Ingmar Meinecke

Syndecan-4 regulates ADAMTS-5 activation and cartilage breakdown in osteoarthritis

Aggrecan cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 5 (ADAMTS-5) is crucial for the breakdown of cartilage matrix during osteoarthritis, a degenerative joint disease that leads to the progressive destruction of articular structures. The mechanisms of ADAMTS-5 activation and their links to the pathogenesis of osteoarthritis remain poorly understood, but syndecans have been shown to be involved in the activation of ADAMTS-4 (ref. 3). Here we show that syndecan-4 is specifically induced in type X collagen–producing chondrocytes both in human osteoarthritis and in murine models of the disease. The loss of syndecan-4 in genetically modified mice and intra-articular injections of syndecan-4–specific antibodies into wild-type mice protect from proteoglycan loss and thereby prevent osteoarthritic cartilage damage in a surgically induced model of osteoarthritis. The occurrence of less severe osteoarthritis-like cartilage destruction in both syndecan-4–deficient mice and syndecan-4–specific antibody–treated wild-type mice results from a marked decrease in ADAMTS-5 activity. Syndecan-4 controls the activation of ADAMTS-5 through direct interaction with the protease and through regulating mitogen-activated protein kinase (MAPK)-dependent synthesis of matrix metalloproteinase-3 (MMP-3). Our data suggest that strategies aimed at the inhibition of syndecan-4 will be of great value for the treatment of cartilage damage in osteoarthritis.

Modification of nuclear PML protein by SUMO-1 regulates Fas-induced apoptosis in rheumatoid arthritis synovial fibroblasts

The small ubiquitin-like modifier (SUMO)-1 is an important posttranslational regulator of different signaling pathways and involved in the formation of promyelocytic leukemia (PML) protein nuclear bodies (NBs). Overexpression of SUMO-1 has been associated with alterations in apoptosis, but the underlying mechanisms and their relevance for human diseases are not clear. Here, we show that the increased expression of SUMO-1 in rheumatoid arthritis (RA) synovial fibroblasts (SFs) contributes to the resistance of these cells against Fas-induced apoptosis through increased SUMOylation of nuclear PML protein and increased recruitment of the transcriptional repressor DAXX to PML NBs. We also show that the nuclear SUMO-protease SENP1, which is found at lower levels in RA SFs, can revert the apoptosis-inhibiting effects of SUMO-1 by releasing DAXX from PML NBs. Our findings indicate that in RA SFs overexpression of SENP1 can alter the SUMO-1-mediated recruitment of DAXX to PML NBs, thus influencing the proapoptotic effects of DAXX. Accumulation of DAXX in PML NBs by SUMO-1 may, therefore, contribute to the pathogenesis of inflammatory disorders.

Gene Transfer of Tissue Inhibitor of Metalloproteinases-3 Reverses the Inhibitory Effects of TNF-α on Fas-Induced Apoptosis in Rheumatoid Arthritis Synovial Fibroblasts

Apart from counteracting matrix metalloproteinases, tissue inhibitor of metalloproteinases-3 (TIMP-3) has proapoptotic properties. These features have been attributed to the inhibition of metalloproteinases involved in the shedding of cell surface receptors such as the TNFR. However, little is known about effects of TIMP-3 in cells that are not susceptible to apoptosis by TNF-α. In this study, we report that gene transfer of TIMP-3 into human rheumatoid arthritis synovial fibroblasts and MRC-5 human fetal lung fibroblasts facilitates apoptosis and completely reverses the apoptosis-inhibiting effects of TNF-α. Although TNF-α inhibits Fas/CD95-induced apoptosis in untransfected and mock-transfected cells, fibroblasts ectopically expressing TIMP-3 are sensitized most strongly to Fas/CD95-mediated cell death by TNF-α. Neither synthetic MMP inhibitors nor glycosylated bioactive TIMP-3 are able to achieve these effects. Gene transfer of TIMP-3 inhibits the TNF-α-induced activation of NF-κB in rheumatoid arthritis synovial fibroblasts and reduces the up-regulation of soluble Fas/CD95 by TNF-α, but has no effects on the cell surface expression of Fas. Collectively, our data demonstrate that intracellularly produced TIMP-3 not only induces apoptosis, but also modulates the apoptosis-inhibiting effects of TNF-α in human rheumatoid arthritis synovial fibroblast-like cells. Thus, our findings may stimulate further studies on the therapeutic potential of gene transfer strategies with TIMP-3.

Altered expression of membrane-bound and soluble CD95/Fas contributes to the resistance of fibrotic lung fibroblasts to FasL induced apoptosis.

BackgroundAn altered susceptibility of lung fibroblasts to Fas-induced apoptosis has been implicated in the pathogenesis of pulmonary fibrosis; however, the underlying mechanism is not completely understood. Here, we studied the susceptibility of lung fibroblasts, obtained from patients with (f-fibs) and without pulmonary fibrosis (n-fibs), to FasL- (CD95L/APO-1) induced apoptosis in relation to the expression and the amounts of membrane-bound and soluble Fas. We also analysed the effects of tumor necrosis factor-β on FasL-induced cell death.MethodsApoptosis was induced with recombinant human FasL, with and without prior stimulation of the fibroblasts with tumor necrosis factor-α and measured by a histone fragmentation assay and flow cytometry. The expression of Fas mRNA was determined by quantitative PCR. The expression of cell surface Fas was determined by flow cytometry, and that of soluble Fas (sFas) was determined by enzyme-linked immunosorbent assay.ResultsWhen compared to n-fibs, f-fibs were resistant to FasL-induced apoptosis, despite significantly higher levels of Fas mRNA. F-fibs showed lower expression of surface-bound Fas but higher levels of sFas. While TNF-α increased the susceptibility to FasL-induced apoptosis in n-fibs, it had no pro-apoptotic effect in f-fibs.ConclusionsThe data suggest that lower expression of surface Fas, but higher levels of apoptosis-inhibiting sFas, contribute to the resistance of fibroblasts in lung fibrosis against apoptosis, to increased cellularity and also to increased formation and deposition of extracellular matrix.

The role of synovial fibroblasts in mediating joint destruction in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease with systemic involvement that affects about 1% of the Western population. The progressive destruction of affected joints is a major characteristic of the disease and distinguishes RA from other acute and chronic arthritides. The etiology of RA is unknown, and a variety of genetic and environmental factors are being discussed as potential causes of the disease. However, in contrast to our incomplete understanding of the etiology, the knowledge about molecular mechanisms leading to joint destruction has advanced considerably over the past years. Thus, a large number of studies have investigated the presence and interplay of several types of cells in rheumatoid synovium, such as lymphocytes, macrophages and fibroblasts. They have led to the understanding that cells in the rheumatoid synovium form a network, which interacts through direct cell-to cell contacts as well as the release of a multitude of cytokines. The use of novel molecular techniques together with the development of new animal models has revised our concept on the pathogenesis of RA and specifically on the role of fibroblasts in initiation and progression of joint destruction. This article will review current data and hypotheses on disease mechanisms by which fibroblasts are involved in the destruction of joints in RA.

The small ubiquitin‐like modifier mediates the resistance of prosthesis‐loosening fibroblast‐like synoviocytes against fas‐induced apoptosis

OBJECTIVE To study the expression of small ubiquitin-like modifier 1 (SUMO-1) in aseptic loosening of prosthesis implants and to investigate its role in regulating the susceptibility of prosthesis-loosening fibroblast-like synoviocytes (FLS) to Fas-induced apoptosis. METHODS Specimens of aseptically loosened tissue were obtained at revision surgery, and the expression of SUMO-1 was analyzed by in situ hybridization. SUMO-1 levels in FLS were determined by quantitative polymerase chain reaction and Western blot analysis. Immunohistochemistry and confocal microscopy were used to study the subcellular localization of SUMO-1. The functional role of SUMO-1 in Fas-induced apoptosis of prosthesis-loosening FLS was investigated by small interfering RNA-mediated knockdown of SUMO-1 and by gene transfer of the nuclear SUMO-specific protease SENP1. RESULTS SUMO-1 was expressed strongly in aseptically loosened tissue and was found prominently at sites adjacent to bone. Prosthesis-loosening FLS expressed levels of SUMO-1 similar to the levels expressed by rheumatoid arthritis (RA) FLS, with SUMO-1 being found mainly in promyelocytic leukemia protein nuclear bodies. Knockdown of SUMO-1 had no effect on spontaneous apoptosis but significantly increased the susceptibility of prosthesis-loosening FLS to Fas-induced apoptosis. Gene transfer of the nuclear SUMO-specific protease SENP1 reverted the apoptosis-inhibiting effects of SUMO-1. CONCLUSION These data suggest that SUMO-1 is involved in the activation of both RA FLS and prosthesis-loosening FLS by preventing these cells from undergoing apoptosis. Modification of nuclear proteins by SUMO-1 contributes to the antiapoptotic effects of SUMO-1 in prosthesis-loosening FLS, providing evidence for the specific activation of sumoylation during their differentiation. Therefore, SUMO-1 may be an interesting target for novel strategies to prevent aseptic prosthesis loosening.

Epidemiologie und Historie der Sprunggelenksarthrose

Die Epidemiologie beschaftigt sich mit den Ursachen und Folgen sowie der Verbreitung von Krankheiten in der Bevolkerung bzw. in deren Populationen. Zum einen werden dabei statistische Daten erhoben und ausgewertet, die im Zusammenhang mit bestimmten Gesundheitszustanden stehen, auf der anderen Seiten wird auch mit beobachtenden und experimentellen Studien gearbeitet. Mathematische Modelle und Kennzahlen der Epidemiologie beschreiben dabei bestimmte Zustande und Wahrscheinlichkeiten vom Auftreten, der Ausbreitung und den Folgen von Krankheiten. Die wichtigsten Begriffe sind die Pravalenz, die die Anzahl der erkrankten Individuen in der betrachteten Population angibt, und die Inzidenzrate, die das Auftreten neuer Krankheitsfalle bezogen auf alle Mitglieder der Population beschreibt (Brandt et al. 1998).

Matrix remodeling during enchondral ossification and aggrecan cleavage in osteoarthritic cartilage depends on syndecan-4

Chondrocyte differentiation into hypertrophic chondrocytes is essential for enchondral ossification of long bones during limb development, but plays a role also in pathologic situations such as osteoarthritis (OA). However, the mechanisms that link chondrocyte hypertrophy to cartilage remodeling are poorly understood. Based on recent data that have implicated transmembrane heparan sulfate proteoglycans in matrix turnover and cell differentiation, we analyzed the role of syndecan-4 during limb development in mice and studied its expression and function in OA. Syndecan-4 promoter activity was detected in whole embryos by staining for β-galactosidase in syndecan-4-/- LacZ knockin mice. For cellular localization of syndecan-4 expression within cartilage, immunohistochemistry with antibodies against syndecan-4 and type X collagen was performed. Alizarin red S staining was carried out to analyze the mineralization of bones in wild-type and syndecan-4-/- littermates at days E13.5 and E14.5. To study syndecan-4 in OA, we compared its expression in normal and OA articular cartilage by Northern blot analysis and immunohistochemistry. Blocking antibodies against syndecan-4 were generated and used to analyze the role of syndecan-4 in IL-1-mediated proteoglycan loss in vitro. For functional analysis in vivo, osteoarthritic changes were induced in syndecan-4-/- mice and in wild-type controls by surgically achieved joint instability, and the loss of proteoglycans was assessed by safranin-orange staining. Staining for syndecan-4 and ADAMTS-generated aggrecan neo-epitopes was performed in the knees of these mice. β-Galactosidase-staining of syndecan-4-/- mice at E12.0 showed a strong activity of the syndecan-4 promoter at sites of cartilage condensations. In later stages, syndecan-4 was detected in the growth plates of long bones. In wild-type embryos, syndecan-4 protein was also found mainly in chondrocytes of the hypertrophic zone, where it colocalized with type X collagen. The loss of syndecan-4 was associated with a significant retardation in the mineralization of axial and appendicular bones. Interestingly, there was a clear upregulation of syndecan-4 in human OA cartilage both at the mRNA and the protein level. In the cartilage explant model, blocking antibodies to syndecan-4 substantially significantly reduced the IL-1-induced loss of proteoglycans in wild-type cartilage. Analysis of OA-like changes in mice revealed a strong and early induction of syndecan-4, and there was a significant reduction of proteoglycan loss in the syndecan-4-/- mice compared with their wild-type controls. This was accompanied by a significantly reduced staining for ADAMTS-generated aggrecan neo-epitopes in syndecan-4-/- mice. Our data show that syndecan-4 is induced in hypertrophic chondrocytes both during embryogenesis and in OA cartilage. By promoting ADAMTS-mediated cleavage of aggrecans, syndecan-4 facilitates enchondral ossification but is involved also in cartilage degradation by hypertrophic chondrocytes in OA. Inhibition of syndecan-4 may therefore constitute a promising strategy to interfere with osteoarthritic cartilage damage.

Ätiologie und Pathogenese der Sprunggelenksarthrose

Das Sprunggelenk besteht aus dem oberen und unteren Sprunggelenk. Das obere Sprunggelenk wird durch die Malleolengabel und dem Talus gebildet. Die Malleolengabel ist durch die Syndesmose fest miteinander verbunden. Zudem ist das obere Sprunggelenk durch Seitenbander, die von den Malleoli zum Talus, Kalkaneus und teilweise auch zum Os naviculare ziehen, gesichert. Das obere Sprunggelenk ist somit ein Scharniergelenk und hat nur einen Freiheitsgrad der Beweglichkeit (Extension/Flexion). Die Trochlea tali artikuliert dabei mit der distalen Tibia und seitlich mit den Malleoli. Zwischen dem oberen und dem unteren Sprunggelenk nimmt der Talus eine Schlusselstellung ein. Er nimmt die Last des oberen Sprunggelenks auf und leitet sie uber das untere Sprunggelenk an die anschliesenden Knochen des Fuses weiter.