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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 regeneration 1 , 2 , 3 , 4 , 5 . However, myostatin deficiency also increases bone formation, mainly through loading-associated effects on bone 6 , 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 RA 12 . 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.

Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.

Bone resorption is highly dependent on the dynamic rearrangement of the osteoclast actin cytoskeleton to allow formation of actin rings and a functional ruffled border. Hem1 is a hematopoietic-specific subunit of the WAVE-complex which regulates actin polymerization and is crucial for lamellipodia formation in hematopoietic cell types. However, its role in osteoclast differentiation and function is still unknown. Here, we show that although the absence of Hem1 promotes osteoclastogenesis, the ability of Hem1 -/- osteoclasts to degrade bone was severely impaired. Global as well as osteoclast-specific deletion of Hem1 in vivo revealed increased femoral trabecular bone mass despite elevated numbers of osteoclasts in vivo. We found that the resorption defect derived from the morphological distortion of the actin-rich sealing zone and ruffled border deformation in Hem1-deficient osteoclasts leading to impaired vesicle transport and increased intracellular acidification. Collectively, our data identify Hem1 as a yet unknown key player in bone remodeling by regulating ruffled border formation and consequently the resorptive capacity of osteoclasts.

Transient receptor potential channel 1 (TRPC1) is a widely expressed mechanosensitive ion channel located within the endoplasmic reticulum membrane, crucial for refilling depleted internal calcium stores during activation of calcium-dependent signaling pathways. Here, we have demonstrated that TRPC1 activity is protective within cartilage homeostasis in the prevention of cellular senescence–associated cartilage breakdown during mechanical and inflammatory challenge. We revealed that TRPC1 loss is associated with early stages of osteoarthritis (OA) and plays a nonredundant role in calcium signaling in chondrocytes. Trpc1 –/– mice subjected to destabilization of the medial meniscus–induced OA developed a more severe OA phenotype than WT controls. During early OA development, Trpc1 –/– mice displayed an increased chondrocyte survival rate; however, remaining cells displayed features of senescence including p16 INK4a expression and decreased Sox9. RNA-Seq identified differentially expressed genes related to cell number, apoptosis, and extracellular matrix organization. Trpc1 –/– chondrocytes exhibited accelerated dedifferentiation, while demonstrating an increased susceptibility to cellular senescence. Targeting the mechanism of TRPC1 activation may be a promising therapeutic strategy in OA prevention.

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/d ColVI-Cre) but not in myeloid cells (ActβAd/d LysM-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/d LysM-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.

BackgroundThe ubiquitously expressed transmembrane heparan sulfate proteoglycan Syndecan-4 (Scd4) is crucial in inflammatory diseases, like rheumatoid arthritis. Depending on the tissue, it can either protect or promote an inflammatory process. By its binding of molecules, such as cytokines and growth factors, it can initiate signalling pathways and it has been implicated in cell-matrix adhesion, cell migration, differentiation as well as proliferation. However, the involvement of Sdc4 in intestinal inflammation is unknown so far. Our group revealed a protective function of Sdc4 in experimental intestinal inflammation.Material and methodsWe monitored the course of DSS-induced colitis in Scd4-/- and C57BL/6 WT mice and analysed the changes in body weight, colon length, histology and inflammatory cellular infiltrate. We also evaluated Sdc4 protein- and mRNA-level by immunofluorescence staining (IF) and quantitative real-time PCR. Colon-permeability was examined in vivo by using the Evans Blue method and measuring the clearance for Citrobacter rodentium in vivo. Wound healing effects of Scd4 were analysed in vitro by scratch assay analysis with human epithelial colon cell line (T-84) and in vivo by mechanically induced wounds in colonoscopies of Scd4-/- compared to WT mice.ResultsThe expression of Scd4 is decreased upon the course of colitis and increased during remission. The course of colitis was markedly aggravated in Scd4-/- mice, reflected by dramatically loss of body weight, increased mortality rates and histological damage, emphasised by increased invasion of macrophages and granulocytes into the colon. Also colonic epithelial permeability of DSS-treated Scd4-/- mice was enhanced associated with an altered expression of tight junction proteins. Furthermore, Sdc4 deficiency resulted in a prolonged intestinal wound healing in vitro and in vivo due to reduced proliferation rates in vitro.ConclusionsOur data indicates that Scd4 is crucial in experimental intestinal inflammation. It exerts protective effects by maintaining epithelial barrier integrity and regeneration. Further studies are needed to explore the mechanisms of Sdc4-signalling in colitis.

Abstract Background The transmembrane heparan sulfate proteoglycan Syndecan-4 (Sdc4) plays an important role in the regulation of various inflammatory disorders. However, the involvement of Sdc4 in intestinal inflammation remains unknown. Therefore, we assessed the impact of Sdc4 deficiency on experimental colitis and epithelial wound healing in vitro and in vivo. Methods Dextran sulfate sodium (DSS)-induced colitis was monitored in wild type and Sdc4-deficient (Sdc4-/-) mice by assessment of body weight, histology, inflammatory cellular infiltration, and colon length. Syndecan-4 expression was measured by immunohistochemistry, Western blot, and quantitative real-time PCR. Epithelial permeability was evaluated by Evans blue measurements, Western blot, and immunohistological analysis of tight junction protein expression. Impact of Sdc4 on epithelial wound healing was determined by scratch assay in vitro and by colonoscopy following mechanical wounding in vivo. Results In Sdc4-/- mice, colitis-like symptoms including severe weight loss, shortened colon length, histological damage, and invasion of macrophages and granulocytes were markedly aggravated compared with wild type (WT) animals. Moreover, colonic epithelial permeability in Sdc4-/- mice was enhanced, while tight junction protein expression decreased. Furthermore, Sdc4-/- colonic epithelial cells had lower cell proliferation and migration rates which presented in vivo as a prolonged intestinal wound healing phenotype. Strikingly, in WT animals, Sdc4 expression was reduced during colitis and was elevated during recovery. Conclusions The loss of Sdc4 aggravates the course of experimental colitis, potentially through impaired epithelial cell integrity and regeneration. In view of the development of current treatment approaches involving Sdc4 inhibition for inflammatory disorders like arthritis, particular caution should be taken in case of adverse gastrointestinal side-effects.

BackgroundThe ubiquitously expressed transmembrane heparan sulfate proteoglycan Syndecan-4 (Scd4) is crucial in inflammatory diseases, like rheumatoid arthritis. Depending on the tissue, it can either protect or promote an inflammatory process. By its binding of molecules, such as cytokines and growth factors, it can initiate signalling pathways and it has been implicated in cell-matrix adhesion, cell migration, differentiation as well as proliferation. However, the involvement of Sdc4 in intestinal inflammation is unknown so far. Our group revealed a protective function of Sdc4 in experimental intestinal inflammation.Material and methodsWe monitored the course of DSS-induced colitis in Scd4-/- and C57BL/6 WT mice and analysed the changes in body weight, colon length, histology and inflammatory cellular infiltrate. We also evaluated Sdc4 protein- and mRNA-level by immunofluorescence staining (IF) and quantitative real-time PCR. Colon-permeability was examined in vivo by using the Evans Blue method and measuring the clearance for Citrobacter rodentium in vivo. Wound healing effects of Scd4 were analysed in vitro by scratch assay analysis with human epithelial colon cell line (T-84) and in vivo by mechanically induced wounds in colonoscopies of Scd4-/- compared to WT mice.ResultsThe expression of Scd4 is decreased upon the course of colitis and increased during remission. The course of colitis was markedly aggravated in Scd4-/- mice, reflected by dramatically loss of body weight, increased mortality rates and histological damage, emphasised by increased invasion of macrophages and granulocytes into the colon. Also colonic epithelial permeability of DSS-treated Scd4-/- mice was enhanced associated with an altered expression of tight junction proteins. Furthermore, Sdc4 deficiency resulted in a prolonged intestinal wound healing in vitro and in vivo due to reduced proliferation rates in vitro.ConclusionsOur data indicates that Scd4 is crucial in experimental intestinal inflammation. It exerts protective effects by maintaining epithelial barrier integrity and regeneration. Further studies are needed to explore the mechanisms of Sdc4-signalling in colitis.

BackgroundPsoriatic-Arthritis (PsA) is a type of inflammatory chronic arthritis with a seronegative spondyloarthropathy and associated psoriasis. The Danger-Associated Molecular Pattern molecules (DAMPs) S100A8 and S100A9 are both antimicrobial proteins with chemotactic activity and are the most abundant DAMPs expressed during many inflammatory disorders. The expression of the S100A8/S100A9-complex is highly elevated in psoriasis and psoriatic arthritis.However, the mechanisms that regulate S100A8/S100A9-complex-activities are poorly understood, which has led us to examine the role of S100A8 and S100A9 under chronic inflammatory conditions.Material and methodsWe crossed S100A9-deficient mice with TTP (tristetraprolin)-deficient mice into a systemic inflammatory model featuring high levels of TNF with an arthritic joint destruction phenotype. Disease progression in TTP-/- x S100A9-/- mice was analysed by immunostaining, immunohistochemistry and the adapted PASI-score. To neutralise TNF in TTP-/- x S100A9-/- mice we used an aTNF-inhibiting monoclonal antibody already in clinical use for therapy of arthritis and psoriasis. To measure altered protein levels we used Western blot analysis. Primary keratinocytes were isolated of the skin from newborn mice and infected with E.coli isolated from the faeces of mice.ResultsTTP/S100A9 deficiency led to highly elevated levels of the S100A9 complex partner S100A8 in the epidermis and to a severe psoriatic phenotype of TTP-/- x S100A9-/- mice. Furthermore the mice showed an accelerated course of arthritis compared to TTP-/- mice, including increased articular cartilage loss and bone destruction. Inhibition of TNF by application of anti-TNF clearly reduced the psoriatic phenotype of TTP-/- x S100A9-/- mice. Additionally, the reduction of the environmental bacterial levels led to a milder phenotype and decelerated pathogenesis. The in vitro infection of isolated keratinocytes with isolated E.coli resulted in a high expression of S100A8.ConclusionsThe data reveal that the S100A8/S100A9-complex acts not only as a systemic danger signal molecule, but is also TNF dependent and is essential for the regulation of inflammation. The loss of S100A9 led to a disregulated inflammatory response and this to a severe psoriasis with enhanced cartilage and bone destruction. Furthermore, an exogenic bacterial factor, such as E. coli, is also demonstrated to be important in the activation of the disease.

BackgroundPsoriatic-Arthritis (PsA) is a type of inflammatory chronic arthritis with a seronegative spondyloarthropathy and associated psoriasis. The Danger-Associated Molecular Pattern molecules (DAMPs) S100A8 and S100A9 are both antimicrobial proteins with chemotactic activity and are the most abundant DAMPs expressed during many inflammatory disorders. The expression of the S100A8/S100A9-complex is highly elevated in psoriasis and psoriatic arthritis.However, the mechanisms that regulate S100A8/S100A9-complex-activities are poorly understood, which has led us to examine the role of S100A8 and S100A9 under chronic inflammatory conditions.Material and methodsWe crossed S100A9-deficient mice with TTP (tristetraprolin)-deficient mice into a systemic inflammatory model featuring high levels of TNF with an arthritic joint destruction phenotype. Disease progression in TTP-/- x S100A9-/- mice was analysed by immunostaining, immunohistochemistry and the adapted PASI-score. To neutralise TNF in TTP-/- x S100A9-/- mice we used an aTNF-inhibiting monoclonal antibody already in clinical use for therapy of arthritis and psoriasis. To measure altered protein levels we used Western blot analysis. Primary keratinocytes were isolated of the skin from newborn mice and infected with E.coli isolated from the faeces of mice.ResultsTTP/S100A9 deficiency led to highly elevated levels of the S100A9 complex partner S100A8 in the epidermis and to a severe psoriatic phenotype of TTP-/- x S100A9-/- mice. Furthermore the mice showed an accelerated course of arthritis compared to TTP-/- mice, including increased articular cartilage loss and bone destruction. Inhibition of TNF by application of anti-TNF clearly reduced the psoriatic phenotype of TTP-/- x S100A9-/- mice. Additionally, the reduction of the environmental bacterial levels led to a milder phenotype and decelerated pathogenesis. The in vitro infection of isolated keratinocytes with isolated E.coli resulted in a high expression of S100A8.ConclusionsThe data reveal that the S100A8/S100A9-complex acts not only as a systemic danger signal molecule, but is also TNF dependent and is essential for the regulation of inflammation. The loss of S100A9 led to a disregulated inflammatory response and this to a severe psoriasis with enhanced cartilage and bone destruction. Furthermore, an exogenic bacterial factor, such as E. coli, is also demonstrated to be important in the activation of the disease.