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Objective: Cartilage in joints such as the hip and knee experiences repeated phases of heavy loading and low load recovery during the 24-h day/night cycle. Our previous work has shown 24 h rhythmic changes in gene expression at transcript level between night and day in wild type mouse cartilage which is lost in a circadian clock knock-out mouse model. However, it remains unknown to what extent circadian rhythms also regulate protein level gene expression in this matrix rich tissue., Methods: We investigated daily changes of protein abundance in mouse femoral head articular cartilage by performing a 48-h time-series LC-MS/MS analysis., Results: Out of the 1,177 proteins we identified across all time points, 145 proteins showed rhythmic changes in their abundance within the femoral head cartilage. Among these were molecules that have been implicated in key cartilage functions, including CTGF, MATN1, PAI-1 and PLOD1 & 2. Pathway analysis revealed that protein synthesis, cytoskeleton and glucose metabolism exhibited time-of-day dependent functions. Analysis of published cartilage proteomics datasets revealed that a significant portion of rhythmic proteins were dysregulated in osteoarthritis and/or ageing., Conclusions: Our circadian proteomics study reveals that articular cartilage is a much more dynamic tissue than previously thought, with chondrocytes driving circadian rhythms not only in gene transcription but also in protein abundance. Our results clearly call for the consideration of circadian timing mechanisms not only in cartilage biology, but also in the pathogenesis, treatment strategies and biomarker detection in osteoarthritis., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cysteine-rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) is an endoplasmic reticulum (ER)-resident chaperone highly activated under ER stress in conditions such as chondrodysplasias; however, its role in healthy skeletal development is unknown. We show for the first time that cartilage-specific deletion of Creld2 results in disrupted endochondral ossification and short limbed dwarfism, whereas deletion of Creld2 in bone results in osteopenia, with a low bone density and altered trabecular architecture. Our study provides the first evidence that CRELD2 promotes the differentiation and maturation of skeletal cells by modulating noncanonical WNT4 signaling regulated by p38 MAPK. Furthermore, we show that CRELD2 is a novel chaperone for the receptor low-density lipoprotein receptor-related protein 1 (LRP1), promoting its transport to the cell surface, and that LRP1 directly regulates WNT4 expression in chondrocytes through TGF-β1 signaling. Therefore, our data provide a novel link between an ER-resident chaperone and the essential WNT signaling pathways active during skeletal differentiation that could be applicable in other WNT-responsive tissues. © 2020 American Society for Bone and Mineral Research. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.., (© 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.)

Background: Osteoarthritis has been associated with a plethora of pathological factors and one which has recently emerged is chondrocyte endoplasmic reticulum (ER) stress. ER stress is sensed by key ER-resident stress sensors, one of which is activating transcription factor 6 (ATF6). The purpose of this study is to determine whether increased ER stress plays a role in OA., Methods: OA was induced in male wild-type (+/+), ColIITg cog (c/c) and Atf6α -/- mice by destabilisation of the medial meniscus (DMM). c/c mice have increased ER stress in chondrocytes via the collagen II promoter-driven expression of ER stress-inducing Tg cog . Knee joints were scored histologically for OA severity. RNA-seq was performed on laser-micro-dissected RNA from cartilage of +/+ and c/c DMM-operated mice., Results: In situ hybridisation demonstrated a correlation between the upregulation of ER stress marker, BiP, and early signs of proteoglycan loss and cartilage damage in DMM-operated +/+ mice. Histological analysis revealed a significant reduction in OA severity in c/c mice compared with +/+ at 2 weeks post-DMM. This chondroprotective effect in c/c mice was associated with a higher ambient level of BiP protein prior to DMM and a delay in chondrocyte apoptosis. RNA-seq analysis suggested Xbp1-regulated networks to be significantly enriched in c/c mice at 2 weeks post-DMM. Compromising the ER through genetically ablating Atf6α, a key ER stress sensor, had no effect on DMM-induced OA severity., Conclusion: Our studies indicate that an increased capacity to effectively manage increases in ER stress in articular cartilage due either to pre-conditioning as a result of prior exposure to ER stress or to genetic pre-disposition may be beneficial in delaying the onset of OA, but once established, ER stress plays no significant role in disease progression.

The unfolded protein response (UPR) is a conserved cellular response to the accumulation of proteinaceous material in endoplasmic reticulum (ER), active both in health and disease to alleviate cellular stress and improve protein folding. Multiple epiphyseal dysplasia (EDM5) is a genetic skeletal condition and a classic example of an intracellular protein aggregation disease, whereby mutant matrilin-3 forms large insoluble aggregates in the ER lumen, resulting in a specific 'disease signature' of increased expression of chaperones and foldases, and alternative splicing of the UPR effector XBP1. Matrilin-3 is expressed exclusively by chondrocytes thereby making EDM5 a perfect model system to study the role of protein aggregation in disease. In order to dissect the role of XBP1 signalling in aggregation-related conditions we crossed a p.V194D Matn3 knock-in mouse model of EDM5 with a mouse line carrying a cartilage specific deletion of XBP1 and analysed the resulting phenotype. Interestingly, the growth of mice carrying the Matn3 p.V194D mutation compounded with the cartilage specific deletion of XBP1 was severely retarded. Further phenotyping revealed increased intracellular retention of amyloid-like aggregates of mutant matrilin-3 coupled with dramatically decreased cell proliferation and increased apoptosis, suggesting a role of XBP1 signalling in protein accumulation and/or degradation. Transcriptomic analysis of chondrocytes extracted from wild type, EDM5, Xbp1-null and compound mutant lines revealed that the alternative splicing of Xbp1 is crucial in modulating levels of protein aggregation. Moreover, through detailed transcriptomic comparison with a model of metaphyseal chondrodysplasia type Schmid (MCDS), an UPR-related skeletal condition in which XBP1 was removed without overt consequences, we show for the first time that the differentiation-state of cells within the cartilage growth plate influences the UPR resulting from retention of a misfolded mutant protein and postulate that modulation of XBP1 signalling pathway presents a therapeutic target for aggregation related conditions in cells undergoing proliferation., Competing Interests: The authors have declared that no competing interests exist.

Motivation: Skeletal diseases are prevalent in society, but improved molecular understanding is required to formulate new therapeutic strategies. Large and increasing quantities of available skeletal transcriptomics experiments give the potential for mechanistic insight of both fundamental skeletal biology and skeletal disease. However, no current repository provides access to processed, readily interpretable analysis of this data. To address this, we have developed SkeletalVis, an exploration portal for skeletal gene expression experiments., Results: The SkeletalVis data portal provides an exploration and comparison platform for analysed skeletal transcriptomics data. It currently hosts 287 analysed experiments with 739 perturbation responses with comprehensive downstream analysis. We demonstrate its utility in identifying both known and novel relationships between skeletal expression signatures. SkeletalVis provides users with a platform to explore the wealth of available expression data, develop consensus signatures and the ability to compare gene signatures from new experiments to the analysed data to facilitate meta-analysis., Availability and Implementation: The SkeletalVis data portal is freely accessible at http://phenome.manchester.ac.uk., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press.)

This review, based on the BSMB Fell-Muir Lecture I presented in July 2018 at the Matrix Biology Europe Conference in Manchester, gives a personal perspective of my own laboratory's contributions to research into type X collagen, metaphyseal chondrodysplasia type Schmid and potential treatments for this disorder that are currently entering clinical trial. I have tried to set the advances made in the context of the scientific technologies available at the time and how these have changed over the more than three decades of this research., (© 2019 The Author. International Journal of Experimental Pathology © 2019 International Journal of Experimental Pathology.)

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) resident protein that can be secreted due to an imperfect KDEL motif. MANF plays a cytoprotective role in several soft tissues and is upregulated in conditions resulting from intracellular retention of mutant protein, including two skeletal diseases, metaphyseal chondrodysplasia, Schmid type (MCDS) and multiple epiphyseal dysplasia (MED). The role of MANF in skeletal tissue homeostasis is currently unknown. Interestingly, cartilage-specific deletion of Manf in a mouse model of MED resulted in increased disease severity, suggesting its upregulation may be chondroprotective. Treatment of MED chondrocytes with exogenous MANF led to a decrease in the cellular levels of BiP (GRP78), confirming MANF's potential to modulate ER stress responses. However, it did not alleviate the intracellular retention of mutant matrilin-3, suggesting that it is the intracellular MANF that is of importance in the pathobiology of skeletal dysplasias. The Col2Cre-driven deletion of Manf from mouse cartilage resulted in a chondrodysplasia-like phenotype. Interestingly, ablation of MANF in cartilage did not have extracellular consequences but led to an upregulation of several ER-resident chaperones including BiP. This apparent induction of ER stress in turn led to dysregulated chondrocyte apoptosis and decreased proliferation, resulting in reduced long bone growth. We have previously shown that ER stress is an underlying disease mechanism for several skeletal dysplasias. The cartilage-specific deletion of Manf described in this study phenocopies our previously published chondrodysplasia models, further confirming that ER stress itself is sufficient to disrupt skeletal growth and thus represents a potential therapeutic target.

Mutations, mostly in the region of the COL10A1 gene encoding the C-terminal non-collagenous domain, cause the dwarfism metaphyseal chondrodysplasia type Schmid (MCDS). In most cases, the disease mechanism involves the misfolding of the mutant protein causing increased endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). However, in an iliac crest biopsy, the COL10A1 p.Y632X mutation was found to produce instability of the mutant mRNA such that little mutant protein may be produced. To investigate the disease mechanism further, a gene-targeted mouse model of the Col10a1 p.Y632X mutation was generated. In this model, the mutant mRNA showed no instability, and in mice heterozygous for the mutation, mutant and wild-type mRNAs were present at equal concentrations. The protein was translated from the mutant allele and retained within the cell, triggering increased ER stress and a UPR. The mutation produced a relatively severe form of MCDS. Nevertheless, treatment of the mice with carbamazepine (CBZ), a drug which stimulates intracellular proteolysis and alleviates ER stress, effectively reduced the disease severity in this model of MCDS caused by a premature stop codon in the Col10a1 gene. Specifically, the drug reduced ER stress in the growth plate, restored growth plate architecture toward the wild-type state, significantly increased bone growth and within 2 weeks of treatment corrected the MCDS-induced hip distortion. These results indicate that CBZ is likely to be effective in ongoing clinical trials against all forms of MCDS whether caused by premature stop codons or substitutions.

Fibrillin microfibrils are extracellular matrix assemblies that form the template for elastic fibres, endow blood vessels, skin and other elastic tissues with extensible properties. They also regulate the bioavailability of potent growth factors of the TGF-β superfamily. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)10 is an essential factor in fibrillin microfibril function. Mutations in fibrillin-1 or ADAMTS10 cause Weill-Marchesani syndrome (WMS) characterized by short stature, eye defects, hypermuscularity and thickened skin. Despite its importance, there is poor understanding of the role of ADAMTS10 and its function in fibrillin microfibril assembly. We have generated an ADAMTS10 WMS mouse model using Clustered Regularly Spaced Interspaced Short Palindromic Repeats and CRISPR associated protein 9 (CRISPR-Cas9) to introduce a truncation mutation seen in WMS patients. Homozygous WMS mice are smaller and have shorter long bones with perturbation to the zones of the developing growth plate and changes in cell proliferation. Furthermore, there are abnormalities in the ciliary apparatus of the eye with decreased ciliary processes and abundant fibrillin-2 microfibrils suggesting perturbation of a developmental expression switch. WMS mice have increased skeletal muscle mass and more myofibres, which is likely a consequence of an altered skeletal myogenesis. These results correlated with expression data showing down regulation of Growth differentiation factor (GDF8) and Bone Morphogenetic Protein (BMP) growth factor genes. In addition, the mitochondria in skeletal muscle are larger with irregular shape coupled with increased phospho-p38 mitogen-activated protein kinase (MAPK) suggesting muscle remodelling. Our data indicate that decreased SMAD1/5/8 and increased p38/MAPK signalling are associated with ADAMTS10-induced WMS. This model will allow further studies of the disease mechanism to facilitate the development of therapeutic interventions.

Whilst the role of ATF6α in modulating the unfolded protein response (UPR) has been well documented, the function of its paralogue ATF6β is less well understood. Using knockdown in cell culture and gene ablation in mice we have directly compared the roles of ATF6α & β in responding to the increased ER stress induced by mutant forms of type X collagen that cause the ER stress-associated metaphyseal chondrodysplasia type Schmid (MCDS). ATF6α more efficiently deals with the disease-associated ER stress in the absence of ATF6β and conversely, ATF6β is less effective in the absence of ATF6α. Furthermore, disease severity in vivo is increased by ATF6α ablation and decreased by ATF6β ablation. In addition, novel functions for each paralogue are described including an ATF6β-specific role in controlling growth plate chondrocyte proliferation. The clear demonstration of the intimate relationship of the two ATF6 isoforms and how ATF6β can moderate the activity of ATF6α and vice versa is of great significance for understanding the UPR mechanism. The activities of both ATF6 isoforms and their separate roles need consideration when deciding how to target increased ER stress as a means of treating MCDS and other ER stress-associated diseases., (Copyright © 2018 University of Manchester. Published by Elsevier B.V. All rights reserved.)

Introduction: Osteoarthritis (OA) is a heterogeneous and complex disease. We have used a network biology approach based on genome-wide analysis of gene expression in OA knee cartilage to seek evidence for pathogenic mechanisms that may distinguish different patient subgroups., Methods: Results from RNA-Sequencing (RNA-Seq) were collected from intact knee cartilage at total knee replacement from 44 patients with OA, from 16 additional patients with OA and 10 control patients with non-OA. Results were analysed to identify patient subsets and compare major active pathways., Results: The RNA-Seq results showed 2692 differentially expressed genes between OA and non-OA. Analysis by unsupervised clustering identified two distinct OA groups: Group A with 24 patients (55%) and Group B with 18 patients (41%). A 10 gene subgroup classifier was validated by RT-qPCR in 16 further patients with OA. Pathway analysis showed increased protein expression in both groups. PhenomeExpress analysis revealed group differences in complement activation, innate immune responses and altered Wnt and TGFβ signalling, but no activation of inflammatory cytokine expression. Both groups showed suppressed circadian regulators and whereas matrix changes in Group A were chondrogenic, in Group B they were non-chondrogenic with changes in mechanoreceptors, calcium signalling, ion channels and in cytoskeletal organisers. The gene expression changes predicted 478 potential biomarkers for detection in synovial fluid to distinguish patients from the two groups., Conclusions: Two subgroups of knee OA were identified by network analysis of RNA-Seq data with evidence for the presence of two major pathogenic pathways. This has potential importance as a new basis for the stratification of patients with OA for drug trials and for the development of new targeted treatments., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The short-limbed dwarfism metaphyseal chondrodysplasia type Schmid (MCDS) is linked to mutations in type X collagen, which increase ER stress by inducing misfolding of the mutant protein and subsequently disrupting hypertrophic chondrocyte differentiation. Here, we show that carbamazepine (CBZ), an autophagy-stimulating drug that is clinically approved for the treatment of seizures and bipolar disease, reduced the ER stress induced by 4 different MCDS-causing mutant forms of collagen X in human cell culture. Depending on the nature of the mutation, CBZ application stimulated proteolysis of misfolded collagen X by either autophagy or proteasomal degradation, thereby reducing intracellular accumulation of mutant collagen. In MCDS mice expressing the Col10a1.pN617K mutation, CBZ reduced the MCDS-associated expansion of the growth plate hypertrophic zone, attenuated enhanced expression of ER stress markers such as Bip and Atf4, increased bone growth, and reduced skeletal dysplasia. CBZ produced these beneficial effects by reducing the MCDS-associated abnormalities in hypertrophic chondrocyte differentiation. Stimulation of intracellular proteolysis using CBZ treatment may therefore be a clinically viable way of treating the ER stress-associated dwarfism MCDS.

Objectives: The circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration., Methods: Clock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2 ::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1 ., Results: Here we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1β but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs., Conclusions: We have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

PhenomeScape is a Cytoscape app which provides easy access to the PhenomeExpress algorithm to interpret gene expression data. PhenomeExpress integrates protein interaction networks with known phenotype to gene associations to find active sub-networks enriched in differentially expressed genes. It also incorporates cross-species phenotypes and associations to include results from animal models of disease. With expression data imported into PhenomeScape, the user can quickly generate and visualise interactive sub-networks. PhenomeScape thus enables researchers to use prior knowledge of a disease to identify differentially regulated sub-networks and to generate an overview of altered biologically processes specific to that disease., Availability and Implementation: Freely available for download at https://github.com/soulj/PhenomeScape CONTACT: jamie.soul@postgrad.manchester.ac.uk or jean-marc.schwartz@manchester.ac.uk., (© The Author 2016. Published by Oxford University Press.)

Objectives: Joint degeneration in osteoarthritis (OA) is characterised by damage and loss of articular cartilage. The pattern of loss is consistent with damage occurring only where the mechanical loading is high. We have investigated using RNA-sequencing (RNA-seq) and systems analyses the changes that occur in damaged OA cartilage by comparing it with intact cartilage from the same joint., Methods: Cartilage was obtained from eight OA patients undergoing total knee replacement. RNA was extracted from cartilage on the damaged distal medial condyle (DMC) and the intact posterior lateral condyle (PLC). RNA-seq was performed to identify differentially expressed genes (DEGs) and systems analyses applied to identify dysregulated pathways., Results: In the damaged OA cartilage, there was decreased expression of chondrogenic genes SOX9, SOX6, COL11A2, COL9A1/2/3, ACAN and HAPLN1; increases in non-chondrogenic genes COL1A1, COMP and FN1; an altered pattern of secreted proteinase expression; but no expression of major inflammatory cytokines. Systems analyses by PhenomeExpress revealed significant sub-networks of DEGs including mitotic cell cycle, Wnt signalling, apoptosis and matrix organisation that were influenced by a core of altered transcription factors (TFs), FOSL1, AHR, E2F1 and FOXM1., Conclusions: Gene expression changes in damaged cartilage suggested a signature non-chondrogenic response of altered matrix protein and secreted proteinase expression. There was evidence of a damage response in this late OA cartilage, which surprisingly showed features detected experimentally in the early response of cartilage to mechanical overload. PhenomeExpress analysis identified a hub of DEGs linked by a core of four differentially regulated TFs., (Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Osteoarthritis (OA) is the most prevalent and debilitating joint disease, and there are currently no effective disease-modifying treatments available. Multiple risk factors for OA, such as aging, result in progressive damage and loss of articular cartilage. Autonomous circadian clocks have been identified in mouse cartilage, and environmental disruption of circadian rhythms in mice predisposes animals to OA-like damage. However, the contribution of the cartilage clock mechanisms to the maintenance of tissue homeostasis is still unclear. Here, we have shown that expression of the core clock transcription factor BMAL1 is disrupted in human OA cartilage and in aged mouse cartilage. Furthermore, targeted Bmal1 ablation in mouse chondrocytes abolished their circadian rhythm and caused progressive degeneration of articular cartilage. We determined that BMAL1 directs the circadian expression of many genes implicated in cartilage homeostasis, including those involved in catabolic, anabolic, and apoptotic pathways. Loss of BMAL1 reduced the levels of phosphorylated SMAD2/3 (p-SMAD2/3) and NFATC2 and decreased expression of the major matrix-related genes Sox9, Acan, and Col2a1, but increased p-SMAD1/5 levels. Together, these results define a regulatory mechanism that links chondrocyte BMAL1 to the maintenance and repair of cartilage and suggest that circadian rhythm disruption is a risk factor for joint diseases such as OA.

Objective: To define how the catabolic cytokines (Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFα)) affect the circadian clock mechanism and the expression of clock-controlled catabolic genes within cartilage, and to identify the downstream pathways linking the cytokines to the molecular clock within chondrocytes., Methods: Ex vivo cartilage explants were isolated from the Cry1-luc or PER2::LUC clock reporter mice. Clock gene dynamics were monitored in real-time by bioluminescence photon counting. Gene expression changes were studied by qRT-PCR. Functional luc assays were used to study the function of the core Clock/BMAL1 complex in SW-1353 cells. NFкB pathway inhibitor and fluorescence live-imaging of cartilage were performed to study the underlying mechanisms., Results: Exposure to IL-1β severely disrupted circadian gene expression rhythms in cartilage. This effect was reversed by an anti-inflammatory drug dexamethasone, but not by other clock synchronizing agents. Circadian disruption mediated by IL-1β was accompanied by disregulated expression of endogenous clock genes and clock-controlled catabolic pathways. Mechanistically, NFкB signalling was involved in the effect of IL-1β on the cartilage clock in part through functional interference with the core Clock/BMAL1 complex. In contrast, TNFα had little impact on the circadian rhythm and clock gene expression in cartilage., Conclusion: In our experimental system (young healthy mouse cartilage), we demonstrate that IL-1β (but not TNFα) abolishes circadian rhythms in Cry1-luc and PER2::LUC gene expression. These data implicate disruption of the chondrocyte clock as a novel aspect of the catabolic responses of cartilage to pro-inflammatory cytokines, and provide an additional mechanism for how chronic joint inflammation may contribute to osteoarthritis (OA)., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Schmid metaphyseal chondrodysplasia (MCDS) involves dwarfism and growth plate cartilage hypertrophic zone expansion resulting from dominant mutations in the hypertrophic zone collagen, Col10a1. Mouse models phenocopying MCDS through the expression of an exogenous misfolding protein in the endoplasmic reticulum (ER) in hypertrophic chondrocytes have demonstrated the central importance of ER stress in the pathology of MCDS. The resultant unfolded protein response (UPR) in affected chondrocytes involved activation of canonical ER stress sensors, IRE1, ATF6, and PERK with the downstream effect of disrupted chondrocyte differentiation. Here, we investigated the role of the highly conserved IRE1/XBP1 pathway in the pathology of MCDS. Mice with a MCDS collagen X p.N617K knock-in mutation (ColXN617K) were crossed with mice in which Xbp1 was inactivated specifically in cartilage (Xbp1CartΔEx2), generating the compound mutant, C/X. The severity of dwarfism and hypertrophic zone expansion in C/X did not differ significantly from ColXN617K, revealing surprising redundancy for the IRE1/XBP1 UPR pathway in the pathology of MCDS. Transcriptomic analyses of hypertrophic zone cartilage identified differentially expressed gene cohorts in MCDS that are pathologically relevant (XBP1-independent) or pathologically redundant (XBP1-dependent). XBP1-independent gene expression changes included large-scale transcriptional attenuation of genes encoding secreted proteins and disrupted differentiation from proliferative to hypertrophic chondrocytes. Moreover, these changes were consistent with disruption of C/EBP-β, a master regulator of chondrocyte differentiation, by CHOP, a transcription factor downstream of PERK that inhibits C/EBP proteins, and down-regulation of C/EBP-β transcriptional co-factors, GADD45-β and RUNX2. Thus we propose that the pathology of MCDS is underpinned by XBP1 independent UPR-induced dysregulation of C/EBP-β-mediated chondrocyte differentiation. Our data suggest that modulation of C/EBP-β activity in MCDS chondrocytes may offer therapeutic opportunities.

Objective: To investigate the in vivo role of the IRE1/XBP1 unfolded protein response (UPR) signaling pathway in cartilage., Design: Xbp1(flox/flox).Col2a1-Cre mice (Xbp1(CartΔEx2)), in which XBP1 activity is ablated specifically from cartilage, were analyzed histomorphometrically by Alizarin red/Alcian blue skeletal preparations and X-rays to examine overall bone growth, histological stains to measure growth plate zone length, chondrocyte organization, and mineralization, and immunofluorescence for collagen II, collagen X, and IHH. Bromodeoxyuridine (BrdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses were used to measure chondrocyte proliferation and cell death, respectively. Chondrocyte cultures and microdissected growth plate zones were analyzed for expression profiling of chondrocyte proliferation or endoplasmic reticulum (ER) stress markers by Quantitative PCR (qPCR), and of Xbp1 mRNA splicing by RT-PCR to monitor IRE1 activation., Results: Xbp1(CartΔEx2) displayed a chondrodysplasia involving dysregulated chondrocyte proliferation, growth plate hypertrophic zone shortening, and IRE1 hyperactivation in chondrocytes. Deposition of collagens II and X in the Xbp1(CartΔEx2) growth plate cartilage indicated that XBP1 is not required for matrix protein deposition or chondrocyte hypertrophy. Analyses of mid-gestation long bones revealed delayed ossification in Xbp1(CartΔEx2) embryos. The rate of chondrocyte cell death was not significantly altered, and only minimal alterations in the expression of key markers of chondrocyte proliferation were observed in the Xbp1(CartΔEx2) growth plate. IRE1 hyperactivation occurred in Xbp1(CartΔEx2) chondrocytes but was not sufficient to induce regulated IRE1-dependent decay (RIDD) or a classical UPR., Conclusion: Our work suggests roles for XBP1 in regulating chondrocyte proliferation and the timing of mineralization during endochondral ossification, findings which have implications for both skeletal development and disease., (Copyright © 2015 Osteoarthritis Research Society International. All rights reserved.)

Mutations in genes encoding cartilage oligomeric matrix protein and matrilin-3 cause a spectrum of chondrodysplasias called multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). The majority of these diseases feature classical endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) as a result of misfolding of the mutant protein. However, the importance and the pathological contribution of ER stress in the disease pathogenesis are unknown. The aim of this study was to investigate the generic role of ER stress and the UPR in the pathogenesis of these diseases. A transgenic mouse line (ColIITgcog) was generated using the collagen II promoter to drive expression of an ER stress-inducing protein (Tgcog) in chondrocytes. The skeletal and histological phenotypes of these ColIITgcog mice were characterised. The expression and intracellular retention of Tgcog induced ER stress and activated the UPR as characterised by increased BiP expression, phosphorylation of eIF2α and spliced Xbp1. ColIITgcog mice exhibited decreased long bone growth and decreased chondrocyte proliferation rate. However, there was no disruption of chondrocyte morphology or growth plate architecture and perturbations in apoptosis were not apparent. Our data demonstrate that the targeted induction of ER stress in chondrocytes was sufficient to reduce the rate of bone growth, a key clinical feature associated with MED and PSACH, in the absence of any growth plate dysplasia. This study establishes that classical ER stress is a pathogenic factor that contributes to the disease mechanism of MED and PSACH. However, not all the pathological features of MED and PSACH were recapitulated, suggesting that a combination of intra- and extra-cellular factors are likely to be responsible for the disease pathology as a whole.

We describe a new method, PhenomeExpress, for the analysis of transcriptomic datasets to identify pathogenic disease mechanisms. Our analysis method includes input from both protein-protein interaction and phenotype similarity networks. This introduces valuable information from disease relevant phenotypes, which aids the identification of sub-networks that are significantly enriched in differentially expressed genes and are related to the disease relevant phenotypes. This contrasts with many active sub-network detection methods, which rely solely on protein-protein interaction networks derived from compounded data of many unrelated biological conditions and which are therefore not specific to the context of the experiment. PhenomeExpress thus exploits readily available animal model and human disease phenotype information. It combines this prior evidence of disease phenotypes with the experimentally derived disease data sets to provide a more targeted analysis. Two case studies, in subchondral bone in osteoarthritis and in Pax5 in acute lymphoblastic leukaemia, demonstrate that PhenomeExpress identifies core disease pathways in both mouse and human disease expression datasets derived from different technologies. We also validate the approach by comparison to state-of-the-art active sub-network detection methods, which reveals how it may enhance the detection of molecular phenotypes and provide a more detailed context to those previously identified as possible candidates.

The small GTPase RhoA is a major regulator of actin reorganization during the formation of stress fibers; thus identifying molecules that regulate Rho activity is necessary for a complete understanding of the mechanisms that determine cell contractility. Here, we have identified Arhgap28 as a Rho GTPase activating protein (RhoGAP) that switches RhoA to its inactive form. We generated an Arhgap28-LacZ reporter mouse that revealed gene expression in soft tissues at E12.5, pre-bone structures of the limb at E15.5, and prominent expression restricted mostly to ribs and limb long bones at E18.5 days of development. Expression of recombinant Arhgap28-V5 in human osteosarcoma SaOS-2 cells caused a reduction in the basal level of RhoA activation and disruption of actin stress fibers. Extracellular matrix assembly studies using a 3-dimensional cell culture system showed that Arhgap28 was upregulated during Rho-dependent assembly of the ECM. Taken together, these observations led to the hypothesis that an Arhgap28 knockout mouse model would show a connective tissue phenotype, perhaps affecting bone. Arhgap28-null mice were viable and appeared normal, suggesting that there could be compensation from other RhoGAPs. Indeed, we showed that expression of Arhgap6 (a closely related RhoGAP) was upregulated in Arhgap28-null bone tissue. An upregulation in RhoA expression was also detected suggesting that Arhgap28 may be able to additionally regulate Rho signaling at a transcriptional level. Microarray analyses revealed that Col2a1, Col9a1, Matn3, and Comp that encode extracellular matrix proteins were downregulated in Arhgap28-null bone. Although mutations in these genes cause bone dysplasias no bone phenotype was detected in the Arhgap-28 null mice. Together, these data suggest that the regulation of Rho by RhoGAPs, including Arhgap28, during the assembly and development of mechanically strong tissues is complex and may involve multiple RhoGAPs.

Pseudoachondroplasia (PSACH) is an autosomal dominant skeletal dysplasia caused by mutations in cartilage oligomeric matrix protein (COMP) and characterised by short limbed dwarfism and early onset osteoarthritis. Mouse models of PSACH show variable retention of mutant COMP in the ER of chondrocytes, however, in each case a different stress pathway is activated and the underlying disease mechanisms remain largely unknown. T585M COMP mutant mice are a model of moderate PSACH and demonstrate a mild ER stress response. Although mutant COMP is not retained in significant quantities within the ER of chondrocytes, both BiP and the pro-apoptotic ER stress-related transcription factor CHOP are mildly elevated, whilst bcl-2 levels are decreased, resulting in increased and spatially dysregulated chondrocyte apoptosis. To determine whether the abnormal chondrocyte apoptosis observed in the growth plate of mutant mice is CHOP-mediated, we bred T585M COMP mutant mice with CHOP-null mice to homozygosity, and analysed the resulting phenotype. Although abnormal apoptosis was alleviated in the resting zone following CHOP deletion, the mutant growth plates were generally more disorganised. Furthermore, the bone lengths of COMP mutant CHOP null mice were significantly shorter at 9 weeks of age when compared to the COMP mutant mice, including a significant difference in the skull length. Overall, these data demonstrate that CHOP-mediated apoptosis is an early event in the pathobiology of PSACH and suggest that the lack of CHOP, in conjunction with a COMP mutation, may lead to aggravation of the skeletal phenotype via a potentially synergistic effect on endochondral ossification.

Mutant matrilin-3 (V194D) forms non-native disulphide bonded aggregates in the rER of chondrocytes from cell and mouse models of multiple epiphyseal dysplasia (MED). Intracellular retention of mutant matrilin-3 causes endoplasmic reticulum (ER) stress and induces an unfolded protein response (UPR) including the upregulation of two genes recently implicated in ER stress: Armet and Creld2. Nothing is known about the role of Armet and Creld2 in human genetic diseases. In this study, we used a variety of cell and mouse models of chondrodysplasia to determine the genotype-specific expression profiles of Armet and Creld2. We also studied their interactions with various mutant proteins and investigated their potential roles as protein disulphide isomerases (PDIs). Armet and Creld2 were up-regulated in cell and/or mouse models of chondrodysplasias caused by mutations in Matn3 and Col10a1, but not Comp. Intriguingly, both Armet and Creld2 were also secreted into the ECM of these disease models following ER stress. Armet and Creld2 interacted with mutant matrilin-3, but not with COMP, thereby validating the genotype-specific expression. Substrate-trapping experiments confirmed Creld2 processed PDI-like activity, thus identifying a putative functional role. Finally, alanine substitution of the two terminal cysteine residues from the A-domain of V194D matrilin-3 prevented aggregation, promoted mutant protein secretion and reduced the levels of Armet and Creld2 in a cell culture model. We demonstrate that Armet and Creld2 are genotype-specific ER stress response proteins with substrate specificities, and that aggregation of mutant matrilin-3 is a key disease trigger in MED that could be exploited as a potential therapeutic target.

Disease mechanisms leading to different forms of chondrodysplasia include extracellular matrix (ECM) alterations and intracellular stress resulting in abnormal changes to chondrocyte proliferation and survival. Delineating the relative contribution of these two disease mechanisms is a major challenge in understanding disease pathophysiology in genetic skeletal diseases and a prerequisite for developing effective therapies. To determine the influence of intracellular stress and changes in chondrocyte phenotype to the development of chondrodysplasia, we targeted the expression of the G2320R mutant form of thyroglobulin to the endoplasmic reticulum (ER) of resting and proliferating chondrocytes. Previous studies on this mutant protein have shown that it induces intracellular aggregates and causes cell stress and death in the thyroid gland. The expression and retention of this exogenous mutant protein in resting and proliferating chondrocytes resulted in a chronic cell stress response, growth plate dysplasia and reduced bone growth, without inducing any alterations to the architecture and organization of the cartilage ECM. More significantly, the decreased bone growth seemed to be the direct result of reduced chondrocyte proliferation in the proliferative zone of growth plates in transgenic mice, without transcriptional activation of a classical unfolded protein response (UPR) or apoptosis. Overall, these data show that mutant protein retention in the ER of resting and proliferative zone chondrocytes is sufficient to cause disrupted bone growth. The specific disease pathways triggered by mutant protein retention do not necessarily involve a prototypic UPR, but all pathways impact upon chondrocyte proliferation in the cartilage growth plate.

Objective: To characterize the circadian clock in murine cartilage tissue and identify tissue-specific clock target genes, and to investigate whether the circadian clock changes during aging or during cartilage degeneration using an experimental mouse model of osteoarthritis (OA)., Methods: Cartilage explants were obtained from aged and young adult mice after transduction with the circadian clock fusion protein reporter PER2::luc, and real-time bioluminescence recordings were used to characterize the properties of the clock. Time-series microarrays were performed on mouse cartilage tissue to identify genes expressed in a circadian manner. Rhythmic genes were confirmed by quantitative reverse transcription-polymerase chain reaction using mouse tissue, primary chondrocytes, and a human chondrocyte cell line. Experimental OA was induced in mice by destabilization of the medial meniscus (DMM), and articular cartilage samples were microdissected and subjected to microarray analysis., Results: Mouse cartilage tissue and a human chondrocyte cell line were found to contain intrinsic molecular circadian clocks. The cartilage clock could be reset by temperature signals, while the circadian period was temperature compensated. PER2::luc bioluminescence demonstrated that circadian oscillations were significantly lower in amplitude in cartilage from aged mice. Time-series microarray analyses of the mouse tissue identified the first circadian transcriptome in cartilage, revealing that 615 genes (∼3.9% of the expressed genes) displayed a circadian pattern of expression. This included genes involved in cartilage homeostasis and survival, as well as genes with potential importance in the pathogenesis of OA. Several clock genes were disrupted in the early stages of cartilage degeneration in the DMM mouse model of OA., Conclusion: These results reveal an autonomous circadian clock in chondrocytes that can be implicated in key aspects of cartilage biology and pathology. Consequently, circadian disruption (e.g., during aging) may compromise tissue homeostasis and increase susceptibility to joint damage or disease., (© 2013 The Authors. Arthritis & Rheumatism is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

Among the compensatory mechanisms restoring circulating blood volume after severe haemorrhage, increased vasopressin secretion enhances water permeability of distal nephron segments and stimulates Na(+) reabsorption in cortical collecting tubules via epithelial sodium channels (ENaC). The ability of vasopressin to upregulate ENaC via a cAMP-dependent mechanism in the medium to long term is well established. This study addressed the acute regulatory effect of cAMP on human ENaC (hENaC) and thus the potential role of vasopressin in the initial compensatory responses to haemorrhagic shock. The effects of raising intracellular cAMP (using 5 mmol/L isobutylmethylxanthine (IBMX) and 50 μmol/L forskolin) on wild-type and Liddle-mutated hENaC activity expressed in Xenopus oocytes and hENaC localisation in oocyte membranes were evaluated by dual-electrode voltage clamping and immunohistochemistry, respectively. After 30 min, IBMX + forskolin had stimulated amiloride-sensitive Na(+) current by 52% and increased the membrane density of Na(+) channels in oocytes expressing wild-type hENaC. These responses were prevented by 5 μmol/L brefeldin A, which blocks antegrade vesicular transport. By contrast, IBMX + forskolin had no effects in oocytes expressing Liddle-mutated hENaC. cAMP stimulated rapid, exocytotic recruitment of wild-type hENaC into Xenopus oocyte membranes, but had no effect on constitutively over-expressed Liddle-mutated hENaC. Extrapolating these findings to the early cAMP-mediated effect of vasopressin on cortical collecting tubule cells, they suggest that vasopressin rapidly mobilises ENaC to the apical membrane of cortical collecting tubule cells, but does not enhance ENaC activity once inserted into the membrane. We speculate that this stimulatory effect on Na(+) reabsorption (and hence water absorption) may contribute to the early restoration of extracellular fluid volume following severe haemorrhage.

Pseudoachondroplasia and multiple epiphyseal dysplasia are genetic skeletal diseases resulting from mutations in cartilage structural proteins. Electron microscopy and immunohistochemistry previously showed that the appearance of the cartilage extracellular matrix (ECM) in targeted mouse models of these diseases is disrupted; however, the precise changes in ECM organization and the pathological consequences remain unknown. Our aim was to determine the effects of matrilin-3 and COMP mutations on the composition and extractability of ECM components to inform how these detrimental changes might influence cartilage organization and degeneration. Cartilage was sequentially extracted using increasing denaturants and the extraction profiles of specific proteins determined using SDS-PAGE/Western blotting. Furthermore, the relative composition of protein pools was determined using mass spectrometry for a non-biased semi-quantitative analysis. Western blotting revealed changes in the extraction of matrilins, COMP and collagen IX in mutant cartilage. Mass spectrometry confirmed quantitative changes in the extraction of structural and non-structural ECM proteins, including proteins with roles in cellular processes such as protein folding and trafficking. In particular, genotype-specific differences in the extraction of collagens XII and XIV and tenascins C and X were identified; interestingly, increased expression of several of these genes has recently been implicated in susceptibility and/or progression of murine osteoarthritis. We demonstrated that mutation of matrilin-3 and COMP caused changes in the extractability of other cartilage proteins and that proteomic analyses of Matn3 V194D, Comp T585M and Comp DelD469 mouse models revealed both common and discrete disease signatures that provide novel insight into skeletal disease mechanisms and cartilage degradation.

Mutations causing metaphyseal chondrodysplasia type Schmid (MCDS) (e.g., Col10a1p.N617K) induce the pathology by a mechanism involving increased endoplasmic reticulum (ER) stress triggering an unfolded protein response (UPR) in hypertrophic chondrocytes (Rajpar et al. 2009). Here we correlate the expression of mutant protein with the onset of the UPR and disease pathology (hypertrophic zone [HZ] expansion) in MCDS and ColXTg(cog) mouse lines from E14.5 to E17.5. Embryos homozygous for the Col10a1p.N617K mutation displayed a delayed secretion of mutant collagen X accompanied by a UPR at E14.5, delayed ossification of the primary center at E15.5, and an expanded HZ at E17.5. Heterozygote embryos expressed mutant collagen X from E14.5 but exhibited no evidence of a UPR or an HZ expansion until after E17.5. Embryos positive for the ER stress-inducing ColXTg(cog) allele expressed Tg(cog) at E14.5, but the onset of the UPR was not apparent until E15.5 in homozygous and E17.5 in hemizygous embryos. Only homozygous embryos exhibited an HZ expansion at E17.5. The differential onset of the UPR and pathology, dependent on mutation type and gene dosage, indicates that hypertrophic chondrocytes have a latent capacity to deal with ER stress, which must be exceeded to trigger the UPR and HZ expansion.

Objective: To use an in vitro model of chondrogenesis to identify microRNAs (miRNAs) with a functional role in cartilage homeostasis., Methods: The expression of miRNAs was measured in the ATDC5 cell model of chondrogenesis using microarray and was verified using quantitative reverse transcription-polymerase chain reaction. MicroRNA expression was localized by in situ hybridization. Predicted miRNA target genes were validated using 3'-untranslated region-Luc reporter plasmids containing either wild-type sequences or mutants of the miRNA target sequence. Signaling through the Smad pathway was measured using a (CAGA)(12) -Luc reporter., Results: The expression of several miRNAs was regulated during chondrogenesis. These included 39 miRNAs that are coexpressed with miRNA-140 (miR-140), which is known to be involved in cartilage homeostasis and osteoarthritis (OA). Of these miRNAs, miR-455 resides within an intron of COL27A1 that encodes a cartilage collagen. When human OA cartilage was compared with cartilage obtained from patients with femoral neck fractures, the expression of both miR-140-5p and miR-455-3p was increased in OA cartilage. In situ hybridization showed miR-455-3p expression in the developing limbs of chicks and mice and in human OA cartilage. The expression of miR-455-3p was regulated by transforming growth factor β (TGFβ) ligands, and miRNA regulated TGFβ signaling. ACVR2B, SMAD2, and CHRDL1 were direct targets of miR-455-3p and may mediate its functional impact on TGFβ signaling., Conclusion: MicroRNA-455 is expressed during chondrogenesis and in adult articular cartilage, where it can regulate TGFβ signaling, suppressing the Smad2/3 pathway. Diminished signaling through this pathway during the aging process and in OA chondrocytes is known to contribute to cartilage destruction. We propose that the increased expression of miR-455 in OA exacerbates this process and contributes to disease pathology., (Copyright © 2012 by the American College of Rheumatology.)

Objective: Mutations in matrilin 3 can result in multiple epiphyseal dysplasia (MED), a disease characterized by delayed and irregular bone growth and early-onset osteoarthritis. Although intracellular retention of the majority of mutant matrilin 3 was previously observed in a murine model of MED caused by a Matn3 V194D mutation, some mutant protein was secreted into the extracellular matrix. Thus, it was proposed that secretion of mutant matrilin 3 may be dependent on the formation of hetero-oligomers with matrilin 1. The aim of this study was to investigate the hypothesis that deletion of matrilin 1 would abolish the formation of matrilin 1/matrilin 3 hetero-oligomers, eliminate the secretion of mutant matrilin 3, and influence disease severity., Methods: Mice with a Matn3 V194D mutation were crossed with Matn1-null mice, generating mice that were homozygous for V194D and null for matrilin 1. This novel mouse was used for in-depth phenotyping, while cartilage and chondrocytes were studied both histochemically and biochemically., Results: Endochondral ossification was not disrupted any further in mice with a double V194D mutation compared with mice with a single mutation. A similar proportion of mutant matrilin 3 was present in the extracellular matrix, and the amount of retained mutant matrilin 3 was not noticeably increased. Retained mutant matrilin 3 formed disulfide-bonded aggregates and caused the co-retention of matrilin 1., Conclusion: We showed that secretion of matrilin 3 V194D mutant protein is not dependent on hetero-oligomerization with matrilin 1, and that the total ablation of matrilin 1 expression has no impact on disease severity in mice with MED. Mutant matrilin 3 oligomers form non-native disulfide-bonded aggregates through the misfolded A domain., (Copyright © 2012 by the American College of Rheumatology.)