1,720,964 research outputs found
Identification of a novel regulator of adamts-5-mediated aggrecan degradation in chondrocytes
No full textPurpose: there are currently no therapies to reduce cartilage loss in osteoarthritis. Our group has been studying the molecular mechanisms regulating the activity of proteases involved in osteoarthritis pathogenesis to identify new therapeutic targets. To do this, we have focused on proteins required for formation and function of an organelle called the primary cilium. We have previously seen that a hypomorphic mutation affecting the ciliary protein IFT88 resulted in elevated matrix catabolism in vitro, which was possibly due to disrupted regulation of protease activity by LRP-1-mediated endocytosis. Here, we aimed to identify other ciliary proteins that regulate proteolytic matrix degradation, determine the mechanism by which these proteins may be acting, and investigate whether expression of the genes encoding these proteins is altered in osteoarthritis.Methods: proteins with different roles in cilia formation and function were depleted in a mouse chondrocyte line using siRNAs. Proteolytic matrix degradation was measured by culturing cells with purified aggrecan and subsequently detecting the neoepitope AGEG on aggrecan fragments by western blot. qPCR, western blotting and microscopy were used to investigate mechanisms of protease activity regulation.Results: siRNA-mediated knockdown of the ciliary kinase TTBK2 resulted in increased AGEG-positive aggrecan fragment production relative to control cells treated with non-targeting siRNA. Elevated AGEG in TTBK2 knockdown cells was abolished with combined knockdown of TTBK2 and ADAMTS-5. TTBK2 knockdown was associated with a 20% increase in the expression of Adamts5 mRNA. Expression of targets of the Hedgehog signalling pathway, which is dependent on the cilium and has previously been linked to regulation of cartilage matrix degradation, was not affected in TTBK2 knockdown cells. Activation of the Hedgehog pathway with recombinant Indian Hedgehog did not affect AGEG production. Extracellular levels of the endogenous protease inhibitor TIMP-3 were unaffected by TTBK2 knockdown.Conclusions: these Results indicate that TTBK2 is a novel regulator of ADAMTS-5-mediated aggrecan degradation in chondrocytes. The mechanism by which TTBK2 regulates ADAMTS-5 activity likely does not involve Hedgehog signalling or TIMP-3, and work is ongoing to determine the contribution of transcriptional changes to elevated ADAMTS-5 activity. Current studies are also addressing whether TTBK2 regulates ADAMTS-5 endocytosis or its extracellular activation. Further investigation of TTBK2 in more clinically relevant contexts is now required, particularly as exploration of global gene expression datasets from RNA-sequencing in normal and osteoarthritic knee cartilage showed that TTBK2 expression is statistically significantly downregulated in osteoarthritis
The primary cilium orchestrates chondrocyte mechanotransduction
No full textPurpose: to understand the role of the chondrocyte primary cilium in mechanotransduction. The primary cilium is a singular organelle protruding outward from the mature centriole into the extracellular space, decorated with many receptors and integrins. The tubulin scaffold, ciliary membrane and proteome are constructed and maintained by dynein motors and intraflagellar transport cargos. In many cell types such as kidney epithelia, vascular endothelium and osteocytes, primary cilia are involved in mechanotransduction, often through the activities of polycystin 1 and 2 located on the cilium. Physiologically cartilage experiences mechanical signals, altering cell behaviour, that include compression and fluid flow. It is thought aberrant mechanotransduction may be one factor in the development of tissue pathology. Here we test the hypothesis that the cilium is essential for chondrocyte mechanotransduction and the up-regulation of proteoglycan synthesis through an established purinergic pathway involving the release of ATP and subsequent activation of Ca2+ signalling.Methods: to test this we used a hypomorphic mutation of Tg737, which encodes for IFT88, and abolishes genesis and growth of the cilium and which has been shown in vivo to result in murine matrix patterning defects. A well-established 3D agarose culture system was implemented to allow compressive loading of murine WT and Tg737 chondrocytes in culture followed by the quantification of ATP release with a luciferase assay, calcium transients by means of Fluo-4 imaging, and matrix production by qPCR and biochemical assay. Additionally expression of purinergic receptors (P2R) and polycystins (PC) 1 and 2 were assessed by western blot and immunocytochemistry.Results: compression of WT chondrocytes increased calcium transients (p<0.05) and matrix production at gene and protein levels (p<0.05) however these mechanosensitive responses were not present in Tg737 chondrocytes. Mechanosensitive ATP release (p<0.01) was maintained between WT and Tg737 cells implying that the cilium is required for ATP reception or transduction. Indeed, exogenous addition of ATP up-regulated Ca2+ transients in WT (p<0.001) but did not in Tg737 cells, although there were no differences in P2R expression. In Tg737 cells PC-1 expression was altered such that the full size protein product was absent.Conclusions: we conclude that the primary cilium is essential for chondrocyte mechanotransduction through the regulation of purinergic Ca2+ signalling. We speculate that this may be attributed to a role for the cilia protein polycystin-1. This demonstrates the central role for the chondrocyte primary cilium in cartilage physiology in the context of the chondrocyte response to mechanical stimuli
Exploring and exploiting a ciliome-protease-matrix regulatory axis disturbed in osteoarthritis
No full textPurpose: in osteoarthritis (OA) cartilage homeostasis is disturbed sufficiently such that tissue is degraded and destroyed. A carefully balanced relationship between anabolic and catabolic activity is upset within the complex cartilage matrix environment. Mechanics, at the level of the whole joint, tissue and at the cellular level, is proposed to be a major contributor to this imbalance. During cartilage development, mechanical and biological cues are partially transduced by the primary cilium and/or its associated machinery (ciliome). Learning from its roles in development, we wish to understand the post-developmental influence of the ciliome. We have a particular focus on the interaction between the ciliome and protease activity in OA and have recently discovered a novel ciliome-protease regulatory axis acting through LRP-1. We hypothesise that roles of the ciliome alter into adulthood, but influence is maintained, in part, through a ciliome-endocytosis-protease-matrix continuum that is disturbed in OA.Methods: intraflagellar transport proteins (IFTs) carry cargoes to and within the cilium such as the regulatory GTPase arl13b. IFT88 is a core component of the ciliome, which is also physically and mechanistically associated with the centrioles. We have created a cartilage-specific, adult mouse IFT88 deletion model by crossing IFT88fl/fl x Acan-CreER2. This mouse was phenotyped using histology and μCT. We are also using a double transgenic model (ARL13bmCherry; Centrin 2-GFP) that enables in situ visualisation of the cilia and centrioles in cartilage. Chondrocyte polarity and cilia were analysed in situ following confocal imaging, using ImageJ software. In vitro we measured protease activity in both tissue co-culture and recombinant matrix overlay systems, quantifying production of neoepitopes by chondrocytes.Results: IFT88 exerts strong influence in the mouse joint into adolescence with a less acute, but nevertheless sustained influence into adulthood. This includes a disturbed chondrocyte orientation, and changes to the cartilage matrix profile and the bone beneath. In vitro, IFT88 modulates matrix catabolic activity of chondrocytes through LRP-1-mediated endocytosis. Cartilage has a complex organisation that is disturbed in OA. Chondrocyte polarity, and thus a putative ciliome-LRP-1-matrix axis, is highly organised in relation to this.Conclusions: the ciliome is physiologically relevant in adult joints and potentially has a pathophysiological role in OA, possibly through the regulation of protease activity via an LRP-1-protease axis. Currently, we are investigating the rescue of pathophysiological protease activity in vitro by modulating ciliome and peri-ciliary components. In vivo,we are using ageing and pre-clinical disease models to further understand the physiological and pathophysiological relevance of IFT and the ciliome
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
The primary cilia protein IFT88, exerts a profound and evolving influence in the post-natal joint, through joint maturation, adult life and in disease
No full textPurpose: Mechanical forces are critical for joint development, homeostasis and disease. During skeletal development mechanics contributes to formation of the joint, endochondral ossification during long bone elongation and growth plate closure. Physiological loading during adult life underpins articular cartilage health, continuity and thickness. Pathological loading of the joint leads to cartilage degradation and osteoarthritis (OA). However, we don’t yet understand the relative contributions of the cartilage, bone and synovium or cells vs matrix in healthy or pathological mechanotransduction. In both chondrocytes and osteocytes, proteins critical to the assembly and function of the primary cilium have been shown to be important to the response to mechanics. Recently we have shown they regulate cartilage turnover. The cilium is a microtubule-based organelle most famously associated with transduction of extracellular cues including the hedgehog ligand and mechanics. As such mutations to ciliary proteins results in a group of human congenital diseases known as ciliopathies including skeletal disorders, further emphasising the cilium’s critical role in musculoskeletal development. However, little is known about the cilium’s post-natal roles within the joint. A core component of the cilium is IFT88, disruption of which alters mechanically induced matrix production and catabolism in chondrocytes. We hypothesise that the crucial role of the cilium and associated machinery persists into adulthood, is important for the joints response to physiological loading and by extension, cartilage health.Methods: We have created an adult, cartilage-specific, inducible deletion model of IFT88 (ACAN;IFT88fl/flCreERT2) and a pan tissue (ROSA26;IFT88fl/flCreERT2). These were validated by qPCR, western blot, in vitro and a ROSA26tdtomato reporter. Phenotyping of this mouse was conducted using histology, MicroCT and imageJ. The surgical destabilisation of the medial meniscus model (DMM) was used to challenge the joint with aberrant mechanics to induce OA followed by double-blinded OARSI scoring.Results: The reporter mouse reveals that aggrecan cre is driving in the articular surface, growth plate, menisci and some populations within the bone. qPCR indicates an approximate halving of IFT88 mRNA in the ACAN;IFT88fl/flCreERT2, termed cKO hereafter. Activation of the ROSA26;IFT88fl/flCreERT2 also results in a halving of IFT88 mRNA in multiple tissues and reduction of protein in cartilaginous tissue. Between 4 and 10 weeks of age in mice, endochondral ossification is nearing completion and growth plate closure begins. Deletion of IFT88 at 4 weeks of age results in a longer growth plate including enlarged bi-lateral growth plate cartilage at 6 weeks. Deletion at 6 weeks also results in a longer growth plate at 8 weeks; in each case the cKO growth plate remains at the length it was 2 weeks earlier prior to deletion. Between 8 and 10 weeks of age, cKO of IFT88 also results in longer growth plates as growth plate closure is inhibited. Between 6 and 10 weeks of age the articular surface thickens at a linear rate from ∼85μm to 115μm. In the cKO, this thickening over each 2 week period is inhibited. Two weeks after deletion, cartilage is ∼15μm thinner at every time point in the cKO. Further analysis of histology reveals a reduction in the calcified cartilage region, whereas no change was seen in the non-calcified region. At 12 weeks post-DMM there is a significant difference in OARSI score between with higher scores in the cKO. In older mice (6 months of age) there is a pronounced long bone phenotype with loss of trabeculae and total bone volume.Conclusions: We have successfully depleted chondrocytes of IFT88 using an aggrecan Cre system, however potentially only in a subset of the population. IFT88 maintains influence through adolescence and early adulthood. Our data suggests IFT88/the cilium continues to regulate growth plate dynamics during and beyond developmental stages, potentially regulating both the Hh-PTHrP-Hh feedback loop during endochondral ossification and growth plate closure mechanisms. We propose this is by dysregulation of upstream mechanotransduction. IFT88 maintains influence during articular cartilage maturation and in early adulthood. Upon knock out of IFT88, articular cartilage thickness is reduced and associated with changes to the calcified region. This is in some agreement with changes seen in small models of cartilage atrophy, where the mechanism is not yet understood. On the introduction of pathological mechanical load using DMM, IFT88 deletion leads to exacerbation of articular cartilage degradation, potentially owing to loss of physiological mechanotransduction prior to DMM challenge. On-going work is analysing the effects at earlier time points in disease progression (8 weeks post-DMM) and importantly the naive articular surface during adulthood. Collectively, with our previous in vitro work, we propose IFT88 regulates cartilage turnover and potentially ossification of deep zone articular cartilage. We are now testing these hypotheses, targeting other elements of the ciliome and exploring the molecular mechanisms downstream to IFT88 loss in mature and mechanically challenged cartilage to compare with changes seen in atrophy and OA.</p
Chondrocyte dedifferentiation down regulates mechano-responsiveness and hedgehog signalling associated with changes in primary cilia structure
Full text linkPurpose: with the limited availability of human chondrocytes for tissue engineering applications, passaging is used to increase cell yield. However, this 2D expansion leads to a loss of chondrogenic phenotype associated with changes in actin organisation. Primary cilia are microtubule structures that act as a signalling hub controlling a variety of signalling pathways including mechanotransduction and hedgehog signalling, both of which are involved in differentiation and osteoarthritis. Primary cilia structure and function are known to be regulated by changes in actin tension. Thus the present study tests the hypothesis that chondrocyte dedifferentiation during monolayer expansion leads to alterations in mechano-responsiveness and hedgehog signalling mediated by changes in primary cilia structure.Methods: chondrocytes were isolated from the metacarpophalangeal joints of 18-24 month old steers. Primary chondrocytes at passage 0 (P0) were cultured in monolayer up to passage 5 (P5) using DMEM containing 10% FBS, 1.9 mM L-glutamine, 96 U/ml penicillin, 96 mg/ml streptomycin, 20 mM HEPES buffer, and 0.74 mM L-ascorbic acid.For mechano-responsiveness, primary (P0) and dedifferentiated (P5) chondrocytes were cultured on collagen I-coated Bioflex plates and subjected to cyclic tensile strain (10%, 0.33 Hz, 1 hour) via the Flexcell 4000T system, prior to analysis of collagen II, aggrecan, ADAMTS5, and MMP13 gene expression. For hedgehog signalling, P0 and P5 chondrocytes were treated with 1 μg/ml Indian hedgehog (Ihh) for 24 hours. Hedgehog pathway activation was then quantified by analysis of Gli1 and Ptch1 gene expression. Controls were maintained in parallel for both studies. Total RNA was isolated using RNeasy Kit (Qiagen). Quantitect Reverse Transcription Kit (Qiagen) was used to covert 1 μg RNA to cDNA and real-time PCR was performed using KAPA SYBR® FAST qPCR Kit (KAPA Biosystems).Cells at P0 to P5 were also fixed with 4% paraformaldehyde and labelled for acetylated α-tubulin and Ki67, with DAPI counterstaining. Confocal microscopy was used to determine the percentage of Ki67 positive cells as well as primary cilia prevalence and length.Results: at P0, cyclic tensile strain significantly upregulated aggrecan, type II collagen and ADAMTS5 gene expression and downregulated MMP13 expression (Fig. 1). This response to loading was completely lost in the P5 chondrocytes. When chondrocytes at P0 were treated with Ihh, pathway activation was confirmed by a 5-fold increase in Gli1 and Ptch1 gene expression (Fig. 2). However both Gli1 and Ptch1 responses to Ihh were significantly reduced at P5 with a complete suppression of Ptch1 up-regulation.Cell proliferation, as indicated by Ki67, remained less than 30% although there was a significant increase by passage 3 (Fig. 3A). The percentage of ciliated cells increased significantly after the first passage and remained constant at 40-50% with subsequent passaging (Fig. 3B). Chondrocyte primary cilia length increased continuously with passage, doubling in length from P0 to P5 (Fig. 3C and D).Conclusions: changes in primary cilia length affects cilia function including mechanotransduction and hedgehog signalling and may also affect other cilia signalling pathways. As hypothesized, the reduction in mechano-responsiveness and hedgehog signalling with extended 2D culture is due to the associated elongation of primary cilia. To further identify the underpinning mechanisms, ongoing studies are using super resolution microscopy to examine whether cilia elongation at P5 is associated with alterations in cilia expression of ARL13B, and putative mechanoreceptors polycystin 2 and TRPV4. The observed reductions in mechano-responsiveness and ligand-induced hedgehog signalling with passage have important implications for the success of cartilage tissue engineering. Future work will investigate whether modulation of cilia length can maintain or rescue mechano- and hedgehog-responsiveness during expansion of chondrocytes for cartilage tissue engineering
The ciliary protein intraflagellar transport 88 is required for the maturation, homeostasis and mechanoadaptation of articular cartilage
No full textPurpose: The development, maturation and maintenance of articular cartilage depends on the integration of external cues, such as mechanics, with intrinsic cell signalling programmes, such as hedgehog (Hh) signalling. Aberrant mechanics and the post-natal activation of Hh signalling have both been associated with the development of murine and human OA. It remains to be fully elucidated how chondrocytes transduce and integrate these cues in vivo. Chondrocytes assemble a primary cilium, a microtubule-based organelle with a devoted trafficking machinery, IntraFlagellar Transport or IFT. In vitro studies indicate ciliary IFT helps tune the chondrocyte response to Hh ligand and the anabolic matrix response to cell compression. Recently, we have shown loss of function of IFT88, inhibiting ciliogenesis in chondrocytes, impairs LRP-1 mediated endocytotic clearance of proteases, resulting in increased aggrecanolysis. While constitutive and peri-natal disruption of ciliary proteins, Hh signalling and altered mechanics, all drastically alter joint development in vivo, the influence of IFT in adult cartilage homeostasis remains unknown.Methods: IFT88 was targeted using a cartilage-specific, inducible mouse line (ACANCreERT2;IFT88fl/fl : cKO hereafter). Cre activity was validated by qPCR, and using a ROSA26tdtomato reporter line. IFT88fl/fl mice, also receiving I.P injections of tamoxifen, were used as controls. Tibial articular cartilage was assessed 2, 14 or 26 weeks post tamoxifen, at 8, 10, 22 and 34 weeks of age respectively, using histomorphometric analyses, including measurements of articular cartilage thickness, relative calcification, and OARSI score and by means of immunohistochemistry (IHC). The DMM model, which destabilising the joint, was performed at 10 weeks of age. Means ± S.D are quoted throughout, Mann-Whitney U-tests used for statistical comparisons.Results: The Tdtomato reporter demonstrated ACANcre activity in hip and throughout knee cartilage. Tamoxifen treatment of cKO mice resulted in a 50% reduction of IFT88 mRNA in articular cartilage (p=0.02, n=6 ctrl, 14 cKO). In control mice, tibial articular cartilage thickens between 8 and 22 weeks of age, most notably on the medial plateau. Calcified cartilage (below the tidemark) progressively increases on both plateaus, between 6 and 22 weeks of age. IFT88 depletion (cKO) resulted in thinner medial articular cartilage (MC), compared with controls, at all 5 time-points. Fig 1.A shows MC thickness in adult control (crosses) and cKO (circles). In control mice, MC thickness increased from 99.0 +/- 9.2 μm at 8 weeks of age to 108.9 +/- 7.2 μm at 10 weeks of age. Tamoxifen treatment, at 8 weeks of age, inhibited this increase in cKO mice (MC thickness at 10 weeks was 96.2 +/- 7.4 μm, p=0.02, compared with 10 week ctrl, n=7). By 22 weeks of age mean MC thickness in cKO was 90.2 +/- 3.3 μm compared with 111.6 +/- 10.1 μm in control animals (p=0.0002, n= 7 and 10 respectively). By 34 weeks MC had continued to thin, but this was now associated with surface damage and osteophyte formation. In the most extreme case, MC was completely lost (Fig.1B). In contrast, lateral plateau thickness and OARSI score was unaffected. At all time-points thinning was attributable to loss of calcified cartilage. IHC analyses revealed no striking differences in collagen X expression, NITEGE neoepitope staining or the expression of LRP-1β. 12 weeks post DMM, OARSI scores were statistically significantly higher in cKO mice.Conclusions: Progressive thickening and calcification in the mouse medial compartment illustrates the continued adaptation of adolescent and adult articular cartilage, in the medial loading environment. IFT88 deletion inhibits MC thickening, leading to atrophy, which then predisposes the joint to spontaneous OA as the mouse ages. The lateral compartment is relatively unaffected. We propose this may be due, in part, to disruption of mechanotransduction and downstream anabolic remodelling in medial cartilage. Deletion of IFT88 impairs the progressive calcification of articular cartilage, in both compartments, which may be due to disruption of intrinsic cartilage Hh signalling. Mechanistic experiments, dissecting the relative roles and integration of IFT, mechanics and Hh in the context of adult cartilage are on-going. We conclude that IFT88 maintains a profound influence in post-natal articular cartilage homeostasis and protection from OA.</div
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