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ObjectiveThe aim of this study was to assess the extent and the mechanism by which activin A contributes to progressive joint destruction in experimental arthritis and which activin A-expressing cell type is important for disease progression.MethodsLevels of activin A in synovial tissues were evaluated by immunohistochemistry, cell-specific expression and secretion by PCR and ELISA, respectively. Osteoclast (OC) formation was assessed by tartrat-resistant acid phosphatase (TRAP) staining and activity by resorption assay. Quantitative assessment of joint inflammation and bone destruction was performed by histological and micro-CT analysis. Immunoblotting was applied for evaluation of signalling pathways.ResultsIn this study, we demonstrate that fibroblast-like synoviocytes (FLS) are the main producers of activin A in arthritic joints. Most significantly, we show for the first time that deficiency of activin A in arthritic FLS (ActβAd/dColVI-Cre) but not in myeloid cells (ActβAd/dLysM-Cre) reduces OC development in vitro, indicating that activin A promotes osteoclastogenesis in a paracrine manner. Mechanistically, activin A enhanced OC formation and activity by promoting the interaction of activated Smad2 with NFATc1, the key transcription factor of osteoclastogenesis. Consistently, ActβAd/dLysM-Cre hTNFtg mice did not show reduced disease severity, whereas deficiency of activin A in ColVI-Cre-expressing cells such as FLS highly diminished joint destruction reflected by less inflammation and less bone destruction.ConclusionsThe results highly suggest that FLS-derived activin A plays a crucial paracrine role in inflammatory joint destruction and may be a promising target for treating inflammatory disorders associated with OC formation and bone destruction like rheumatoid arthritis.

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/β-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation., Fibroblast-like synoviocytes are important mediators of joint pathology in rheumatoid arthritis (RA). Here the authors show that Lasp1 is epigenetically regulated and highly expressed by these cells in RA and its deletion can limit joint pathology in a mouse model of inflammatory arthritis.

Background Activin A and follistatin exhibit immunomodulatory functions, thus affecting autoinflammatory processes as found in rheumatoid arthritis (RA). The impact of both proteins on the behavior of synovial fibroblasts (SF) in RA as well as in osteoarthritis (OA) is unknown. Methods Immunohistochemical analyses of synovial tissue for expression of activin A and follistatin were performed. The influence of RASF overexpressing activin A on cartilage invasion in a SCID mouse model was examined. RASF and OASF were stimulated with either IL-1β or TNFα in combination with or solely with activin A, activin AB, or follistatin. Protein secretion was measured by ELISA and mRNA expression by RT-PCR. Smad signaling was confirmed by western blot. Results In human RA synovial tissue, the number of activin A-positive cells as well as its extracellular presence was higher than in the OA synovium. Single cells within the tissue expressed follistatin in RA and OA synovial tissue. In the SCID mouse model, activin A overexpression reduced RASF invasion. In human RASF, activin A was induced by IL-1β and TNFα. Activin A slightly increased IL-6 release by unstimulated RASF, but decreased protein and mRNA levels of follistatin. Conclusion The observed decrease of cartilage invasion by RASF overexpressing activin A in the SCID mouse model appears to be mediated by an interaction between activin/follistatin and other local cells indirectly affecting RASF because activin A displayed certain pro-inflammatory effects on RASF. Activin A even inhibits production and release of follistatin in RASF and therefore prevents itself from being blocked by its inhibitory binding protein follistatin in the local inflammatory joint environment. Electronic supplementary material The online version of this article (10.1186/s13075-019-1926-7) contains supplementary material, which is available to authorized users.

Although the impact of osteoblast-osteoclast crosstalk in bone remodelling has been intensively studied, the importance of osteocytes, descendants of osteoblasts, in this process has for a long time been neglected. During their embedding phase, osteocytes undergo considerable phenotypic transformation, from a cuboidal, highly metabolically active osteoblast secreting extracellular matrix to a small, stellate, quiescent osteocyte with numerous long dendrites. Osteocytes are encysted in cavities (lacunae) and their dendritic extensions are located in tunnels (canaliculi) forming a remarkable, highly branched, lacunar-canalicular signalling network that spans the entire bone matrix. Osteocytes and their dendrites can communicate directly with each other and through the release of effector proteins such as sclerostin and nuclear factorkappa;B ligand (RANKL), influence osteoblast and osteoclast formation. This allows osteocytes embedded within the bone matrix to communicate and coordinate activity of cells on the bone surface to adapt to mechanical needs and hormonal changes. Besides their importance in sustaining physiological bone homeostasis, accumulating evidence suggests that dysregulated osteocyte function and alterations in the osteocyte lacunar-canalicular network structure are characteristics of skeletal diseases. This review highlights some aspects of osteocyte communication with osteoclasts and mesenchymal stromal cells, the importance of blood vessel-osteocyte interaction and describes central functions of these cells in rheumatoid arthritis, osteoarthritis, osteomyelitis and osteoporosis. Within the last decade new technologies and tools have facilitated the study of osteocyte biology and the search for therapeutic targets to address bone fragility in the near future.

Synovial joints are unique functional elements of the body and provide the ability for locomotion and for physical interaction with the environment. They are composed of different connective tissue structures, of which the synovial membrane is one central component. It shows a number of peculiarities that makes it different from other membranes in our body, while several lines of evidence suggest that synovial fibroblasts, also termed fibroblast-like synoviocytes (FLS) critically contribute to these peculiarities. This becomes evident particularly under disease conditions such as in rheumatoid arthritis and osteoarthritis, where the synovium is a key pathophysiological component. Therefore, an in-depth knowledge of FLS biology is not only important for understanding key features of articular function but also provides explanations for important characteristics of both degenerative and inflammatory joint diseases. This article reviews the structure, biochemical composition and functions of the synovial membrane and by focusing on the role of synovial fibroblasts explains key features of articular tissue remodelling particularly under disease conditions.

Career situation of first and presenting author Student for a master or a PhD. Introduction Activins and inhibins belong to the transforming growth factor β family. Activins are disulphide-linked homo- or heterodimers consisting of two inhibin β chains (βA, βB) that are expressed in many cell types. However, activin A (βA βA) is the only activin that is expressed in bone and cartilage. Moreover, activin A has been demonstrated not only to stimulate receptor activator of NF-κB ligand (RANKL)-induced osteoclast (OC) differentiation but also to inhibit osteoblast differentiation. Objectives Here we investigate the impact of activin A on joint destruction in rheumatoid arthritis. Methods Synovial tissue samples from rheumatoid arthritis (RA) and osteoarthritis (OA) patients were analysed by immunohistochemical staining. For in vitro experiments, synovial fibroblasts (FLS) were isolated from hind paws of WT mice. Effects of cytokines on the secretion of activin A by mouse FLS were evaluated by ELISA. Bone marrow-derived macrophages (BMM) were isolated from femurs and tibias of WT mice and differentiated into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL with or without activin A. OC differentiation was characterised by TRAP staining. Resorption activity was determined by quantification of osteoclast-mediated pit formation on a calcium phosphate-coated plate. Furthermore, osteoclast-specific gene expression as well as the activation of SMAD2 in BMMs, OCs and FLS were analysed by immunoblotting. The interaction of phospho-SMAD2 with NFATc1 was evaluated by co-immunoprecipitation using Dynabeads. Results We demonstrate that activin A is highly abundant in the synovium of RA but not of OA patients. In vitro, activin A secretion by FLS was strongly enhanced by pro-inflammatory cytokines. Furthermore, activin A strongly enhanced the RANKL-mediated differentiation of BMMs into mature OCs, reflected by a significantly increased OC number and OC size. Moreover, concomitant administration of activin A led to a significant increase of the total resorption area as well as resorption area per pit, indicating an increased activity of individual OCs. Furthermore, activin A enhanced the RANKL-induced expression OC differentiation markers, but alone was not able to induce OC differentiation. Analyses of signaling pathways revealed that activin A induce the activation of SMAD2 in BMMs and OCs. Finally, upon co-stimulation with RANKL, activin A resulted in an increased interaction between activated SMAD2 and NFATc1. Conclusions The data strongly suggest that increased expression of activin A in the arthritic joint is associated with enhanced osteoclast formation, promoting joint destruction in rheumatoid arthritis. Disclosure of Interest None declared.

Career situation of first and presenting author Student for a master or a PhD. Introduction Sclerostin is a Wnt inhibitor and has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Sclerostin loss-of-function leads to a higher bone mass and bone strength. Therefore, inhibition of sclerostin is currently considered as a promising treatment for osteoporosis. Surprisingly, in a TNFα-dependent arthritis mouse model (hTNFtg) the genetic deficiency of sclerostin caused a deterioration of disease severity. hTNFtg mice lacking sclerostin displayed enhanced and bone erosion associated with an elevated number of osteoclasts within the joint. Objectives In order to understand the underlying mechanisms, we aimed to investigate the direct and indirect impact of sclerostin on osteoclast differentiation and bone erosion in arthritis. Methods Sclerostin knockout (sost-/-) mice were crossbred with hTNFtg mice to obtain sost-/-/hTNFtg mice, from which synovial fibroblasts (SF) were isolated. Cocultures of synovial fibroblasts and green fluorescent protein (GFP+) bone marrow-derived macrophages (BMM) were performed and osteoclastogenesis was analysed. Receptor activator of NF-kB ligand (RANKL) and macrophage colony stimulating factor (MCSF) expression was measured by ELISA and IL-1a expression by Western Blot. Viability of osteoclasts precursors was measured by MTT Assay. Results In cocultures of SF and GFP+ BMM, osteoclast formation was enhanced by sost-/-/hTNFtg SF compared to hTNFtg SF. Expression of RANKL and MCSF, two crucial factors for osteoclast differentiation, was not different between the genotypes. Interestingly, stimulation of wildtype BMM with conditioned media (CM) from hTNFtg or sost-/-/hTNFtg synovial fibroblasts showed no TRAP+ cells at all. However, CM supplemented with RANKL lead to an elevated number of osteoclasts using sost-/-/hTNFtg CM compared to hTNFtg CM, pointing to a secreted factor promoting osteoclast development. In this regard, the osteogenic factor IL-1α, was higher expressed in sost-/-/hTNFtg SF than in hTNFtg SF. Moreover, the treatment of cocultures with recombinant sclerostin lead to a decreased number of osteoclasts in both genotypes. Accordingly, sclerostin inhibited osteoclastogenesis in monocultures when administered in the pre-differentiation phase, whereas no effect was observed in the differentiation phase, indicating an inhibitory effect of sclerostin mainly on osteoclast precursors. Conclusions Sclerostin deficiency in hTNFtg SF promotes RANKL-mediated osteoclast differentiation, which is likely dependent on the inhibitory effect of sclerostin itself and/or on the promoting effect of higher levels of IL-1α. Disclosure of Interest None declared.

Introduction Activins and inhibins belong to the transforming growth factor β family. Activins are disulphide-linked homo- or heterodimers consisting of two inhibin β chains (βA, βB) that are expressed in many cell types. However, activin A (βA βA) is the only activin that is expressed in bone and cartilage. Moreover, activin A has been demonstrated not only to stimulate receptor activator of NF-κB ligand (RANKL)-induced osteoclast (OC) differentiation but also to inhibit osteoblast differentiation. Objectives Here we investigate the impact of activin a on joint destruction in rheumatoid arthritis. Methods Synovial tissue samples from rheumatoid arthritis (RA) and osteoarthritis (OA) patients were analysed by immunohistochemical staining. For in vitro experiments, bone marrow-derived macrophages (BMM) were isolated from femurs and tibias of WT mice and differentiated into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL with or without activin A. OC differentiation was characterised by TRAP staining. Resorption activity was determined by quantification of osteoclast-mediated pit formation on a calcium phosphate-coated plate. Proliferation of BMMs was evaluated using the CyQUANT Cell Proliferation Assay Kit. Furthermore, osteoclast-specific gene expression as well as the activation of SMAD2, MAPK and NF-kB signalling were analysed by immunoblotting. The interaction of phospho-SMAD2 with NFATc1 was evaluated by co-immunoprecipitation using Dynabeads. Results We demonstrate that activin A is highly abundant in the synovium of RA but not of OA patients. In vitro, activin A strongly enhanced the RANKL-mediated differentiation of BMMs into mature OCs, reflected by a significantly increased OC number, OC size and number of nuclei per OC compared to the conventional treatment with RANKL alone. Moreover, concomitant administration of activin A led to a significant increase of the total resorption area as well as resorption area per pit, indicating an increased activity of individual OCs. Effects of activin A on BMMs were not caused by increased proliferation since no effect on M-CSF-driven proliferation was observed. Furthermore, activin A alone was not able to induce the expression of OC differentiation markers, but the RANKL-induced expression was enhanced by activin A. After stimulation with activin A, BMMs showed an activation of SMAD2, but not of MAPK p38, ERK, JNK or NF-kB. Finally, co-stimulation of RANKL and activin A resulted in an increased interaction of activated SMAD2 with NFATc1. Conclusions The data strongly suggest that increased expression of activin A in the arthritic joint is most likely associated with enhanced osteoclast formation, promoting joint destruction in rheumatoid arthritis. Disclosure of interest None declared

Introduction Sclerostin is a Wnt inhibitor and has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. The lack of sclerostin in humans and mice leads to a higher bone mass and bone strength known as sclerosteosis. Therefore, inhibition of sclerostin is currently investigated as a treatment against osteoporosis. Surprisingly, the genetic deficiency or pharmacological inhibition of sclerostin causes a deterioration of disease severity in a TNFα-dependent arthritis mouse model (hTNFtg). hTNFtg mice lacking sclerostin displayed enhanced joint inflammation, cartilage loss and bone erosion associated with an elevated number of osteoclasts within the joint. Objectives We want to investigate the impact of sclerostin on osteoclast differentiation and bone erosion in arthritis. Methods Sclerostin knockout (sost -/- ) mice were crossbred with hTNFtg mice to obtain sost -/- /hTNFtg synovial fibroblasts. Co-cultures of synovial fibroblasts and wildtype bone marrow macrophages were analysed by TRAP staining. RANKL expression was measured by ELISA and cytokine expression by array analysis and Western Blot. Moreover, the influence of sclerostin on osteoclastogenesis was additionally analysed in mono-cultures. Results In our co-culture system of synovial fibroblasts and bone marrow derived macrophages, fibroblasts from sost -/- /hTNFtg mice strongly promote osteoclastogenesis in comparison to hTNFtg synovial fibroblasts. Notably, no increased expression of receptor activator of NF-kB ligand (RANKL) was detectable in sost -/- /hTNFtg fibroblasts even after stimulation with inflammatory cytokines. Interestingly, basal secretion of IL-1α, which is known to stimulate osteoclastogenesis, was higher in sost -/- /hTNFtg compared to hTNFtg fibroblasts. Accordingly, sclerostin inhibited osteoclastogenesis when administered in the pre-differentiation phase, whereas no effect was observed in the differentiation phase, indicating an inhibitory effect of sclerostin on osteoclast precursors. Conclusions Sclerostin deficiency in hTNFtg synovial fibroblasts promotes RANKL-mediated osteoclastogenesis, which is most likely dependent on IL-1α. Disclosure of interest None declared

Background Myostatin, a secreted member of the transforming growth factor-β (TGF-β) family, is predominantly expressed in skeletal muscle and negatively regulates muscle growth. We previously demonstrated that myostatin deficiency leads to reduced joint inflammation and joint destruction in arthritic human TNF transgenic mice (Mstn-/- hTNFtg). Additionally, myostatin was highly expressed in rheumatoid arthritis (RA) synovial tissues with most prominent staining of synovial fibroblasts (FLS). Moreover, further studies have shown that myostatin participates in the complex interplay of inflammatory cytokines, increasing tissue and systemic levels of IL1, IL6 and TNFα. Here we investigate the regulatory role of myostatin on joint inflammation and inflammatory cytokine expression in the development of RA. Material and methods Immunophenotyping of isolated spleen and bone marrow derived cells of wt, Mstn-/-, hTNFtg and Mstn-/-hTNFtg mice was performed by FACS analysis. Moreover, hTNFtg mice with a Col1α2-specific deletion of myostatin (Col1α2-Cre/Mstnfl/fl hTNFtg) were generated and clinical severity and histological changes were determined by μCT and histomorphometry. Effects of myostatin on the secretion of IL-6 by human and mouse FLS and the expression of Interleukin-17 receptor C (IL-17RC) were evaluated by ELISA and western blotting. The secretion of various cytokines/chemokines by FLS of Mstn-/-hTNFtg and hTNFtg mice was investigated using a specific antibody array. Results In vivo, the bone marrow of Mstn-/-hTNFtg mice showed a decreased number of B-cells compared to control mice, which might influence the development of the disease. Moreover, comparison of hTNFtg mice, which exhibit a Col1α2-specific deletion of myostatin, with hTNFtg control mice, lead to decreased bone destruction and inflammation examined by µCT and histomorphometric analysis. In vitro, stimulation of FLS with myostatin enhanced the IL-17-mediated secretion of IL-6 by FLS through upregulation of the IL-17RC. Furthermore, analyses of IL-17-mediated cytokine regulation revealed differences in the secretion profile of cytokines/chemokines between FLS of Mstn-/-hTNFtg and FLS of hTNFtg control mice, suggesting that myostatin is involved in the regulation of joint inflammation. Conclusion These data suggest that myostatin could be an indirect regulator of cytokine expression by FLS, associated with enhanced inflammation and thus promote joint destruction during chronic inflammatory arthritis.

Background Rheumatoid arthritis is a common autoimmune disease characterised by the hyperplastic transformation of synovium, its infiltration with γ inflammatory cells and by stimulation of bone resorption through osteoclast activation leading to joint destruction. Posttranslational modification by SUMOylation has been described for various proteins and is involved in a wide variety of cellular processes, including protein localization and stability, transcriptional regulation, cell survival and death. Here, we investigated the role of SUMO-1 in osteoclastogenesis and studied the skeletal phenotype of SUMO-1-/- mice. Materials and methods The skeletal phenotype of 8-week old SUMO-1-/- and wild- type (WT) mice was investigated by µCT-analysis and by van Kossa and TRAP stainings. For in vitro experiments, bone marrow-derived macrophages (BMMs) were isolated from SUMO-1-/- and WT mice, and differentiated into osteoclasts in presence of M-CSF and RANKL. Osteoclast differentiation was characterised by TRAP staining and by real-time PCR analysis of osteoclast marker genes. Activation of p38 and p44/42 MAPK as well as NF-κB signalling was investigated by western blotting. The osteoclast resorption capacity was determined using a calcium phosphate bone resorption assay. Cell motility was analysed by live cell imaging. Results SUMO-1-/- mice demonstrated 20% higher trabecular bone volume compared with WT mice. Moreover, trabecular thickness was increased and trabecular separation was decreased in SUMO-1-/- mice. In addition, SUMO-1-/- mice display a significant reduction in osteoclasts. Of note, SUMO-1 loss was associated with impaired in vitro osteoclast differentiation and resorption capacity. Expression of DC-STAMP, cathepsin K and integrin s3 was decreased in SUMO-1-/- osteoclasts compared to WT osteoclasts. However, no differences in activation of p38 and p44/42 MAPK as well as the NF-κB signalling pathway in BMMs and osteoclasts of SUMO-1-/- and WT mice was observed. Conversely, cell migration and fusion seemed to be affected in SUMO-1-/- osteoclasts. Conclusions We found that SUMO-1-/- mice have a higher bone mass owing to a decreased number of functional osteoclasts. Our data suggest that SUMO-1 is involved in the regulation of bone mass by osteoclast formation and activity, and therefore may be an interesting target for treating diseases associated with bone loss.

Item does not contain fulltext Sclerostin, an inhibitor of the Wnt/beta-catenin pathway, has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Mutations in the human sclerostin gene (SOST) lead to sclerosteosis with progressive skeletal overgrowth, whereas sclerostin-deficient (Sost(-/-)) mice exhibit increased bone mass and strength. Therefore, antibody-mediated inhibition of sclerostin is currently being clinically evaluated for the treatment of postmenopausal osteoporosis in humans. We report that in chronic TNFalpha (tumor necrosis factor alpha)-dependent arthritis, fibroblast-like synoviocytes constitute a major source of sclerostin and that either the lack of sclerostin or its antibody-mediated inhibition leads to an acceleration of rheumatoid arthritis (RA)-like disease in human TNFalpha transgenic (hTNFtg) mice with enhanced pannus formation and joint destruction. Inhibition of sclerostin also failed to improve clinical signs and joint destruction in the partially TNFalpha-dependent glucose-6-phosphate isomerase-induced arthritis mouse model, but ameliorated disease severity in K/BxN serum transfer-induced arthritis mouse model, which is independent of TNF receptor signaling, thus suggesting a specific role for sclerostin in TNFalpha signaling. Sclerostin effectively blocked TNFalpha- but not interleukin-1-induced activation of p38, a key step in arthritis development, pointing to a previously unrealized protective role of sclerostin in TNF-mediated chronic inflammation. The possibility of anti-sclerostin antibody treatment worsening clinical RA outcome under chronic TNFalpha-dependent inflammatory conditions in mice means that caution should be taken both when considering such treatment for inflammatory bone loss in RA and when using anti-sclerostin antibodies in patients with TNFalpha-dependent comorbidities.

In osteoarthritis (OA), hepatocyte growth factor (HGF) is supposed to play a role in cartilage repair. Because the development of osteophytes is a major characteristic of OA and thought to be part of an attempted repair process, the purpose of this study was to determine whether HGF may be involved in osteophyte formation. HGF levels in synovial fluids from 41 patients assessed by enzyme immunosorbant assay were correlated with disease severity and osteophyte formation, evaluated by anteroposterior weight-bearing radiographs. Detection of HGF, c-Met, and CD68 in cartilage and synovial tissues was assessed by immunohistochemistry. Effects of HGF on the secretion of TGF-beta1 and BMP-2 by chondrocytes, fibroblast-like synovial cells (FLS), and macrophages as well as HGF-induced secretion of MCP-1 by FLS and chondrocytes were determined by ELISA. HGF was detected in all synovial fluids and concentrations correlated highly with disease severity and osteophyte formation (p < 0.001). Immunohistochemistry revealed weak synovial staining for HGF, whereas increasing numbers of HGF expressing chondrocytes were detected depending on disease severity. In addition, an increased number of macrophages in synovial specimens was observed, which was likewise severity dependent. In a series of subsequent in vitro studies, HGF remarkable induced MCP-1 secretion by FLS in a dose-dependent manner. No effect on TGF-beta1 and BMP-2 secretion by FLS and chondrocytes was evident upon HGF stimulation, whereas secretion of these growth factors by PMA-differentiated THP-1 cells was significantly increased by HGF. The results indicate that HGF may facilitate osteophyte development by promoting MCP-1-mediated entry of monocytes/macrophages into the OA-affected joint and/or by stimulating macrophage-derived growth factors.

Myostatin is shown to directly promote osteoclast differentiation, and its inhibition improves arthritic bone loss in two mouse models. Myostatin (also known as growth and differentiation factor 8) is a secreted member of the transforming growth factor-β (TGF-β) family that is mainly expressed in skeletal muscle, which is also its primary target tissue. Deletion of the myostatin gene (Mstn) in mice leads to muscle hypertrophy, and animal studies support the concept that myostatin is a negative regulator of muscle growth and regeneration1,2,3,4,5. However, myostatin deficiency also increases bone formation, mainly through loading-associated effects on bone6,7,8,9,10,11. Here we report a previously unknown direct role for myostatin in osteoclastogenesis and in the progressive loss of articular bone in rheumatoid arthritis (RA). We demonstrate that myostatin is highly expressed in the synovial tissues of RA subjects and of human tumor necrosis factor (TNF)-α transgenic (hTNFtg) mice, a model for human RA12. Myostatin strongly accelerates receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast formation in vitro through transcription factor SMAD2-dependent regulation of nuclear factor of activated T-cells (NFATC1). Myostatin deficiency or antibody-mediated inhibition leads to an amelioration of arthritis severity in hTNFtg mice, chiefly reflected by less bone destruction. Consistent with these effects in hTNFtg mice, the lack of myostatin leads to increased grip strength and less bone erosion in the K/BxN serum-induced arthritis model in mice. The results strongly suggest that myostatin is a potent therapeutic target for interfering with osteoclast formation and joint destruction in RA.

Purpose: The purpose of this study was to correlate expression of CD44v5 in osteoarthritic synovium, cartilage, and synovial fluid with radiographical, histomorphological, and biochemical data. Methods: Cartilage and synovia specimens of 27 patients with osteoarthritis were histomorphologically assessed according to Mankin and Pelletier, respectively. Extended weight-bearing antero-posterior radiographs were evaluated according to Kellgren and Ahlback. Expression of membrane-bound CD44v5 was analyzed by immunohistochemistry and levels of soluble CD44v5 were determined by ELISA. Results: Expression of CD44v5 in cartilage and synovia was detected in 67% and 59% of the patients, respectively. Immunohistochemical findings in cartilage correlated significantly with structural cartilage changes (p

Emerging data suggest that urokinase-type plasminogen activator (UPA),beyond its role in pericellular proteolysis, may also act as a mitogen. We investigated the function of endogenous UPA in mediating the mitogenic effects of platelet-derived growth factor (PDGF) and basic fibroblast growth factor(bFGF) on human vascular smooth muscle cells (SMC). Growth-arrested SMC constitutively expressed UPA, but UPA expression and secretion increased several times upon stimulation with either PDGF or bFGF. Inhibition of endogenous UPA with a polyclonal antibody significantly reduced DNA synthesis and proliferation of PDGF or bFGF stimulated SMC, this effect already being evident when the cells entered S-phase. The proliferative activity of endogenous UPA was dependent on a functional catalytic domain as demonstrated by inhibition experiments with a specific monoclonal antibody (394OA) and p-aminobenzamidine, respectively. In contrast, neither plasmin generation nor binding of UPA to its receptor (CD87) were required for UPA-mediated mitogenic effects. The results demonstrate that endogenous UPA is not only overexpressed in SMC upon stimulation with PDGF/bFGF, but also mediates the mitogenic activity of the growth factors in a catalytic-domain-dependent manner. Specific inhibition of this UPA domain may represent an attractive target for pharmacological interventions in atherogenesis and restenosis after angioplasty.

Background and objectives Wnt-inhibitor sclerostin has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Loss of sclerostin expression results in high bone mass and bone strength in patients with sclerosteosis and sclerostin knockout mice. Therefore, antibody-mediated inhibition of sclerostin is currently evaluated for the treatment of osteoporosis in humans. Since it has been shown that sclerostin is upregulated by TNFα, we studied its impacton inflammatory arthritis using RA mouse models such as the human TNF transgenic (hTNFtg) model, the G6PI-induced arthritis model and the K/BxN serum transfer-induced arthritis model. Materials and methods Sclerostin knockout ( sost -/- ) mice were crossed with hTNFtg mice to generate sost -/ - /hTNFtg. Mice with serum transfer-induced arthritis were generated by injection of arthritogenic serum collected from K/BxN mice in sost -/- and wild type (WT) mice. Arthritis was induced in DEREG mice by immunisation with recombinant glucose-6-phosphate isomerase (G6PI). To switch to the non-remitting G6PI-induced arthritis, Foxp3 + regulatory T-cells were depleted by diphtheria toxin. hTNFtg and G6PI-induced arthritis mice were treated with a neutralising antibody against human and murine sclerostin. Clinical disease severity, bone erosion, cartilage destruction and pannus formation were evaluated by histomorphometric, x-ray and micro-CT analysis. Sclerostin expression and p38 activation was assessed by immunohistochemistry, western-blot-analysisor RT-PCR. Knockdown of LRP5 and LRP6 was performed by transfection of fibroblast-like synoviocytes (FLS) with siRNA. Results This study showed for the first time that TNFα induces sclerostin expressionin RA-FLS. Surprisingly, the lack of sclerostin and its antibody-mediated inhibition led to deterioration of RA-like disease in hTNFtgmice with enhanced pannus formation and joint destruction. Suggesting a specific role for sclerostin in TNFα signalling, inhibition of sclerostin also failed to improve clinical signs and joint destruction in the partially TNFα-dependent G6PI-induced arthritis, but ameliorated disease severity in K/BxN serum transfer-induced arthritis,in which TNFα plays only a minor role. FLS from sost -/- /hTNFtg mice displayed increased TNFα-mediated p38 activation, a key step in arthritis development. In turn, sclerostin effectively blocked TNFα-induced but not IL-1-induced activation of p38 with participation of the canonical Wnt receptor LRP6. Conclusion Collectively, these data demonstrated that sclerostin appears to have a protective function in TNF-mediated chronic inflammation.

Objective Synovial fibroblasts (SFs) contribute to several aspects of the pathogenesis of rheumatoid arthritis (RA) and have been implicated most prominently in the progressive destruction of articular cartilage. Targeting the invasive phenotype of RASFs has therefore gained increasing attention, but the precise measurement of their invasive capacity and the evaluation of potential treatment effects constitute a challenge that needs to be addressed. This study used a novel in vitro invasion assay based on the breakdown of transepithelial electrical resistance to determine the course of fibroblast invasion into extracellular matrix. Methods A matrix-associated transepithelial resistance invasion (MATRIN) assay was used to assess SFs from patients with RA in comparison with SFs from patients with osteoarthritis (OA). The SFs were grown on a commercially available collagen mix that was placed onto the upper side of a Transwell polycarbonate membrane. In addition, freshly isolated cartilage extracts were studied to assess the conditions in vivo. Under this membrane, a monolayer of MDCK-C7 cells was seeded to create a high electrical resistance. Results Invasion of fibroblasts into the matrix affected the integrity of the MDCK-C7 monolayer and led to a measurable decrease and subsequent breakdown of electrical resistance. Unlike in the assay with OASFs, which did not achieve a breakdown of resistance up to 72 hours, RASFs exhibited a pronounced invasiveness in this assay, with a 50% breakdown after 42 hours. Treatment of fibroblasts with either a matrix metalloproteinase inhibitor or antibodies against β1 integrin significantly reduced the invasiveness of RASFs. Conclusion The MATRIN assay is a valuable and sensitive biologic assay system that can be used to determine precisely the invasive potential of RASFs in vitro, and thus would be suitable for screening anti-invasion compounds.

Myeloma cells express basic fibroblast growth factor (bFGF), an angiogenic cytokine triggering marrow neovascularization in multiple myeloma (MM). In solid tumors and some lymphohematopoietic malignancies, angiogenic cytokines have also been shown to stimulate tumor growth via paracrine pathways. Since interleukin-6 (IL-6) is a potent growth and survival factor for myeloma cells, we have studied the effects of bFGF on IL-6 secretion by bone marrow stromal cells (BMSCs) and its potential reverse regulation in myeloma cells. Both myeloma-derived cell lines and myeloma cells isolated from the marrow of MM patients were shown to express and secrete bFGF. Cell-sorting studies identified myeloma cells as the predominant source of bFGF in MM marrow. BMSCs from MM patients and control subjects expressed high-affinity FGF receptors R1 through R4. Stimulation of BMSCs with bFGF induced a time- and dose-dependent increase in IL-6 secretion (median, 2-fold; P

Rheumatoid arthritis (RA) is the prototype for an inflammatory arthritis, characterised by chronic inflammation and progressive joint destruction. In this context, tumour necrosis factor α (TNFα) is a key cytokine in promoting destruction of bone by increasing the number of bone-resorbing osteoclasts and decreasing the number of bone-forming osteoblasts, thereby leading to an overall bias toward bone resorption. Myostatin (GDF-8) is a member of the transforming growth factor β family that is expressed in skeletal muscle, which is also its primary target tissue. Since several lines of evidence suggest a role for myostatin also in bone homeostasis, the authors investigated the specific contribution of myostatin to pathological bone remodelling and damage in RA. Methods In vivo, myostatin expression was studied by immunohistochemistry in synovial tissues of RA and osteoarthritis (OA) patients as well as in arthritic human TNF transgenic (hTNFtg) mice. Effects of myostatin on osteoclast differentiation were evaluated by tartrate-resistant acid phosphatase (TRAP) staining and signalling pathways as well as expression of osteoclast-specific genes were analysed by immunoblotting. To assess the functional role of myostatin in vivo, myostatin knockout ( Mstn −/− ) mice were crossed with hTNFtg mice and the clinical severity of disease was assessed. In addition, histological changes including bone erosion and inflammation were evaluated by histomorphometric analyses. Results Myostatin was highly expressed in RA synovial tissues with most prominent staining of synovial fibroblasts. In contrast, only very few myostatin expressing cells were found in OA synovial tissues. In line with these results, strong expression of myostatin in joints of hTNFtg mice was observed, whereas only negligible staining was found in wild type animals. In vitro, myostatin dramatically accelerated receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast development. TRAP staining revealed the appearance of very large osteoclasts with a huge cytoplasmatic compartment and a very high number of nuclei already after 3 days. In contrast, the conventional setting of osteoclast differentiation (macrophage colony-stimulating factor and RANKL) did not generate osteoclasts during this short time period. In addition, myostatin-deficient BMMs showed a reduced RANKL-induced osteoclast formation, suggesting that myostatin is not only a paracrine stimulator of osteoclastogenesis. Histomorphometric analyses revealed that the lack of myostatin not only ameliorate the clinical severity of arthritis in hTNFtg mice but improve joint damage in this mouse model of RA. Mstn −/− /hTNFtg mice displayed less bone erosion and synovial inflammation compared to hTNFtg mice. Conclusions This data suggest that the expression of myostatin in arthritic joints is associated with an enhanced osteoclast formation and thus, significantly involved in bone destruction during chronic inflammatory arthritis.

Vascular endothelial growth factor (VEGF), a multifunctional cytokine, potently stimulates angiogenesis including tumor neovascularization. Although well established in solid tumors, the role of VEGF in bone marrow neoangiogenesis and paracrine tumor-stromal cell interactions in lymphohematopoietic malignancies has not been fully elucidated. In multiple myeloma (MM), marrow neovascularization parallels disease progression. This parallel prompted us to investigate the expression and secretion of VEGF by myeloma cells and its potential effects in myeloma-marrow stroma interactions. The biologically active splice variants VEGF165 and VEGF121 were expressed and secreted by myeloma cell lines and plasma cells isolated from the marrow of patients with MM. As shown by immunocytochemistry or RT-PCR, myeloma cells did not express or weakly expressed the VEGF receptors FLT-1 and FLK-1/KDR, indicating that autocrine stimulation is unlikely. In contrast, FLK-1/KDR was abundantly expressed by marrow stromal cells. Therefore, we studied the effects of VEGF on marrow stroma, focusing on the secretion of interleukin-6 (IL-6), a potent growth factor for myeloma cells and an inhibitor of plasma cell apoptosis. Exposure of stromal and microvascular endothelial cells to recombinant human (rh) VEGF165 or VEGF121 induced a time- and dose-dependent increase in IL-6 secretion (14- to 27-fold at 50 ng/mL after 24 hours, P

Background Directed monocyte migration, cell-cell contacts and fusion to multinucleated cells are necessary events in the process of osteoclast differentiation. Since transient receptor potential canonical channel 1 (TRPC1) has a pivotal role in these specific processes in other cell types, we investigated the role of TRPC1 in osteoclastogenesis and bone remodeling. Objectives To analyse the effect of TRPC1 knockout on in-vitro osteoclast differentiation and to study the skeletal phenotype of TRPC1-/- mice under physiological conditions as well as using an animal model of postmenopausal osteoporosis. Methods For all in-vitro experiments, bone marrow macrophages were isolated from TRPC1-/- mice and corresponding wild type (WT) controls and were cultured in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand. Osteoclast-like cells were characterized by staining for tartrate-resistant acid phosphatase (TRAP). Using quantitative real-time PCR mRNA levels of TRPC1 and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATC1) were analysed. Time-lapse videomicroscopy was used to study osteoclast migration and fusion. The intracellular Ca 2+ concentration of osteoclasts was measured ratiometrically with the fluorescent Ca 2+ dye Fura-2. The skeletal phenotype of 16-week old mice was investigated by μCT-analyses of trabecular bone in the lumbar spine. Ovariectomy was performed on 12-week old sex- and aged-matched littermates as a model for human postmenopausal bone loss. Results PCR analyses revealed that TRPC1 is hardly expressed in monocytes and pre-osteoclasts, but gets upregulated during osteoclast-differentiation. In osteoclast formation assays, the loss of TRPC1 is associated with a significantly impaired osteoclast differentiation. As seen in μCT analysis, TRPC1-/- mice showed no differences in bone phenotype compared to WT mice under physiological conditions. However, in the ovariectomy model of estrogen-deficiency mediated bone loss, TRPC1-/- mice exhibited a reduced loss of trabecular bone volume (-28,9% in WT compared to -13,1% in TRPC1-/-) and bone tissue density (-3,2% in WT compared to +0,9% in TRPC1-/-) in LW5. Analysing the underlying signaling pathways, we found no differences in Ca 2+ -oscillations and store-operated calcium entries and no differences in NFATC1 expression. In time-lapse microscopy, however, we observed a reduced capacity of TRPC1-/- osteoclast precursors to migrate and to fuse, due to a reduced velocity, translocation and a reduced increase of cell area over time. Conclusions These results provide the first evidence that TRPC1 is necessary for migration and fusion of osteoclasts and that the loss of TRPC1 while not affecting physiological bone turnover has a clear effect on accelerated bone loss as seen in estrogen deficiency. Thus, our data implicate that TRPC1 may represent a potential target for treating rapid osteoporotic bone loss. Disclosure of Interest None Declared

Background The Fibroblast Activation Protein alpha (FAPα) is an integral membrane serine protease that plays a major role in migration, wound healing, and metastasis. Based on recent studies that have implicated membrane-bound serine proteases in osteoclast migration, we studied the expression of FAPα in rheumatoid arthritis (RA) and analysed its role in osteoclast development under inflammatory conditions. Methods FAPα expression in vivo was determined by immunohistochemistry in human synovial tissues of patients with RA and osteoarthritis (OA) as well as in hind paws of tumour necrosis factor-alpha transgenic (hTNFtg) mice, which develop a RA-like destructive arthritis. In vitro expression and regulation of FAPα was analysed in differentiated osteoclasts cocultured with osteoblasts, RA or OA synovial fibroblasts (SF) by PCR. To scrutinise the role of FAPα in osteoclastogenesis, we analysed the in vitro differentiation of osteoclasts from wildtype (WT) and FAP -/- mice (Oncology Research, Boehringer Ingelheim RCV, Vienna) by TRAP staining. In order to assess the role of FAP in arthritis severity, we crossed FAP -/- and hTNFtg mice and performed TRAP staining. Results RA synovial tissues demonstrated a high expression of FAPα throughout the tissue whereas in OA samples FAPα was expressed only in the lining layer. In vitro, no expression of FAPα was found in differentiated preosteoclasts and osteoclasts, but coculture experiments showed that RASF, but not OASF or osteoblasts, induce the expression of FAPα in preosteoclasts and osteoclasts. Consistent with the selective induction of FAPα in osteoclasts by RASF, FAPα expression was detected in osteoclasts at the invasion front of the hyperplastic synovial tissues in joints of hTNFtg mice. FAP -/- mice show a severely diminished osteoclast formation compared to WT. We also found a lesser amount of osteoclasts in the hind paws of FAP -/- hTNFtg compared to hTNFtg. Conclusions The disease-dependent expression of FAPα by osteoclasts in human RA and hTNFtg mice suggests an important role of FAPα in joint destruction in RA. The selective induction of FAPα in preosteoclasts and osteoclasts by RASF indicate that FAPα may be regulated through the interaction with the pannus tissue. The fact, that under inflammatory conditions the loss of FAP led to a reduced number of osteoclasts in the hind paws and FAP deficient bone marrow derived macrophages showed a reduced osteoclast formation, suggest a role of this serine protease in macrophage/osteoclast precursor migration and differentiation.

The formation of osteoclasts involves a sequence of cellular events including monocyte migration towards each other, cell-cell contacts and fusion to multinucleated cells. Given that TRPC1has a pivotal role in these specific processes in other cell types, the authors investigated the role of TRPC1 in osteoclastogenesis and studied the skeletal phenotype of TRPC1 −/− mice under physiological conditions as well as using an animal model of postmenopausal osteoporosis. For all in vitro experiments, bone marrow macrophages were isolated from TRPC1 −/− mice and corresponding wild type (WT) controls and were cultured in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor κ-B ligand. Osteoclast-like cells were characterised by staining for tartrate-resistant acid phosphatase (TRAP). Using quantitative real-time PCR mRNA levels of TRPC1 and NFATC1 were analysed. Time-lapse videomicroscopy was used to study osteoclast migration and fusion. The intracellular Ca 2+ concentration of osteoclasts was measured ratiometrically with the fluorescent Ca 2+ dye Fura-2. The skeletal phenotype of 16-week old mice was investigated by μCT-analyses of trabecular bone in the lumbar spine. Ovariectomy was performed on 12-week old sex- and aged-matched littermates as a model for human postmenopausal bone loss. PCR analyses indicate that TRPC1 is hardly expressed in monocytes and preosteoclasts but gets upregulated during osteoclast-differentiation. In osteoclast formation assays, the loss of TRPC1 leads to impaired osteoclast differentiation with a 75% reduction in large osteoclasts. While µCT analysis revealed no differences in bone phenotype of TRPC1 −/− mice compared to WT mice under physiological conditions, there were significant effects in the ovariectomy model of estrogen-deficiency mediated bone loss. In this model, TRPC1 −/− mice exhibited a reduced loss of trabecular bone volume (−28.9% in WT compared to −13.1% in TRPC1 −/− ) and BTD (−3.2% in WT compared to +0.9% in TRPC1 −/− ) in LW5. Analysing the underlying signaling pathways, the authors found no differences in Ca 2+ -oscillations and store-operated calcium entries and no differences in NFATC1 expression. In time-lapse microscopy, however, the authors observed a reduced capacity of TRPC1 −/− osteoclast precursors to migrate and to fuse, due to a reduced velocity, translocation and a reduced increase of cell area over time. These results indicate that TRPC1 dependent pathways contribute to the migration and fusion of osteoclasts and that the loss of TRPC1 while not affecting physiological bone turnover has a clear effect on accelerated bone loss as seen in estrogen deficiency. Therefore, these data suggest that TRPC1 may be a new therapeutic target for rapid osteoporotic bone loss.

Background Syndecan-4 (sdc4) is a transmembrane heparan sulfate proteoglycan that in addition to functioning as an adhesion a molecule can bind cytokines and impact on inflammation. Modulation of inflammatory signals by sdc4 may occur either through mere binding of cytokines, in which case sdc4 acts as decoy receptor or through initiation of sdc-dependent signalling following sdc4 complex formation. While arthritic cartilage damage is decreased in sdc4-deficient mice most likely due to reduced sdc4 signaling, osteopontin- mediated liver damage has been shown to be increased in these mice due to the lack of scd4 decoy receptor function. Based on this dual and in part opposing effects of sdc4, the aim of this study was to investigate if the loss of sdc4 changes the natural course of murine experimental colitis. Methods Experimental Colitis was induced in sdc4-/- mice and in C57BL/6 WT mice by DSS. The course of colitis was monitored by weight loss as well as assessment of colon length and blinded histological scoring of colonic changes at the end of the experiment. In addition, sdc4-/- and C57BL/6 WT mice were orally gavaged with 5×108 colony-forming units (CFU) of Citrobacter rodentium. Fecal excretion of C rodentium and changes of body weight were monitored. At day 21 postinfection, inflammatory changes of the colon were evaluated histologically. Results Beginning from day 5 after start of DSS-administration, sdc4-/- mice lost dramatically more body weight compared to WT animals (day 8: 24.8%±1.9 vs 9.2%±3.1; p=0.008). In accordance with the increased loss of body weight, the colon length of sdc4-/- mice was significantly shortened (63.3 mm±2.4 vs 74.8±2.3; p=0.01) and the histological damage according to the Dieleman-Score was markedly aggravated (16AU±3.7 vs 3.4AU±0.2; p=0.016). At day 19 postinfection, the fecal excretion of C rodentium in sdc4-/- was prolonged compared to WT animals (2.5×105±6.8×103 vs 9.6×103±6.5×102; p=0.01). Histological damage of colonic mucosa, reflected by lengthening of crypts, was increased in Sdc4-/- mice (14.3AU±1.3 vs 10.2AU±0.9; p=0.03). Conclusions The data suggest that like in inflammatory liver damage, sdc4 exerts protective effects in intestinal inflammation. Future studies are needed to explore the underlying mechanisms and to determine if these effects are due to a decoy receptor function of sdc4 or whether sdc4 complex formation and signalling is involved.

A genomic clone containing 2.3 kilobases (kb) of the 5' flanking region of the human follicle-stimulating hormone receptor (FSHR) plus the translated region of exon 1 and subsequent sequences of intron A has been isolated and characterized. This portion of the 5' flanking region has neither a TATA nor a CCAAT box and shows features of promoters seen in "housekeeping" genes. Using RNAse protection multiple transcriptional start sites could be identified, the major ones clustered between -114 and -79 bp. Chimeras containing 1486 bp of the 5' flanking region, or deletions thereof, expressed significant chloramphenicol acetyltransferase (CAT) activity when transiently transfected into Chinese hamster ovary (CHO), primary rat Sertoli and human granulosa-lutein cells. Deletion analyses indicated that a proximal promoter can be allocated to the region from -225 to -1 bp.

Background In rheumatoid arthritis (RA), progressive joint destruction is a hallmark of disease and results from both increased bone resorption and the lack of repair mechanisms. tumour necrosis factor α (TNFα) contributes to both aspects of pathologic joint remodelling by increasing the number of bone-resorbing osteoclasts and decreasing the number of bone-forming osteoblasts. Sclerostin is a potent inhibitor of osteoblast development by antagonising the Wnt/β-catenin signalling pathway. Based on recent data that have shown increased expression of sclerostin under inflammatory conditions, the authors studied its expression in human RA and in human TNF transgenic (hTNFtg) mice, which develop a RA-like destructive arthritis. Moreover, the authors analysed the effects of sclerostin deficiency on the development and severity of the arthritis in these mice. Methods Expression of sclerostin was determined by immunohistochemistry, western blot and reverse transcriptase PCR. To assess the functional role of sclerostin in vivo, sclerostin knockout ( SOST −/− ) mice were crossed with hTNFtg mice. In addition to determining the clinical severity of disease in SOST −/− /hTNFtg and hTNFtg mice, histological changes including bone erosion, cartilage destruction and inflammation were evaluated by histomorphometric analyses. The number of osteoclasts was quantified using tartrate-resistant acid phosphatase staining. Immunohistochemistry was performed to analyse the expression of molecules of the Wnt pathway. Results Immunohistochemistry and western blot analyses revealed a strong overexpression of sclerostin in synovial tissue of RA compared to osteoarthritis patients. Likewise, ankle joints of hTNFtg mice showed high levels of sclerostin, especially in the infiltrating pannus, whereas only negligible staining was observed in wild-type animals. In vitro, expression of sclerostin was only found in osteoblasts and osteoclasts, but could be induced in RA synovial fibroblasts by TNFα. Surprisingly, the lack of sclerostin not only increased the clinical severity of arthritis in hTNFtg mice but most dramatically accelerated joint damage in this mouse model of RA. SOST −/− /hTNFtg mice displayed significant higher bone erosion, synovial hyperplasia and osteoclast numbers compared to hTNFtg mice. Moreover, immunohistochemistry revealed higher levels of dickkopf 1 (DKK-1) and Wnt-5a in joints of SOST-deficient hTNFtg mice. Conclusions The authors hypothesise that under inflammatory conditions, higher levels of DKK-1 and Wnt-5a in joints of sclerostin-deficient arthritic mice counteract the beneficial effect of sclerostin deficiency by increasing osteoclast development through enhanced blockade of the Wnt-3a pathway by DKK-1 as well as by promoting synovial hyperplasia through Wnt-5a-mediated synoviocyte activation. These results may have an impact on the use of sclerostin inhibitors in inflammatory joint diseases.

The matrix metalloproteinases (MMPs) and their endogenous regulators, the tissue inhibitors of MMPs (TIMPs 1–4) are responsible for the physiological remodelling of the extracellular matrix (ECM) by maintaining the balance between matrix destruction and formation. An imbalance between MMPs and TIMPs leads to excess MMP activity and is associated with ECM destruction in various inflammatory conditions such as rheumatoid arthritis and malignant tumours. Among all TIMPs, TIMP3 appears to play …

Joints affected by rheumatoid arthritis (RA) display signs of bone resorption and usually lack signs of repair which contributes to rapid and progressive loss of joint structures. Sclerostin is a potent inhibitor of osteoblast development by antagonising the Wnt/β-catenin signalling pathway, a key regulator of bone turnover. Since it has been shown that sclerostin is expressed under inflammatory conditions, we analysed the …

Introduction Inflammatory destructive arthritis, like rheumatoid arthritis (RA), is characterized by invasion of synovial fibroblasts (SF) into the articular cartilage and erosion of the underlying bone, leading to progressive joint destruction. Because fibroblast activation protein alpha (FAP) has been associated with cell migration and cell invasiveness, we studied the function of FAP in joint destruction in RA. Methods Expression of FAP in synovial tissues and fibroblasts from patients with osteoarthritis (OA) and RA as well as from wild-type and arthritic mice was evaluated by immunohistochemistry, fluorescence microscopy and polymerase chain reaction (PCR). Fibroblast adhesion and migration capacity was assessed using cartilage attachment assays and wound-healing assays, respectively. For in vivo studies, FAP-deficient mice were crossed into the human tumor necrosis factor transgenic mice (hTNFtg), which develop a chronic inflammatory arthritis. Beside clinical assessment, inflammation, cartilage damage, and bone erosion were evaluated by histomorphometric analyses. Results RA synovial tissues demonstrated high expression of FAP whereas in OA samples only marginal expression was detectable. Consistently, a higher expression was detected in arthritis SF compared to non-arthritis OA SF in vitro. FAP-deficiency in hTNFtg mice led to less cartilage degradation despite unaltered inflammation and bone erosion. Accordingly, FAP−/− hTNFtg SF demonstrated a lower cartilage adhesion capacity compared to hTNFtg SF in vitro. Conclusions These data point to a so far unknown role of FAP in the attachment of SF to cartilage, promoting proteoglycan loss and subsequently cartilage degradation in chronic inflammatory arthritis.