Objectives Articular cartilage undergoes cyclical heavy loading and low load recovery during the 24-hour day/night cycle. We investigated the daily changes of protein abundance in mouse femoral head articular cartilage by performing 24-hour time-series proteomics study. Methods Tandem mass spectrometry analysis was used to quantify proteins extracted from mouse cartilage. Bioinformatics analysis was performed to quantify rhythmic changes in protein abundance. Primary chondrocytes were isolated and cultured for independent validation of selected rhythmic proteins. Results 145 rhythmic proteins were detected. Among these were key cartilage molecules including CCN2, MATN1, PAI-1 and PLOD1 & 2. Pathway analysis revealed that proteins related to protein synthesis, cytoskeleton and glucose metabolism exhibited time-of-day dependent peaks in their abundance. Meta-analysis of published proteomics datasets from articular cartilage revealed that numerous rhythmic proteins were dysregulated in osteoarthritis and/or ageing. Conclusions Our circadian proteomics study revealed that articular cartilage is a much more dynamic tissue than previously thought. Chondrocytes exhibit circadian rhythms not only in gene expression but also in protein abundance. Our results clearly call for the consideration of circadian timing in understanding cartilage biology, osteoarthritis pathogenesis, treatment strategies and biomarker detection.

Metaphyseal chondrodysplasia, Schmid type (MCDS) is an orphan disease with highly abnormal endochondral ossification causing shortening and deformities of the limbs, impairment of mobility and chronic pain. As for most skeletal dysplasias, current treatment options remain symptomatic due to the lack in causal therapeutic measures. In the actual study the authors took advantage of the well-described pathogenesis in MCDS: In contrast to other collagenopathies, collagen X mutations do not directly lead to structural deficits but induce ER-stress by accumulation of misfolded protein. The authors hypothesize that by pharmacologically upregulating autophagy, enhanced protein clearance would ameliorate ER-stress and improve associated pro-apoptotic and differentiation-inhibiting effects.

Objective: The purpose of this study was to determine if serum microRNA (miRNA) signatures were biomarkers of early cartilage degeneration in preclinical mouse models of post-traumatic osteoarthritis (OA) and inflammatory arthritis.Methods: Cartilage degeneration was induced in 10-12 week old male C57BL6 mice by destabilization of the medial meniscus (DMM) or intra-articular injection of methylated-bovine-serum-albumin (AIA), with sham-operated or saline-injected control animals (n = 6/treatment/time). Total serum RNA and knee joints were isolated at 1, 4 and 16 weeks post-induction. Cartilage degeneration was scored histologically. Serum miRNA expression profiling was performed using Agilent microarrays and validated by qPCR.Results: DMM-operated and AIA mice had characteristic cartilage degeneration (proteoglycan loss, chondrocyte hypertrophy, structural damage), that increased significantly with time compared with controls, and with distinct temporal differences between arthritis models. However, expression profiling revealed no statistically significant dysregulation of serum miRNAs between AIA vs saline-injected or DMM vs sham-operated control mice at the critical early disease stages. The inability to detect DMM or AIA serum miRNA signatures compared with controls was not due to the insensitivity of the expression profiling approach since significant changes were observed in miRNA expression between the arthritis models and between time points.Conclusion: While distinct patterns of progressive cartilage degradation were induced in the arthritis models, we were unable to identify any serum miRNAs that were significantly dysregulated in early stages of disease compared with controls. This suggests circulating serum miRNAs may not be useful as cartilage biomarkers in distinguishing the early or progressive stages of arthritis cartilage degeneration. (C) 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Purpose: Osteoarthritis (OA) is the most common form of degenerative joint disease and it results in considerable disability and health care expenditure worldwide. OA is a disease that impacts on all tissues of the joint, however, it is primarily characterized by the progressive breakdown of articular cartilage (AC). Whilst many disease targets and pathological pathways are being continuously associated with OA, there are still no disease modifying OA drugs that have demonstrated dramatic success. Clearly, there is still more work needed to fully understand the complex mechanisms responsible and thus improve on the development of more effective therapeutic approaches. Intriguingly, an increasing number of microRNAs (miRNAs) are being identified as novel regulators of OA disease initiation and progression, making them exciting candidates for therapeutic targets and diagnostic biomarkers. The purpose of this study is to identify and characterize the expression profile of miRNAs in cartilage of normal, early and progressive stages of OA in a mouse model of post-traumatic OA. Methods: OA was induced in 10-12 week old male wild type mice by bilateral surgical destabilization of the medial meniscus (DMM). RNA from the medial tibial AC of DMM and sham-operated mice was isolated by laser microdissection at 1 and 6 weeks post-surgery. Four mice per time point per group were used for this experiment. miRNA expression profiling was performed using Agilent miRNA microarrays. The severity of chondrocyte hypertrophy, proteoglycan (PG) loss, and structural damage in the tibial AC of the contralateral medial femoro-tibial joint was scored by one observer blinded to surgical intervention and post-operative time. Results: Chondrocyte hypertrophy was considerably more extensive in the tibial AC of DMM compared with sham-operated joints at both 1 and 6 weeks (p = 0.002 and 0.004, respectively), but did not differ with time in either surgical group. There was no AC PG loss in either surgical group at 1 week, but significant PG loss was observed in the DMM group with time (p < 0.001) and in DMM versus sham at 6 weeks (p < 0.001). Similarly maximal and total AC structural damage scores did not differ between DMM and sham at 1 week, but both scores progressed with time in DMM (p < 0.003) and in DMM versus sham at 6 weeks (p < 0.001). miRNA expression profiling identified 122 and 74 miRNAs in AC that were differentially expressed (adj.p.value < 0.05) in DMM versus sham-operated mice at 1 and 6 weeks post-surgery, respectively. Moreover, 57 dysregulated miRNAs were found to be common to both 1 and 6 week time points. Amongst the 57, several miRNAs were identified with a FC > 2.0 and adj.p.value < 0.05, including miR-6931-5p, miR-3082-5p, miR-1187, miR-669n, miR-468-3p, miR-669l-5p, miR-669e-5p and miR-672-5p. In addition, differentially expressed miRNAs were also found to be exclusively associated with either 1 or 6 weeks post-surgery (65 miRNAs at 1 week and 17 miRNAs at 6 weeks). Conclusions: We demonstrated typical OA pathological features in the AC of DMM-operated mice compared with sham, some of which were time dependent (PG loss and structural damage). Our microarray data revealed dynamic changes in miRNA expression profiles between normal (Sham) and OA cartilage (DMM). Interestingly, we observed changes in miRNA expression that mirrored the temporal changes in PG loss and structural damage. Similarly, we also demonstrated changes in miRNA expression which were independent of time, reflecting the chondrocyte hypertrophy scores which were also static with time. In conclusion, we have identified potential miRNA regulators of OA initiation and progression in AC which have not been previously associated with OA. Future studies are now focused on determining their role in OA disease progression and identifying miRNA gene targets.

OBJECTIVE:Scottish fold cats, named for their unique ear shape, have a dominantly inherited osteochondrodysplasia involving malformation in the distal forelimbs, distal hindlimbs and tail, and progressive joint destruction. This study aimed to identify the gene and the underlying variant responsible for the osteochondrodysplasia. DESIGN:DNA samples from 44 Scottish fold and 54 control cats were genotyped using a feline DNA array and a case-control genome-wide association analysis conducted. The gene encoding a calcium permeable ion channel, transient receptor potential cation channel, subfamily V, member 4 (TRPV4) was identified as a candidate within the associated region and sequenced. Stably transfected HEK293 cells were used to compare wild-type and mutant TRPV4 expression, cell surface localisation and responses to activation with a synthetic agonist GSK1016709A, hypo-osmolarity, and protease-activated receptor 2 stimulation. RESULTS:The dominantly inherited folded ear and osteochondrodysplasia in Scottish fold cats is associated with a p.V342F substitution (c.1024G>T) in TRPV4. The change was not found in 648 unaffected cats. Functional analysis in HEK293 cells showed V342F mutant TRPV4 was poorly expressed at the cell surface compared to wild-type TRPV4 and as a consequence the maximum response to a synthetic agonist was reduced. Mutant TRPV4 channels had a higher basal activity and an increased response to hypotonic conditions. CONCLUSIONS:Access to a naturally-occurring TRPV4 mutation in the Scottish fold cat will allow further functional studies to identify how and why the mutations affect cartilage and bone development.

Purpose: Osteoarthritis (OA) is a degenerative joint disease characterized by the progressive breakdown of articular cartilage. While cartilage degradation remains the hallmark of OA, it is clear that all joint tissues contribute to the pathological process, however, the molecular mechanisms that drive these degenerative events remain poorly understood. This highlights the critical need to perform parallel molecular studies on articular cartilage and other OA-affected joint tissues, such as the synovium (SYN) and underlying subchondral bone (SCB). Intriguingly, an increasing number of miRNAs are being identified as novel regulators of OA disease initiation and progression, making them exciting candidates for therapeutic targets and diagnostic biomarkers. The purpose of this study is to investigate previously published candidate miRNAs, known to be dysregulated in human OA, and determine their role in extra-cartilaginous OA joint tissues. Methods: OA was induced in 10–12 week old male wild type mice by bilateral surgical destabilization of the medial meniscus (DMM). RNA from SCB from DMM and sham-operated mice was isolated by laser microdissection at 1 and 6 weeks post-surgery. RNA was also extracted from the SYN of the same mice. miRNA expression profiling of SCB and SYN was performed using Agilent miRNA microarrays. Histological measurements examining the severity of OA in the contralateral joint (including: SCB sclerosis; osteophyte size and maturity; anterior and posterior synovitis - panus presence and bone erosion, sub-synovial inflammatory cell infiltration, synoviocyte hyperplasia, and exudate) were scored by one observer blinded to surgical intervention. Results: There was no difference in SCB sclerosis between surgeries at 1 week but DMM > sham at 6 weeks (p = 0.003), and DMM-6wk > DMM-1wk (p = 0.04). There was no osteophyte development at 1 week but at 6 weeks they had formed in DMM only, being larger (p = 0.002) and more mature (p = 0.001) than sham. Anterior and posterior synovitis decreased with post-operative time after sham and DMM (1wk > 6wk, p < 0.01 for all comparisons). There was no difference between surgeries in the individual synovitis parameters in the anterior aspect of the joint at either time, other than more panus in DMM at 1 week (p = 0.049). However, more severe joint inflammation in DMM compared with sham was evident by significantly higher synovitis scores in the posterior region of the joint (distant from the surgical incision) at both 1 and 6 weeks (p = 0.01 and 0.03, respectively). miRNA expression analysis revealed 584 miRNAs to be differentially expressed between SYN and SCB samples (adj.p.value < 0.05). Moreover, 384 and 164 miRNAs were dysregulated between 1 and 6 week time points (adj.p.value < 0.05) in SYN and SCB, respectively. However, there were no changes in miRNA expression between DMM and sham mice at both 1 and 6 weeks post-surgery in either the SCB or SYN. Additionally, promising candidate miRNAs previously identified in human OA cartilage (for example, miR-140, miR-483, miR-16 and miR-25) were not dysregulated in our data set. Conclusions: We demonstrated typical OA pathology in both SCB (sclerosis, osteophytosis) and SYN (synovial hyperplasia, sub-synovial inflammatory cell infiltration) that differed significantly with post-operative time and between DMM and sham surgeries. Dynamic changes in miRNA expression were observed between joint tissues (SYN v SCB) and time points (1 week v 6 weeks post-surgery), consistent with the temporal changes in pathology severity. However, in contrast to previously published data, we saw no associations with miRNAs and OA (i.e. DMM v sham) in either SYN or SCB joint tissues. Our data demonstrates that miRNAs in SYN and SCB of OA joints are unlikely to be pathological contributors to post-traumatic OA disease progression and our future studies are now focused on miRNAs in articular cartilage.

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.

Objective: To define how the catabolic cytokines (Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF alpha)) 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. NF kappa B pathway inhibitor and fluorescence live-imaging of cartilage were performed to study the underlying mechanisms.Results: Exposure to IL-1 beta 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-1 beta was accompanied by disregulated expression of endogenous clock genes and clock-controlled catabolic pathways. Mechanistically, NF kappa B signalling was involved in the effect of IL-1 beta on the cartilage clock in part through functional interference with the core Clock/BMAL1 complex. In contrast, TNF alpha 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-1 beta (but not TNF alpha) 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 proinflammatory cytokines, and provide an additional mechanism for how chronic joint inflammation may contribute to osteoarthritis (OA). (C) 2015 The Authors. Published by Elsevier Ltd and Osteoarthritis Research Society International.

Objectives: To investigate the regulation of sclerostin (SOST) in osteoarthritis (OA) and its potential effects on articular cartilage degradation.Methods: SOST and other Wnt-beta-catenin components were immuno-localised in osteochondral sections of surgically-induced OA in knees of sheep and mice, and human OA samples obtained at arthroplasty. Regulation of SOST mRNA and protein expression by ovine chondrocytes in response to interleukin-1 alpha (IL-1 alpha) or tumour necrosis factor-alpha (TNF alpha) was examined in explant cultures. The effect of 25 or 250 ng/ml recombinant SOST alone or in combination With IL-1 alpha, on ovine articular cartilage explant aggrecan degradation, and chondrocyte gene expression of Wnt-beta-catenin pathway proteins, metalloproteinases and their inhibitors, and cartilage matrix proteins was quantified.Results: Contrary to being an osteocyte-specific protein, SOST was expressed by articular chondrocytes, and mRNA levels were upregulated in vitro by IL-1 alpha but not TNF alpha. Chondrocyte SOST staining was significantly increased only in the focal area of cartilage damage in surgically-induced OA in sheep and mice, as well as end-stage human OA. In contrast, osteocyte SOST was focally decreased in the subchondral bone in sheep OA in association with bone sclerosis. SOST was biologically active in chondrocytes, inhibiting Wnt-beta-catenin signalling and catabolic metalloproteinase [matrix metalloproteinases (MMP) and distintegrin and metalloproteinase with thrombospndin repeats (ADAMTS)] expression, but also decreasing mRNA levels of aggrecan, collagen II and tissue inhibitors of metalloproteinaes (TIMPs). Despite this mixed effect, SOST dose-dependently inhibited IL-1 alpha-stimulated cartilage aggrecanolysis in vitro.Conclusions: These results implicate SOST in regulating the OA disease processes, but suggest opposing effects by promoting disease-associated subchondral bone sclerosis while inhibiting degradation of cartilage. (C) 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Purpose: To explore the molecular mechanisms of osteoarthritis (OA) initiation and progression, global gene expression profiling was performed on cartilage from mice with surgically-induced OA. We used WT mice, and mice lacking ADAMTS-5 activity (ADAMTS-5 Dcat). By comparing the gene expression during OA in these mice we have distinguished early events leading to aggrecan degradation (pre-fibrillated changes) from the downstream progressive phases that follow loss of aggrecan. Methods: Mechanical instability was introduced into 10 week old male mouse knee joints by surgical destabilization of the medial meniscus (DMM). In this model focal degeneration, exemplified by loss of aggrecan in the non-calcified articular cartilage, occurs in the medial tibial plateau 2 weeks after surgery in WT mice. After 6 weeks, aggrecan loss had progressed and cartilage fibrillation was evident. In ADAMTS-5 Dcat mice aggrecan loss and cartilage erosion is significantly reduced. Noncalcified cartilage from the developing lesion in DMM or sham-operated joints at 1, 2 and 6 weeks post-surgery was harvested by laser microdissection. Chondrocyte total RNA was extracted and cRNA generated by linear amplification (MessageAmp, Ambion), labeled with Cy3/Cy5 and hybridized to 44k whole genome microarrays (Agilent). Data was validated by quantitative PCR of selected genes. Results: There was an early upregulation (1 week) in WT mice of cartilage hypertrophy markers, such as collagen X. However in the ADAMTS-5 Dcat mice, these were not upregulated until 6 weeks, suggesting that the hypertrophic response of chondrocytes is not an initiating event, but an event downstream of the onset of ADAMTS-5 activity and/or cartilage degeneration. Of particular interest was the upregulation of the inflammatory mediator Ptgs2 (Cox-2) early (1 and 2 weeks) in WT OA mice. Upregulation of Cox-2 was not seen in ADAMTS-5 Dcat mice. Dysregulation of several Bmps (eg Bmp7), inflammatory cytokines, Fgfs, members of the Wnt/b-catenin pathway were apparent in WT mice. Expression profiling also showed numerous changes in gene expression that followed the same pattern in both WT and ADAMTS-5 Dcat mice, and thus are likely to be independent of ADAMTS-5-mediated aggrecan degradation. These changes include upregulation of several cathepsins (eg cathepsin S), Bmps (eg Bmp7) lumican and fibronectin. Conclusions: This well controlled OA model combined with precise microdissection of cartilage from developing cartilage lesions and microarray technology provides new insights into gene expressed during the initiation and progression of OA. Furthermore the comparison of OA gene expression in WT, ADAMTS-5 Dcat and other mouse models will allow us to prioritize candidate genes for functional analysis.

Poster Presentations -Cartilage/Chondrocyte Biology S75 were treated with H-89, a PKA inhibitor, W-7, a calmodulin inhibitor or LY294002, a PI3K inhibitor prior to 4a-PDD stimulation in SOX9dependent reporter assay.To examine the chondrogenic differentiation, ATDC5 cells were co-stimulated with 0 to 120 nM 4a-PDD and 10 mg/ml insulin for 10 day, then cells were stained with alcian blue.The amount of SOX9 protein was estimated by Western Blot analysis using anti-SOX9 antibody. Results:We have screened 120,000 cDNA clones and identified 46 genes that activated SOX9-dependent reporter activity.In cDNA microarray analysis, the mRNA levels of Sox5, Sox6, Ifitm5, Myd116, Mef2c and TRPV4 genes were elevated during chondrogenic differentiation of ATDC5 cells treated with insulin.TRPV4, a cation channel molecule was further investigated in this study since it had a strong effect on SOX9dependent transcription.mRNA expression of TRPV4 gene was observed in ATDC5, C3H10T1/2, murine primary chondrocytes prepared from the rib cages and hind limb buds in embryonic day12 embryos, but not in NIH3T3.When ATDC5 cells or C3H10T1/2 cells were treated with various concentration of 4a-PDD, SOX9-dependent transcription was elevated in dose dependent manner and this effect was abolished by the addition of ruthenium red, a TRPV antagonist.In ATDC5 cells, H-89 and W-7 inhibited SOX9-dependent reporter activity caused by the stimulation with 4a-PDD while LY294002 did not.When ATDC5 cells were co-stimulated with 4a-PDD and insulin, GAG accumulation was significantly increased as compared with insulin alone whereas 4a-PDD alone showed no effect.Similar result was obtained in C3H10T1/2 cells co-stimulated with 4a-PDD and BMP-2.Co-stimulation with 4a-PDD demonstrated further elevations of mRNAs for type II collagen and aggrecan in ATDC5 cells when compared to insulin alone.4a-PDD stimulation increased the amount of SOX9 protein in both ATDC5 and C3H10T1/2 cells.Conclusions: We have identified TRPV4 by its ability to activate SOX9dependent transcription.Activation of TRPV4 promoted chondrogenic differentiation of ATDC5 cells in cooperation with insulin and C3H10T1/2 cells in corporation with BMP-2 in vitro.The protein level of SOX9 was increased by the stimulation with 4a-PDD.These observations suggest that TRPV4 may correlate the process of chondrogenesis.

Purpose:To explore the molecular mechanisms of osteoarthritis (OA) initiation and progression, global gene expression profiling was performed on cartilage from mice with surgically-induced OA.We used WT mice, and mice lacking ADAMTS-5 activity (ADAMTS-5 Dcat).By comparing the gene expression during OA in these mice we have distinguished early events leading to aggrecan degradation (pre-fibrillated changes) from the downstream progressive phases that follow loss of aggrecan.Methods: Mechanical instability was introduced into 10 week old male mouse knee joints by surgical destabilization of the medial meniscus (DMM).In this model focal degeneration, exemplified by loss of aggrecan in the non-calcified articular cartilage, occurs in the medial tibial plateau 2 weeks after surgery in WT mice.After 6 weeks, aggrecan loss had progressed and cartilage fibrillation was evident.In ADAMTS-5 Dcat mice aggrecan loss and cartilage erosion is significantly reduced.Noncalcified cartilage from the developing lesion in DMM or sham-operated joints at 1, 2 and 6 weeks post-surgery was harvested by laser microdissection.Chondrocyte total RNA was extracted and cRNA generated by linear amplification (MessageAmp, Ambion), labeled with Cy3/Cy5 and hybridized to 44k whole genome microarrays (Agilent).Data was validated by quantitative PCR of selected genes.Results: There was an early upregulation (1 week) in WT mice of cartilage hypertrophy markers, such as collagen X. However in the ADAMTS-5 Dcat mice, these were not upregulated until 6 weeks, suggesting that the hypertrophic response of chondrocytes is not an initiating event, but an event downstream of the onset of ADAMTS-5 activity and/or cartilage degeneration.Of particular interest was the upregulation of the inflammatory mediator Ptgs2 (Cox-2) early (1 and 2 weeks) in WT OA mice.Upregulation of Cox-2 was not seen in ADAMTS-5 Dcat mice.Dysregulation of several Bmps (eg Bmp7), inflammatory cytokines, Fgfs, members of the Wnt/b-catenin pathway were apparent in WT mice.Expression profiling also showed numerous changes in gene expression that followed the same pattern in both WT and ADAMTS-5 Dcat mice, and thus are likely to be independent of ADAMTS-5-mediated aggrecan degradation.These changes include upregulation of several cathepsins (eg cathepsin S), Bmps (eg Bmp7) lumican and fibronectin.Conclusions: This well controlled OA model combined with precise microdissection of cartilage from developing cartilage lesions and microarray technology provides new insights into gene expressed during the initiation and progression of OA.Furthermore the comparison of OA gene expression in WT, ADAMTS-5 Dcat and other mouse models will allow us to prioritize candidate genes for functional analysis.

OBJECTIVE:Dominant mutations in the three collagen VI genes cause Bethlem myopathy, a disorder characterized by proximal muscle weakness and commonly contractures of the fingers, wrists, and ankles. Although more than 20 different dominant mutations have been identified in Bethlem myopathy patients, the biosynthetic consequences of only a subset of these have been studied, and in many cases, the pathogenic mechanisms remain unknown.METHODS:We have screened fourteen Bethlem myopathy patients for collagen VI mutations and performed detailed analyses of collagen VI biosynthesis and intracellular and extracellular assembly.RESULTS:Collagen VI abnormalities were identified in eight patients. One patient produced around half the normal amount of alpha1(VI) messenger RNA and reduced amounts of collagen VI protein. Two patients had a previously reported mutation causing skipping of COL6A1 exon 14, and three patients had novel mutations leading to in-frame deletions toward the N-terminal end of the triple-helical domain. These mutations have different and complex effects on collagen VI intracellular and extracellular assembly. Two patients had single amino acid substitutions in the A-domains of COL6A2 and COL6A3. Collagen VI intracellular and extracellular assembly was normal in one of these patients.INTERPRETATION:The key to dissecting the pathogenic mechanisms of collagen VI mutations lies in detailed analysis of collagen VI biosynthesis and assembly. The majority of mutations result in secretion and deposition of structurally abnormal collagen VI. However, one A-domain mutation had no detectable effect on assembly, suggesting that it acts by compromising collagen VI interactions in the extracellular matrix of muscle.

WARP is a novel member of the von Willebrand factor A domain superfamily of extracellular matrix proteins that is expressed by chondrocytes. WARP is restricted to the presumptive articular cartilage zone prior to joint cavitation and to the articular cartilage and fibrocartilaginous elements in the joint, spine, and sternum during mouse embryonic development. In mature articular cartilage, WARP is highly specific for the chondrocyte pericellular microenvironment and co-localizes with perlecan, a prominent component of the chondrocyte pericellular region. WARP is present in the guanidine-soluble fraction of cartilage matrix extracts as a disulfide-bonded multimer, indicating that WARP is a strongly interacting component of the cartilage matrix. To investigate how WARP is integrated with the pericellular environment, we studied WARP binding to mouse perlecan using solid phase and surface plasmon resonance analysis. WARP interacts with domain III-2 of the perlecan core protein and the heparan sulfate chains of the perlecan domain I with KD values in the low nanomolar range. We conclude that WARP forms macromolecular structures that interact with perlecan to contribute to the assembly and/or maintenance of “permanent” cartilage structures during development and in mature cartilages.

Schmid metaphyseal chondrodysplasia (SMCD) is a dominantly inherited cartilage disorder caused by mutations in the gene for the hypertrophic cartilage extracellular matrix structural protein, collagen X (COL10A1). Thirty heterozygous mutations have been described, about equally divided into two mutation types, missense mutations, and mutations that introduce premature termination signals. The COL10A1 mutations are clustered (33/36) in the 3′ region of exon 3, which codes for the C‐terminal NC1 trimerization domain. The effect of COL10A1 missense mutations have been examined by in vitro expression and assembly assays and cell transfection studies, which suggest that a common consequence is the disruption of collagen X trimerization and secretion, with consequent intracellular degradation. The effect of COL10A1 nonsense mutations in cartilage tissue has been examined in two patients, demonstrating that the mutant mRNA is completely removed by nonsense mediated mRNA decay. Thus for both classes of mutations, functional haploinsufficiency is the most probable cause of the clinical phenotype in SMCD. Hum Mutat 25:525–534, 2005. © 2005 Wiley‐Liss, Inc.

Characteristic deformities of skull shape occur as a result of different patterns of sutural fusion, while compen- satory skull expansion occurs at unaffected sutures to accommodate the growing brain. Premature fusion of the sagittal suture, for example, results in anterior to posterior elongation of the skull known as scaphocephaly. 2 Sagittal synostosis is the most common type of craniosynostosis, occurring in 40-58% of cases reported in large neurosurgical surveys, and shows a male predominance. 3-6 Most of the cases in these reports are ''non-syndromic'' instances of sagittal synostosis, while familial cases represent 2-9% of the total. Mental retardation is uncommon in isolated sagittal synostosis, but is more common in cases with associated malformations. 4-6