J. Sherwood

WNT-3A modulates articular chondrocyte phenotype by activating both canonical and noncanonical pathways

A single Wnt can simultaneously activate different pathways with distinct and independent outcomes and reciprocal regulation in human articular chondrocytes.

A homeostatic function of CXCR2 signalling in articular cartilage

Objective ELR+ CXC chemokines are heparin-binding cytokines signalling through the CXCR1 and CXCR2 receptors. ELR+ CXC chemokines have been associated with inflammatory arthritis due to their capacity to attract inflammatory cells. Here, we describe an unsuspected physiological function of these molecules in articular cartilage homeostasis. Methods Chemokine receptors and ligands were detected by immunohistochemistry, western blotting and RT-PCR. Osteoarthritis was induced in wild-type and CXCR2−/− mice by destabilisation of the medial meniscus (DMM). CXCR1/2 signalling was inhibited in vitro using blocking antibodies or siRNA. Chondrocyte phenotype was analysed using Alcian blue staining, RT-PCR and western blotting. AKT phosphorylation and SOX9 expression were upregulated using constitutively active AKT or SOX9 plasmids. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Results CXCL6 was expressed in healthy cartilage and was retained through binding to heparan sulfate proteoglycans. CXCR2−/− mice developed more severe osteoarthritis than wild types following DMM, with increased chondrocyte apoptosis. Disruption of CXCR1/2 in human and CXCR2 signalling in mouse chondrocytes led to a decrease in extracellular matrix production, reduced expression of chondrocyte differentiation markers and increased chondrocyte apoptosis. CXCR2-dependent chondrocyte homeostasis was mediated by AKT signalling since forced expression of constitutively active AKT rescued the expression of phenotypic markers and the apoptosis induced by CXCR2 blockade. Conclusions Our study demonstrates an important physiological role for CXCR1/2 signalling in maintaining cartilage homeostasis and suggests that the loss of ELR+ CXC chemokines during cartilage breakdown in osteoarthritis contributes to the characteristic loss of chondrocyte phenotypic stability.

WNT16 antagonises excessive canonical WNT activation and protects cartilage in osteoarthritis.

Objective Both excessive and insufficient activation of WNT signalling results in cartilage breakdown and osteoarthritis. WNT16 is upregulated in the articular cartilage following injury and in osteoarthritis. Here, we investigate the function of WNT16 in osteoarthritis and the downstream molecular mechanisms. Methods Osteoarthritis was induced by destabilisation of the medial meniscus in wild-type and WNT16-deficient mice. Molecular mechanisms and downstream effects were studied in vitro and in vivo in primary cartilage progenitor cells and primary chondrocytes. The pathway downstream of WNT16 was studied in primary chondrocytes and using the axis duplication assay in Xenopus. Results WNT16-deficient mice developed more severe osteoarthritis with reduced expression of lubricin and increased chondrocyte apoptosis. WNT16 supported the phenotype of cartilage superficial-zone progenitor cells and lubricin expression. Increased osteoarthritis in WNT16-deficient mice was associated with excessive activation of canonical WNT signalling. In vitro, high doses of WNT16 weakly activated canonical WNT signalling, but, in co-stimulation experiments, WNT16 reduced the capacity of WNT3a to activate the canonical WNT pathway. In vivo, WNT16 rescued the WNT8-induced primary axis duplication in Xenopus embryos. Conclusions In osteoarthritis, WNT16 maintains a balanced canonical WNT signalling and prevents detrimental excessive activation, thereby supporting the homeostasis of progenitor cells.

Decreased levels of nucleotide pyrophosphatase phosphodiesterase 1 are associated with cartilage calcification in osteoarthritis and trigger osteoarthritic changes in mice

Objective To analyse the function of nucleotide pyrophosphatase phosphodiesterase (NPP1), a member of the pyrophosphate pathway, in osteoarthritis (OA). Methods mRNA expression of NPP1, ANK ankylosing protein and tissue non-specific alkaline phosphatase was assessed by quantitative PCR. NPP1 protein levels were analysed in mouse and human cartilage samples. Bone metabolism was analysed by F18-positron emission tomography-scanning and µCT in ttw/ttw mice. Ttw/ttw mice are mice carrying a loss-of-function mutation in NPP1. Calcification of articular cartilage was assessed using von Kossa staining and OA severity using the Mankin score. Cartilage remodelling was investigated by type X collagen immunohistochemistry. Results Expression of NPP1, but not the other members of this pathway, inversely correlated with cartilage calcification and OA severity in mouse and humans. Proinflammatory cytokines downregulated the expression of NPP1, demonstrating an influence of inflammation on matrix calcification. Ttw/ttw mutant mice, carrying a loss-of-function mutation in NPP1, exhibit increased bone formation process in joints compared with wild types. Ttw/ttw mice also developed spontaneous OA-like changes, evaluated by histological analysis and in vivo imaging. Ectopic calcifications were associated with increased expression of collagen X in the cartilage. Conclusion The authors conclude that OA is characterised by the reactivation of molecular signalling cascades involving proinflammatory cytokines, thereby regulating the pyrophosphate pathway which consequently leads to cartilage ossification, at least in part resembling endochondral ossification.

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.

Cellular and molecular mechanisms of cartilage damage and repair.

Cartilage breakdown is the disabling outcome of rheumatic diseases, whether prevalently inflammatory such as rheumatoid arthritis or prevalently mechanical such as osteoarthritis (OA). Despite the differences between immune-mediated arthritides and OA, common mechanisms drive cartilage breakdown. Inflammation, chondrocyte phenotype and homeostatic mechanisms have recently been the focus of research and will be summarised in this review.

Analyses on the mechanisms that underlie the chondroprotective properties of calcitonin

INTRODUCTION Calcitonin (CT) has recently been shown to display chondroprotective effects. Here, we investigate the putative mechanisms by which CT delivers these actions. METHODS Immortalized C-28/I2 cells or primary adult human articular chondrocytes (AHAC) were cultured in high-density micromasses to investigate: (i) CT anabolic effects using qPCR and immuhistochemistry analysis; (ii) CT anti-apoptotic effects using quantitation of Bax/Bcl gene products ratio, TUNEL assay and caspase-3 expression; (iii) CT effects on CREB, COL2A1 and NFAT transcription factors. RESULTS CT (10(-10)-10(-8)nM) induced significant up-regulation of cartilage phenotypic markers (SOX9, COL2A1 and ACAN), with down-regulation of catabolic (MMP1 and MMP13 and ADAMTS5) gene products both in resting and inflammatory conditions. This was mirrored by an augmented production of type II collagen and accumulation of glycosaminoglycan- and proteoglycan-rich extracellular matrix in vitro. Mechanistic analyses revealed only partial involvement of cyclic AMP formation in these effects of CT. Congruently, using reporter assays for specific transcription factors, there was no indication for CREB activation, whereas the COL2A1 promoter was genuinely and directly activated by cell exposure to CT. Phenotypically, these mechanisms supported the ability of CT, whilst inactive on its own, to counteract the pro-apoptotic effects of IL-1β, demonstrated by TUNEL-positive staining of chondrocytes and ratio of BAX/BCL genes products. CONCLUSION These data may provide a novel lead for the development of CT-based chondroprotective strategies that rely on the engagement of mechanisms that lead to augmented chondrocyte anabolism and inhibited chondrocyte apoptosis.

PPARγ/mTOR signalling: striking the right balance in cartilage homeostasis

Osteoarthritis (OA) is still a highly prevalent and disabling disease for which we do not have a cure. In the last decade, however, the unravelling of molecular mechanisms controlling joint homeostasis, the advances in targeting technologies, and the improvement of animal models allowing the use of mouse genetics has led to the identification of molecular targets that, in animal models,1–4 and possibly also in humans5 can arrest disease or even revert its course. Such strategies include blockade of extracellular matrix-degrading enzymes,6 hypoxia-inducible factor 2α blockade3 ,4 to prevent chondrocyte hypertrophy, the support of parathyroid hormone/parathyroid hormone related protein signalling—an injury-induced homeostatic mechanism affecting both cartilage homeostasis and bone remodelling,2 improving bone turnover using strontium ranelate,5 and blockade of the filamin-core binding factor interaction thereby supporting chondrocytic differentiation using kartogenin.7 Although only strontium ranelate has been tested in the clinic, there is little doubt that the availability of multiple targets will stimulate and instruct the experimentation needed to bridge the gap to the clinic. In this issue of the journal, Vasheghani et al 8 have demonstrated that PPARγ-driven mTOR (mammalian target of rapamycin) inhibition protects cartilage from experimental OA, at least in part by supporting autophagy,8 a process that, by suppressing protein synthesis and enabling the use of cellular components to generate energy, allows cells to escape death in conditions of stress or lack of nutrients.9 The authors previously reported that cartilage-specific disruption of the gene encoding for the transcription factor PPARγ results in spontaneous OA in mice.10 To ensure that this phenotype was not driven by skeletal dysplasia determined by the absence of PPARγ during development, they generated a new mutant in which PPARγ could be deleted postnatally in chondrocytes upon administration of doxycycline. The mice did not develop …

Syndecan-4 Modulates Epithelial Gut Barrier Function and Epithelial Regeneration in Experimental Colitis

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.

A4.11 Syndecan-4 is an important player in regulating the WNT signalling pathway in articular cartilage

Background The heparan sulfate proteoglycan syndecan-4 (Sdc4) is a regulator of various cartilage-related processes including osteoarthritis (OA). Blockade of Sdc4 signalling protects mice from cartilage degradation in experimentally induced OA. OA is characterised by matrix remodelling and hypertrophic differentiation of chondrocytes. Various signalling pathways including the WNT signalling pathway have been implicated to trigger this change of chondrocyte phenotype. Experiments investigating the effect of different WNT3a concentrations on chondrocytes have emphasised a complex dialogue between canonical and non-canonical WNT pathways. We hypothesise that Sdc4 controls the chondrocyte phenotype by specific modulation of WNT signalling pathways. Methods In vitro analyses were performed using neonatal wild type (wt) and Sdc4-/- chondrocytes, or blocking antibodies against Sdc4. The influence of WNT3a on glycosaminoglycan (GAG) production was analysed using alcian blue staining of micromass cultures. Expression of marker genes (e.g. aggrecan, collagen2, MMP13) was measured by quantitative RT-PCR. Effects of WNT3a on canonical and non canonical WNT signalling were analysed using Western Blot (ß-catenin and pCamKII) and luciferase reporter assay (TCF/Lef). In vivorelevance was investigated upon induction of OA using the DMM model. Results Micromass cultures revealed a higher basal GAG production by Sdc4-/- chondrocytes. WNT3a stimulation led to a decrease in GAG production in wt cells, which was absent in Sdc4-/- chondrocytes. qRT-PCR showed a 10x higher basal production of aggrecan and collagen2 in Sdc4-/- chondrocytes. WNT3a increased the expression of both genes in Sdc-4 -/-, whereas decreasing the expression in wt chondrocytes. MMP13 was significantly less expressed in Sdc4-/- chondrocytes and, unlike in wt cells, was not up regulated upon WNT3a stimulation. Western blot showed that ß-catenin is not up regulated upon stimulation with WNT3a in Sdc4-/- chondrocytes. LRP6 was less phosphorylated and TCF/Lef promotor was less activated upon WNT3a stimulation in Sdc4-/- chondrocytes. pCamKII was increased under basal conditions, but decreased upon WNT3a stimulation in Sdc4-/-. The same effects on canonical and non canonical WNT signalling upon WNT stimulation were obtained by using a blocking anti-Sdc-4 antibody. In vivo stainings confirmed in vitro results. Conclusion Sdc4 is a major regulator of cellular response to WNT signals through facilitating the induction of the canonical WNT signalling pathway. The blockade of Sdc4 protects from OA induced changes in chondrocyte phenotype by inhibiting WNT induced differentiation of chondrocytes.