We assessed the subchondral bone and cartilage structure and the expression of cartilage matrix proteases. To assess the involvement of bone soluble factors in cartilage metabolism, supernatant of human bone explants pre-treated with E2 or PAM were transferred to cartilage explants to assess proteoglycan release and aggrecan cleavage. OPG/RANKL mRNA expression was assessed in bone explants by real-time quantitative PCR. The role of osteoprotegerin (OPG) in the bone-cartilage crosstalk was tested using an OPG neutralizing antibody.

Results

Bone mineral density of OP mice and osteoclast number were restored by E2 and PAM (p<0.05). In OP mice, E2 and PAM decreased ADAMTS-4 and -5 expression, while only PAM markedly reduced OA compared to PBS (2.0±0.63 vs 5.2±0.95; p<0.05). OPG/RANKL mRNA was increased in human bone explants treated with both drugs (2.2–3.7-fold). Moreover, supernatants from bone explants cultured with E2 or PAM reduced aggrecan cleavage and cartilage proteoglycan release (73±8.0% and 80±22% of control, respectively, p<0.05). This effect was reversed with osteoprotegerin blockade.

Conclusion

The inhibition of bone resorption by pamidronate in osteopenic mice alleviates the histological OA score with a reduction in the expression of aggrecanases. Bone soluble factors, such as osteoprotegerin, impact the cartilage response to catabolic factors. This study further highlights the importance of subchondral bone in the regulation of joint cartilage damage in OA.     

Degeneration of normal articular cartilage induced by late phase osteoarthritic synovial fluid in beagle dogs

Objective

To investigate the pathogenesis of late phase osteoarthritic (OA) synovial fluid (SF) on normal articular cartilage in vivo and provide an understanding of degenerative cartilage extending in OA joint.

Methods

A random knee, each of 8 beagle dogs, received anterior cruciate ligament transection (ACLT) and was confirmed to have late phase OA degenerative changes at 24 weeks after operation. Thereafter, one random elbow of each canine was injected with autologous late phase OA knee SF. The contralateral elbow was injected with normal saline (NS) of the same volume as SF aspirated from ACLT knee. These two groups of elbows were labeled “SF” and “NS”. 8 other beagle dogs were left intact and placed in Group Control. After aseptic arthrocentesis was performed weekly on both elbows for 24 weeks, morphological changes were observed in the cartilage of the elbows, and expressions of 7 biological etiological factors of chondrocytes of the elbows were determined in Group SF, Group NS and Group Control, respectively.

Results

Morphological changes were observed in articular cartilage of the elbows in Group SF. Levels of unit area of collagen type I in the noncalcified, calcified and full zones of articular cartilage of the elbows in Group SF increased significantly. Level of unit area of collagen type III in the calcified zone of articular cartilage of the elbows in Group SF remained unchanged. Meanwhile, expressions of MMP-1 and MMP-3 of chondrocytes of the elbows in Group SF increased significantly. There was almost no difference between articular cartilage in Group NS and Group Control.

Conclusion

Based on these results, we conclude that OA degeneration of normal articular cartilage can be independently induced by late phase OA SF. Endogenous OA biological etiological factor may be one of the reasons causing degenerative cartilage extending in OA joint.