Svetlana Frank

Cartilage biology, pathology, and repair

Osteoarthritis is one of the most common forms of musculoskeletal disease and the most prominent type of arthritis encountered in all countries. Although great efforts have been made to investigate cartilage biology and osteoarthritis pathology, the treatment has lagged behind that of other arthritides, as there is a lack of effective disease-modifying therapies. Numerous approaches for dealing with cartilage degradation have been tried, but enjoyed very little success to develop approved OA treatments with not only symptomatic improvement but also structure-modifying effect. In this review we discuss the most recent findings regarding the regulation of cartilage biology and pathology and highlight their potential therapeutic values.

Myostatin is a direct regulator of osteoclast differentiation and its inhibition reduces inflammatory joint destruction in mice

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. However, myostatin deficiency also increases bone formation, mainly through loading-associated effects on bone. 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. 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.

Sclerostin inhibition promotes TNF-dependent inflammatory joint destruction

Blocking the Wnt inhibitor sclerostin leads to inflammatory joint destruction through enhanced activation of TNF receptor–mediated signaling, implicating sclerostin as protective in TNFα-dependent chronic inflammation. A surprising role for sclerostin in arthritis Antibodies that block the activity of sclerostin, a bone destruction molecule, are in clinical trials for the treatment of osteoporosis. But these antibodies may not be safe for certain patients: those with inflammatory rheumatoid arthritis (RA). Wehmeyer et al. were surprised to find that sclerostin inhibition did not stop bone loss and actually aggravated disease in an animal model of RA that was dependent on tumor necrosis factor α (TNFα); two other rodent RA models with minimal or no dependence on this inflammatory cytokine were unaffected. The authors found that sclerostin blocks TNFα-induced p38 and NFκB activation—key steps in RA development. Thus, sclerostin appears to have a protective role in TNF-mediated chronic inflammation, and inhibiting it would be contraindicated in a subset of RA patients. This study therefore has immediate implications for current clinical trials involving patients with inflammatory bone loss. Sclerostin, an inhibitor of the Wnt/β-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 TNFα (tumor necrosis factor α)–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 TNFα 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 TNFα-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 TNFα signaling. Sclerostin effectively blocked TNFα- 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 TNFα-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 TNFα-dependent comorbidities.

The loss of α2β1 integrin suppresses joint inflammation and cartilage destruction in mouse models of rheumatoid arthritis.

OBJECTIVE Integrin α2β1 functions as a major receptor for type I collagen on different cell types, including fibroblasts and inflammatory cells. Although in vitro data suggest a role for α2β1 integrin in regulating both cell attachment and expression of matrix-degrading enzymes such as matrix metalloproteinases (MMPs), mice that lack the α2 integrin subunit (Itga2(-/-) mice) develop normally and are fertile. We undertook this study to investigate the effect of Itga2 deficiency in 2 different mouse models of destructive arthritis: the antigen-induced arthritis (AIA) mouse model and the human tumor necrosis factor α (TNFα)-transgenic mouse model. METHODS AIA was induced in the knee joints of Itga2(-/-) mice and wild-type controls. Human TNF-transgenic mice were crossed with Itga2(-/-) mice and were assessed clinically and histopathologically for signs of arthritis, inflammation, bone erosion, and cartilage damage. MMP expression, proliferation, fibroblast attachment, and ERK activation were determined. RESULTS Under arthritic conditions, Itga2 deficiency led to decreased severity of joint pathology. Specifically, Itga2(-/-) mice showed less severe clinical symptoms and dramatically reduced pannus formation and cartilage erosion. Mice lacking α2β1 integrin exhibited reduced MMP-3 expression, both in their sera and in fibroblast-like synoviocytes (FLS), due to impaired ERK activation. Further, both the proliferation and attachment of FLS to cartilage were partially dependent on α2β1 integrin in vitro and in vivo. CONCLUSION Our findings suggest that α2β1 integrin contributes significantly to inflammatory cartilage destruction by promoting fibroblast proliferation and attachment and MMP expression.

Molecular mechanisms of cartilage remodelling in osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease that is characterized primarily by progressive breakdown of articular cartilage. The loss of proteoglycans, the mineralization of the extracellular matrix (ECM) and the hypertrophic differentiation of the chondrocytes constitute hallmarks of the disease. The pathogenesis of OA includes several pathways, which in single are very well investigated and partly understood, but in their complex interplay remain mainly unclear. This review summarises recent data on the underlying mechanisms, specifically with respect to cell-matrix interactions and cartilage mineralization. It points out why these findings are of importance for future OA research and for the development of novel therapeutic strategies to treat OA.

TRPC6 Regulates CXCR2-Mediated Chemotaxis of Murine Neutrophils

Unraveling the mechanisms involved in chemotactic navigation of immune cells is of particular interest for the development of new immunoregulatory therapies. It is generally agreed upon that members of the classical transient receptor potential channel family (TRPC) are involved in chemotaxis. However, the regulatory role of TRPC channels in chemoattractant receptor-mediated signaling has not yet been clarified in detail. In this study, we demonstrate that the TRPC6 channels play a pronounced role in CXCR2-mediated intermediary chemotaxis, whereas N-formyl-methionine-leucine-phenylalanine receptor–mediated end-target chemotaxis is TRPC6 independent. The knockout of TRPC6 channels in murine neutrophils led to a strongly impaired intermediary chemotaxis after CXCR2 activation which is not further reinforced by CXCR2, PI3K, or p38 MAPK inhibition. Furthermore, CXCR2-mediated Ca2+ influx but not Ca2+ store release was attenuated in TRPC6−/− neutrophils. We demonstrate that the TRPC6 deficiency affected phosphorylation of AKT and MAPK downstream of CXCR2 receptor activation and led to altered remodeling of actin. The relevance of this TRPC6-depending defect in neutrophil chemotaxis is underscored by our in vivo findings. A nonseptic peritoneal inflammation revealed an attenuated recruitment of neutrophils in the peritoneal cavity of TRPC6−/− mice. In summary, this paper defines a specific role of TRPC6 channels in CXCR2-induced intermediary chemotaxis. In particular, TRPC6-mediated supply of calcium appears to be critical for activation of downstream signaling components.

Regulation of matrixmetalloproteinase-3 and matrixmetalloproteinase-13 by SUMO-2/3 through the transcription factor NF-κB

Objective Based on previous data that have linked the small ubiquitin-like modifier-1 (SUMO-1) to the pathogenesis of rheumatoid arthritis (RA), we have investigated the expression of the highly homologous SUMO family members SUMO-2/3 in human RA and in the human tumour necrosis factor α transgenic (hTNFtg) mouse model of RA and studied their role in regulating disease specific matrixmetalloproteinases (MMPs). Methods Synovial tissue was obtained from RA and osteoarthritis (OA) patients and used for histological analyses as well as for the isolation of synovial fibroblasts (SFs). The expression of SUMO-2/3 in RA and OA patients as well as in hTNFtg and wild type mice was studied by PCR, western blot and immunostaining. SUMO-2/3 was knocked down using small interfering RNA in SFs, and TNF-α induced MMP production was determined by ELISA. Activation of nuclear factor-κB (NF-κB) was determined by a luciferase activity assay and a transcription factor assay in the presence of the NF-κB inhibitor BAY 11-7082. Results Expression of SUMO-2 and to a lesser extent of SUMO-3 was higher in RA tissues and RASFs compared with OA controls. Similarly, there was increased expression of SUMO-2 in the synovium and in SFs of hTNFtg mice compared with wild type animals. In vitro, the expression of SUMO-2 but not of SUMO-3 was induced by TNF-α. The knockdown of SUMO-2/3 significantly increased the TNF-α and interleukin (IL)-1β induced expression of MMP-3 and MMP-13, accompanied by increased NF-κB activity. Induction of MMP-3 and MMP-13 was inhibited by blockade of the NF-κB pathway. TNF-α and IL-1β mediated MMP-1 expression was not regulated by SUMO-2/3. Conclusions Collectively, we show that despite their high homology, SUMO-2/3 are differentially regulated by TNF-α and selectively control TNF-α mediated MMP expression via the NF-κB pathway. Therefore, we hypothesise that SUMO-2 contributes to the specific activation of RASF.

02.14 Small ubiquitin related modifier-1 (sumo-1) regulates osteoclast differentiation

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 ß3 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.

A10.14 Inhibition of sclerostin accelerates TNFα-mediated bone destruction

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.

A8.12 Small Ubiquitin Related Modifier-1 (SUMO-1) Regulates Osteoclastogenesis

Background and Objectives Rheumatoid arthritis (RA) is a common autoimmune disease characterised by the hyperplastic transformation of synovium, its infiltration with different inflammatory cells and by stimulation of bone resorption through osteoclast activation leading to joint destruction. Posttranslational modification of proteins by SUMO has been shown for a number of target molecules including transcription factors and is involved in a variety of cellular processes, including protein localisation, transcriptional regulation, protein stability, cell survival and death. Previously, we have shown that the increased expression of SUMO-1 contributes to the inflammatory response in RA. Here, we investigated the role of SUMO-1 in osteoclastogenesis and studied the skeletal phenotype of SUMO-1 -/- mice under physiological conditions. Materials and Methods For all in vitro experiments, bone marrow macrophages were isolated from SUMO-1 -/- mice and wild type (WT) controls and were cultured in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor k-B ligand. Osteoclast differentiation was verified by tartrate-resistant acid phosphatase (TRAP) staining. Using real time PCR mRNA levels of DC-STAMP and Cathepsin K were analysed. Proliferation of preosteoclasts was determined using CyQuant proliferation assay. Osteoclast resorption capacity was analysed using a calcium phosphate bone resorption assay. The skeletal phenotype of 8-week old mice was investigated by µCT-analysis of trabecular bone in the lumbar spine and femora. The vertebral bodies L5 from each animal were dehydrated and embedded nondecalcified into methylmetacrylate for sectioning. Sections were stained using van Kossa and for TRAP activity. Results In PCR analyses, we found decreased expression of DC-STAMP and Cathepsin K in SUMO-1 -/- mice compared to wt mice during osteoclast differentiation. Proliferation of preosteoclasts was not affected by loss of SUMO-1. In osteoclast formation assays, the loss of SUMO-1 was associated with impaired osteoclast differentiation and with impaired bone resorption capacity. In addition, histological analyses revealed a reduced number of osteoclasts in SUMO-1 -/- mice. At 8-weeks old, SUMO-1 -/- mice had a 20% higher trabecular bone volume fraction compared with wt mice. Moreover, trabecular thickness was higher and trabecular separation was lower in SUMO-1 -/- mice. Conclusions In our study, we found that SUMO-1 -/- mice have high bone mass owing to a decrease in number, size and function of osteoclasts. Furthermore, osteoclast markers contributing to osteoclast fusion and to osteoclast resorption capacity were decreased. These data suggest that SUMO-1 is involved predominantly 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.