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The JNK pathway (c-jun N-terminal kinase) is activated by a variety of inflammatory and physical stressful stimuli. Recently, we identified a new and an important role for the JNK pathway in the turnover of the cartilage proteoglycan aggrecan. Loss of JNK2 isoform in human chondrocytes and murine cartilage abolished IL1- induced aggrecan degradation and aggrecanase activity. Furthermore, JNK2-null mice were protected from surgically-induced osteoarthritis. Here we show that the JNK2-dependent effect on the aggrecan degradation is linked to an LRP1-dependent endocytic mechanism.

Osteoarthritis is a common degenerative joint disease for which no disease-modifying drugs are currently available. Attempts to treat the disease with small molecule inhibitors of the metalloproteinases that degrade the cartilage matrix have been hampered by a lack of specificity. We aimed to inhibit cartilage degradation by augmenting levels of the endogenous metalloproteinase inhibitor, tissue inhibitor of metalloproteinases 3 (TIMP-3), through blocking its interaction with the endocytic scavenger receptor, low-density lipoprotein receptor-related protein 1 (LRP1). We discovered that suramin (C51H40N6O23S6) bound to TIMP-3 with a KD value of 1.9 ± 0.2 nM and inhibited its endocytosis via LRP1, thus increasing extracellular levels of TIMP-3 and inhibiting cartilage degradation by the TIMP-3 target enzyme, adamalysin with thrombospondin motifs 5 (ADAMTS-5). NF279, a structural analogue of suramin, has increased affinity for TIMP-3 and increased ability to inhibit TIMP- 3 endocytosis and protect cartilage. Suramin is thus a promising scaffold for the development of novel therapeutics to increase TIMP-3 levels and inhibit cartilage degradation in osteoarthritis.

Degradation of the cartilage proteoglycan aggrecan is an early event in the development of osteoarthritis, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 are considered to be the major aggrecan-degrading enzymes. We have recently found that ADAMTS-5 is rapidly endocytosed via low density lipoprotein receptor-related protein 1 (LRP1) and degraded by chondrocytes. Here we report that this regulatory mechanism also applies to ADAMTS-4, although its rate of endocytosis is slower than that of ADAMTS-5. Domain deletion mutagenesis of ADAMTS-4 identified that the cysteine-rich and spacer domains are responsible for binding to LRP1, whereas the thrombospondin 1 and spacer domains are responsible in ADAMTS-5. The estimated tandfrac12; value of ADAMTS-4 endocytosis was about 220 min, whereas that of ADAMTS-5 was 100 min. The difference in half-lives between the two enzymes is explained by the 13-fold lower affinity of ADAMTS-4 for LRP1 compared with that of ADAMTS-5. Studies using soluble ligand binding clusters of LRP1 showed that ADAMTS-4 binds to clusters II and IV with similar KD,app values of 98 and 73 nm, respectively, whereas ADAMTS-5 binds to cluster II, III, and IV with KD,app values of 3.5, 41, and 9 nm, respectively. Thus, ADAMTS-5 competitively inhibits ADAMTS-4 endocytosis but not vice versa. This study highlights that the affinity between a ligand and LRP1 dictates the rate of internalization and suggests that LRP1 is a major traffic controller of the two aggrecanases, especially under inflammatory conditions, where the protein levels of ADAMTS-4 increase, but those of ADAMTS-5 do not.

Objective: Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin‐1 (IL‐1). IL‐1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL‐1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes.Methods: We developed a cell‐based assay combining small interfering RNA (siRNA)–induced knockdown with aggrecan degradation assays. Human articular chondrocytes were overlaid with bovine aggrecan after transfection with siRNAs against molecules of the IL‐1 signaling pathway. After IL‐1 stimulation, released aggrecan fragments were detected with AGEG and ARGS neoepitope antibodies. Aggrecanase activity and tissue inhibitor of metalloproteinases 3 levels were measured by enzyme‐linked immunosorbent assay. Low‐density lipoprotein receptor–related protein 1 (LRP‐1) shedding was analyzed by Western blotting.Results: ADAMTS‐5 is a major aggrecanase in human chondrocytes, regulating aggrecan degradation in response to IL‐1. The tumor necrosis factor receptor–associated 6 (TRAF‐6)/transforming growth factor β–activated kinase 1 (TAK‐1)/MKK‐4 signaling axis is essential for IL‐1–induced aggrecan degradation, while NF‐κB is not. Of the 3 MAPKs (ERK, p38, and JNK), only JNK‐2 showed a significant role in aggrecan degradation. Chondrocytes constitutively secreted aggrecanase, which was continuously endocytosed by LRP‐1, keeping the extracellular level of aggrecanase low. IL‐1 induced aggrecanase activity in the medium in a JNK‐2–dependent manner, possibly by reducing aggrecanase endocytosis, because IL‐1 caused JNK‐2–dependent shedding of LRP‐1.Conclusion: The signaling axis TRAF‐6/TAK‐1/MKK‐4/JNK‐2 mediates IL‐1–induced aggrecanolysis. The level of aggrecanase is controlled by its endocytosis, which may be reduced upon IL‐1 stimulation because of LRP‐1 shedding.

Objective A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen-degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1-MMP), in synovial pannus invasiveness. Methods The expression and localization of MT1-MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1-MMP was analyzed by 3-dimensional (3-D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, or GM6001. The effect of adenoviral expression of a dominant-negative MT1-MMP construct lacking a catalytic domain was also examined. Results MT1-MMP was highly expressed at the pannus–cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3-D collagen matrix in an MT1-MMP–dependent manner. Invasion was blocked by TIMP-2 and GM6001 but not by TIMP-1. Invasion was also inhibited by the overexpression of a dominant-negative MT1-MMP, which inhibits collagenolytic activity and proMMP-2 activation by MT1-MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1-MMP–dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix. Conclusion MT1-MMP serves as an essential collagen-degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1-MMP–dependent invasion may represent a novel therapeutic strategy for RA.

Objectives: The pathogenesis of osteoarthritis (OA) is poorly understood. Loss of the proteoglycan aggrecan from the cartilage is an early event. Recently, we identified a role for the JNK pathway, particularly JNK2, in human articular chondrocytes regulating aggrecan degradation. To understand whether JNK2 plays a similar role in vivo, we investigated the development of OA in JNK2-/- mice by using the surgical model of destabilizing the medial meniscus (DMM). The role of JNK2 in gene expression was also investigated. Methods: Aggrecan fragments were analysed by western blotting. OA was induced by DMM and analysed at 4, 8, and 12 weeks post-surgery. Knee sections were stained with Safranin O. Medial compartments were scored by histological grading for aggrecan loss and cartilage damage. RNA was extracted from JNK2-/- and wild-type knees 6hrs following DMM or after IL1 stimulation of proximal epiphysis. Expression of 33 DMM-regulated genes was analysed by q-PCR customized array cards. Results: Basal and IL-1- or TNF-stimulated release of aggrecanase-generated aggrecan fragments were much reduced in JNK2-/- cartilage. In the OA model, JNK2-/- mice showed significant reduction of aggrecanase-generated fragments and cartilage damage. Of 33 genes investigated, 13 were significantly down-regulated in JNK2-/- mice following DMM compared with wild-type. These included Has1, ADAMTS4, Tnf, IL6, IL18, Inhba, Cd68, Ngf, Ccr2, Wnt16, Tnfaip6 and Il1r, but not ADAMTS-5. Conclusions: JNK2 regulates aggrecan degradation in cultured murine cartilage and surgically-induced OA in vivo following mechanical destabilisation of the knee-joint. This implicates the JNK signalling pathway in OA and suggests novel therapeutic approaches.

International audience

International audience

BACKGROUND: Protozoan parasites of the genus Trypanosoma cause disease in a wide range of mammalian hosts. Trypanosoma brucei brucei, transmitted by tsetse fly to cattle, causes a disease (Nagana) of great economic importance in parts of Africa. T. b. brucei also serves as a model for related Trypanosoma species, which cause human sleeping sickness. MATERIALS AND METHODS: Chalcone and acyl hydrazide derivatives are known to retard the growth of Plasmodium falciparum in vitro and inhibit the malarial cysteine proteinase, falcipain. We tested the effects of these compounds on the growth of bloodstream forms of T. b. brucei in cell culture and in a murine trypanosomiasis model, and investigated their ability to inhibit trypanopain-Tb, the major cysteine proteinase of T. b. brucei. RESULTS: Several related chalcones, acyl hydrazides, and amides killed cultured bloodstream forms of T. b. brucei, with the most effective compound reducing parasite numbers by 50% relative to control populations at a concentration of 240 nM. The most effective inhibitors protected mice from an otherwise lethal T. b. brucei infection in an in vivo model of acute parasite infection. Many of the compounds also inhibited trypanopain-Tb, with the most effective inhibitor having a Ki value of 27 nM. Ki values for trypanopain-Tb inhibition were up to 50- to 100-fold lower than for inhibition of mammalian cathepsin L, suggesting the possibility of selective inhibition of the parasite enzyme. CONCLUSIONS: Chalcones, acyl hydrazides, and amides show promise as antitrypanosomal chemotherapeutic agents, with trypanopain-Tb possibly being one of their in vivo targets.

Involving research users in setting priorities for research is essential to ensure research outcomes are patient-centred and to maximise research value and impact. The Musculoskeletal (MSK) Disorders Research Advisory Group Versus Arthritis led a research priority setting exercise across MSK disorders. The Child Health and Nutrition Research Initiative (CHRNI) method of setting research priorities with a range of stakeholders was utilised, involving four stages and two surveys, to: 1) gather research uncertainties; 2) consolidate these; 3) score uncertainties against importance and impact; and 4) analyse scoring, for prioritisation. 213 and 285 people responded to the first and second surveys respectively, representing clinicians, researchers and people with MSK disorders. Key priorities included developing and testing new treatments, better treatment targeting , early diagnosis, prevention and better understanding and management of pain, with an emphasis on understanding underpinning mechanisms. We present a call to action to researchers and funders to target these priorities.

Extracellular matrix (ECM) is a dynamic 3-dimensional network of macromolecules that provides structural support for the cells and tissues. Accumulated knowledge clearly demonstrated over the last decade that ECM plays key regulatory roles since it orchestrates cell signaling, functions, properties and morphology. Extracellularly secreted as well as cell-bound factors are among the major members of the ECM family. Proteins/glycoproteins, such as collagens, elastin, laminins and tenascins, proteoglycans and glycosaminoglycans, hyaluronan, and their cell receptors such as CD44 and integrins, responsible for cell adhesion, comprise a well-organized functional network with significant roles in health and disease. On the other hand, enzymes such as matrix metalloproteinases and specific glycosidases including heparanase and hyaluronidases contribute to matrix remodeling and affect human health. Several cell processes and functions, among them cell proliferation and survival, migration, differentiation, autophagy, angiogenesis, and immunity regulation are affected by certain matrix components. Structural alterations have been also well associated with disease progression. This guide on the composition and functions of the ECM gives a broad overview of the matrisome, the major ECM macromolecules, and their interaction networks within the ECM and with the cell surface, summarizes their main structural features and their roles in tissue organization and cell functions, and emphasizes the importance of specific ECM constituents in disease development and progression as well as the advances in molecular targeting of ECM to design new therapeutic strategies.

Osteoarthritis (OA) is a chronic, multi-factorial, progressive joint disease. The understanding of OA has evolved away from a wear and tear phenomenon to a complex disease of the joint taken as a whole. A method of stratifying disease, and monitoring progression, is necessary for clinical decision making and for use as end points in future research studies to develop disease modifying OA drugs (DMOADs). The creation of such a tool requires detailed understanding of the disease biopathogenesis. Synovial fluid (SF) is the most likely target to generate the insight required due to its proximity to the intra-articular structures, whose changes are fundamental to the symptomatology of the disease. No work to date has identified any useful single biomarkers of OA and for progress to be made, a consideration of a number of proteins as a panel of markers, whose interrelations describe a signature of different disease states, is required. The emergence of the slow off-rate modified aptamer scan (SOMAscan), a broad spectrum proteomic platform, presents the opportunity to analyse 1310 proteins on a single sample of fluid that measures less than 1 ml. This thesis aims to examine the utility of the SOMAscan in OA biomarker discovery by validation against traditional bench assays, use multivariate statistical analysis methods to identify candidate markers of OA disease states, and contextualise their best clinical timing in the patient pathway by examining the treatment pathways of patients from initial presentation to final treatment. Synovial fluid samples from hip and knee joints were collected from patients with different stages of clinically classified disease including knee injury, early and late OA cohorts, and hip early and late OA cohorts with the early hip OA group requiring the development of an entirely new collection method. The performance of the SOMAscan was validated using ELISA and MSD assays that were performed on a curated list of proteins and compared to the same markers in the SOMAscan showing robust comparison. Multivariate regression analysis showed separation of the knee injury and late OA cohorts using PLS-DA, but the early group were not separable from the injury group. Markers identified by LASSO that were strongly associated with late disease included elevated TSG-6, TIMP-1 and R-spondin 4. These markers were also identified in the PLS-DA model generating potential targets for use in future OA studies. Hip SF analysis showed separation between the early and late OA groups on PLS-DA. LASSO analysis revealed 6 markers: TSG-6, DDR-2, Ephrin-A5, S100A7, MICB, IDS all of which were present in the top 50 marker model produced using PLS-DA. This combined analysis approach has shown differences between the hip groups, differences between hip and knee OA biology and suggested novel targets for future OA studies. An epidemiological study investigating the patient pathways, from presentation in primary care through to eventual arthroplasty, was conducted to identify where a diagnostic test would be best placed in the patient journey. Results showed marked variation in the rates of treatments offered to patients during their disease, with wide variation in analgesia, physiotherapy and intermediate surgical interventions. Most notably it showed that there was a continued increase in the use of opioid analgesia overall, particularly in the most deprived patients, indicating a need to investigate ways to harmonise this patient experience such that patients can be identified reliably at earlier stages of disease. In summary, the works in this thesis have validated the SOMAscan. A new system of biomarker discovery in OA, for use in SF analysis. The SOMAscan analysis of SF has shown a variety of differently expressed markers and pathways including well described OA processes in both knee and hip. It has suggested that early knee OA appears biologically closer to joint injury than to late knee OA, and that hip and knee OA appear to be biologically distinct processes with common thematic pathways as well as differences. This work has collected a new and unique SF cohort of human early hip OA SF to define a biological phenotype of late OA that discriminates between the groups studied. This work has further shown that the patient journey from presentation to definitive treatment is hugely varied, both in duration and treatments delivered, demonstrating that ongoing work is needed not only to identify biomarkers of early OA but to identify means of accessing patients earlier in their clinical disease states. Together these critical elements of disease description and patient identification require optimisation such that future OA biomarkers may be utilised as disease diagnosis, stratification and outcome tools in DMOAD studies.

There are currently no drugs that modify the progression of the degenerative joint disease osteoarthritis, which is characterised by protease-mediated degradation of the cartilage extracellular matrix. Identification of molecules that regulate protease activity could therefore lead to the development of new therapies for osteoarthritis. The Wann group previously showed that mutation of Intraflagellar transport protein 88 (IFT88) increased degradation of the cartilage matrix component aggrecan by proteases in vitro. As IFT88 is required for the assembly of a cell surface organelle called the primary cilium, the aim of my thesis was to investigate whether the primary cilium is involved in the regulation of protease activity. Based on the Wann group’s finding that mutation of IFT88 impaired Low-density lipoprotein receptor-related protein 1 (LRP-1)-mediated endocytosis of extracellular proteases, I hypothesised that the primary cilium is a region of efficient endocytosis and thus regulates the extracellular activity of proteases. To address this aim, I measured protease activity when four genes encoding proteins involved in cilia assembly were knocked down with siRNAs in a chondrocyte cell line. Generation of the AGEG aggrecan neoepitope was used as an indicator of protease activity. I showed that protease activity was not affected by knockdown of three of the ciliary genes tested. However, knockdown of the gene encoding Tau-tubulin kinase 2 (TTBK2) increased the activity of the protease A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5). This increase was not associated with changes in ADAMTS-5 RNA levels during my experiments, constitutive activation of cilia-dependent hedgehog signalling, or reduced levels of the physiological ADAMTS-5 inhibitor, Tissue inhibitor of metalloproteinase 3 (TIMP-3). The effect of knockdown of the TTBK2 gene on ADAMTS-5 endocytosis could not be definitively determined. Together, these results showed that whilst the primary cilium itself may not be required for protease activity regulation, TTBK2 is a novel regulator of ADAMTS-5 activity. Further investigation of the molecular mechanism of ADAMTS-5 regulation by TTBK2 may help to identify new therapeutic targets in osteoarthritis.

Among the hundreds of ligands for heparan sulfate (HS) are chemokines, cytokines, and growth factors, giving this proteoglycan a potentially central role in the regulation of immune responses. The biosynthesis of HS is regulated during inflammation with consequent changes to ligand binding and activity in multiple cell types including myeloid cells. In this thesis, I aimed to comprehensively describe the changes in expression of HS core proteins and biosynthetic enzymes in human primary monocytes and polarised monocyte-derived macrophages, and to investigate the contribution of HS proteoglycans to macrophage functions. Using a custom-designed microfluidic array card, I performed transcriptome analysis of HS-associated gene expression and discovered significant changes at all stages of macrophage maturation and polarisation, revealing that maturation is associated with increased capacity for HS biosynthesis and that pro- and anti-inflammatory macrophages have substantially different HS composition. Of particular interest was the reciprocal regulation observed between the 6-O-sulfotransferase (HS6ST1) and its functional partner the sulfatase (SULF2), which add and remove the 6-O-sulfate group to HS chains, respectively. Using siRNA knockdown of HS6ST1 in monocyte-derived macrophages to ablate 6-O-sulfation of HS, I determined that this modification does not contribute to phagocytosis or migration. Moreover, its absence did not affect macrophage polarisation, whereas total sulfate ablation using sodium chlorate did, suggesting that sulfate modifications at other positions are more important. HS6ST1 knockdown altered post-translational regulation of monocyte chemoattractant protein 1 (MCP-1), indicating the 6-O-sulfate group of HS modulates retention of this and potentially other mediators on the cell surface.