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Frank Zaucke - One of the best experts on this subject based on the ideXlab platform.
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Moderate Cyclic Tensile Strain Alters the Assembly of Cartilage Extracellular Matrix Proteins In Vitro
Journal of biomechanical engineering, 2015Co-Authors: Judith Bleuel, Frank Zaucke, Juliane Heilig, Gert-peter Brüggemann, Marie-louise Wolter, Nina Hamann, Sara Firner, Anja NiehoffAbstract:Mechanical loading influences the structural and mechanical properties of articular cartilage. The cartilage matrix protein collagen II essentially determines the tensile properties of the tissue and is adapted in response to loading. The collagen II network is stabilized by the collagen II-binding cartilage oligomeric matrix protein (COMP), collagen IX, and Matrilin-3. However, the effect of mechanical loading on these extracellular matrix proteins is not yet understood. Therefore, the aim of this study was to investigate if and how chondrocytes assemble the extracellular matrix proteins collagen II, COMP, collagen IX, and Matrilin-3 in response to mechanical loading. Primary murine chondrocytes were applied to cyclic tensile strain (6%, 0.5 Hz, 30 min per day at three consecutive days). The localization of collagen II, COMP, collagen IX, and Matrilin-3 in loaded and unloaded cells was determined by immunofluorescence staining. The messenger ribo nucleic acid (mRNA) expression levels and synthesis of the proteins were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and western blots. Immunofluorescence staining demonstrated that the pattern of collagen II distribution was altered by loading. In loaded chondrocytes, collagen II containing fibrils appeared thicker and strongly co-stained for COMP and collagen IX, whereas the collagen network from unloaded cells was more diffuse and showed minor costaining. Further, the applied load led to a higher amount of COMP in the matrix, determined by western blot analysis. Our results show that moderate cyclic tensile strain altered the assembly of the extracellular collagen network. However, changes in protein amount were only observed for COMP, but not for collagen II, collagen IX, or Matrilin-3. The data suggest that the adaptation to mechanical loading is not always the result of changes in RNA and/or protein expression but might also be the result of changes in matrix assembly and structure.
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COMP does not directly modify the expression of genes involved in cartilage homeostasis in contrast to several other cartilage matrix proteins.
Connective tissue research, 2014Co-Authors: Johannes Ruthard, Frank Zaucke, Gertrud Kühn, Wibke Johannis, Matthias Kamper, Jörg H. Renno, Ute Hillebrand, Stefan Höllriegl, Andreas R. KlattAbstract:AbstractObjective: We investigated whether COMP may modify cartilage metabolism and play a role as an endogenous disease aggravating factor in OA.Materials and methods: Full-length and momomeric COMP was recombinantly expressed in human embryonic kidney cells and purified it via affinity chromatography. Purified COMP was used to stimulate either primary human chondrocytes or cartilage explants. Changes in the expression profiles of inflammatory genes, differentiation markers and growth factors were examined by immunoassay and by quantitative real-time reverse-transcription polymerase chain reaction.Results: Incubation of primary human chondrocytes or cartilage explants in the presence of COMP did not induce statistically significant changes in the expression of IL-6, MMP1, MMP13, collagen I, collagen II, collagen X, TGF-β1 and BMP-2.Conclusions: In contrast to collagen II and Matrilin-3, COMP lacks the ability to trigger a proinflammatory response in chondrocytes, although it carries an RGD motif and can ...
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Analysis of the cartilage proteome from three different mouse models of genetic skeletal diseases reveals common and discrete disease signatures.
Biology open, 2013Co-Authors: Peter A. Bell, Raimund Wagener, Frank Zaucke, David J Thornton, Ray Boot-handford, Manuel Koch, Julian N. Selley, Stacey Warwood, David Knight, Michael D. BriggsAbstract: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.
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Comparative Proteomic Analysis of Normal and Collagen IX Null Mouse Cartilage Reveals Altered Extracellular Matrix Composition and Novel Components of the Collagen IX Interactome
The Journal of biological chemistry, 2013Co-Authors: Bent Brachvogel, Frank Zaucke, John F. Bateman, Jeffrey J. Gorman, Münire Dayakli, Emma L. Norris, Keyur A. Dave, Manuel Koch, Jacek Stermann, Richard WilsonAbstract:BACKGROUND : Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. RESULTS : Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. CONCLUSION : Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome. SIGNIFICANCE : We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, Matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and Matrilin-3, consistent with known interactions. Matrilin-1, Matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas Matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFβ-induced protein βig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations.
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Growth‐related structural, biochemical, and mechanical properties of the functional bone–cartilage unit
Journal of anatomy, 2012Co-Authors: Nina Hamann, Frank Zaucke, Gert-peter Brüggemann, Münire Dayakli, Anja NiehoffAbstract:Articular cartilage and subchondral bone act together, forming a unit as a weight-bearing loading-transmitting surface. A close interaction between both structures has been implicated during joint cartilage degeneration, but their coupling during normal growth and development is insufficiently understood. The purpose of the present study was to examine growth-related changes of cartilage mechanical properties and to relate these changes to alterations in cartilage biochemical composition and subchondral bone structure. Tibiae and femora of both hindlimbs from 7- and 13-week-old (each n = 12) female Sprague-Dawley rats were harvested. Samples were processed for structural, biochemical and mechanical analyses. Immunohistochemical staining and protein expression analyses of collagen II, collagen IX, COMP and Matrilin-3, histomorphometry of cartilage thickness and COMP staining height were performed. Furthermore, mechanical testing of articular cartilage and micro-CT analysis of subchondral bone was conducted. Growth decreased cartilage thickness, paralleled by a functional condensation of the underlying subchondral bone due to enchondral ossification. Cartilage mechanical properties seem to be rather influenced by growth-related changes in the assembly of major ECM proteins such as collagen II, collagen IX and Matrilin-3 than by growth-related alterations in its underlying subchondral bone structure. Importantly, the present study provides a first insight into the growth-related structural, biochemical and mechanical interaction of articular cartilage and subchondral bone. Finally, these data contribute to the general knowledge about the cooperation between the articular cartilage and subchondral bone.
Raimund Wagener - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the cartilage proteome from three different mouse models of genetic skeletal diseases reveals common and discrete disease signatures.
Biology open, 2013Co-Authors: Peter A. Bell, Raimund Wagener, Frank Zaucke, David J Thornton, Ray Boot-handford, Manuel Koch, Julian N. Selley, Stacey Warwood, David Knight, Michael D. BriggsAbstract: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.
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The Matrilin-3 VWA1 domain modulates interleukin-6 release from primary human chondrocytes.
Osteoarthritis and cartilage, 2013Co-Authors: Andreas R. Klatt, Mats Paulsson, Brigitte Paul-klausch, Gabriele Klinger, U. Hillebrand, Gertrud Kühn, Birgit Kobbe, Joerg H. Renno, Wibke Johannis, Raimund WagenerAbstract:Summary Objective We previously demonstrated the ability of Matrilin-3 to modulate the gene expression profile of primary human chondrocytes (PHCs) toward a state favoring cartilage catabolism. The structure within Matrilin-3 responsible for the induction of these catabolic genes is unknown. Here, we investigated the potential of Matrilin-3 (MATN3) and truncated Matrilin-3 proteins, in both monomeric and oligomeric form, to stimulate interleukin (IL)-6 release in PHCs. Methods We expressed full-length Matrilin-3 oligomers, Matrilin-3 von Willebrand factor A (VWA) domain oligomers, Matrilin-3 four epidermal growth factor (EGF) domain oligomers, Matrilin-3 monomers without oligomerization domains, Matrilin-3 VWA domain monomers, and Matrilin-3 4EGF monomers. We then incubated PHCs in the absence or presence of full-length Matrilin-3 or one of the truncated Matrilin-3 proteins and finally determined the release of IL-6 in cell-culture supernatants. Results The addition of full-length Matrilin-3 oligomers, Matrilin-3 VWA domain oligomers, and, less pronounced, Matrilin-3 monomers without oligomerization domains, and Matrilin-3 4EGF-oligomers to the cell-culture medium led to a significant induction of IL-6 in PHCs. Discussion Based on recombinant expression of different Matrilin-3 domains in both monomeric and oligomeric form, this work demonstrated that the VWA1 domain of Matrilin-3 is primarily responsible for the induction of IL-6 release and that the oligomerization of the VWA1 domain markedly promotes its activity.
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The Matrilins: modulators of extracellular matrix assembly.
The international journal of biochemistry & cell biology, 2010Co-Authors: Andreas R. Klatt, Mats Paulsson, Ann-kathrin A. Becker, Cristian Dan Neacsu, Raimund WagenerAbstract:The Matrilins form a family of oligomeric extracellular adaptor proteins that are most strongly expressed in cartilage but also present in many other extracellular matrices. Matrilins bind to different types of collagen fibrils, to other noncollagenous proteins and to aggrecan. They thereby support matrix assembly by connecting fibrillar components and mediating interactions between these and the aggrecan gel. The binding avidity of a Matrilin can be varied by alternative splicing, proteolytic processing and formation of homo- and heterooligomers. Such changes in Matrilin structure may lead to a modulation of extracellular matrix assembly. Some Matrilins bind weakly to α1β1 integrin and cell surface proteoglycans, but even though Matrilins play a role in mechanotransduction and Matrilin-3 activates the expression of osteoarthritis-associated genes the physiological relevance of Matrilin-cell interactions is unclear. Matrilin knockout mice do not display pronounced phenotypes, which points to a redundancy within the protein family or with functionally related proteins. In man, dominant mutations in the von Willebrand factor A like domain of Matrilin-3 lead to a protein retention in the endoplasmic reticulum that causes multiple epiphyseal dysplasia by initiating a cell stress response. In contrast, a mutation in an EGF domain of Matrilin-3 that is associated with hand osteoarthritis and disc degeneration does not interfere with secretion but instead with extracellular assembly of matrix structures. In this review we summarize such information on Matrilin structure and function that we believe is important for the understanding of extracellular matrix assembly and for deciphering pathophysiological mechanisms in diseases causing skeletal malformations or cartilage degeneration.
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a Matrilin 3 mutation associated with osteoarthritis does not affect collagen affinity but promotes the formation of wider cartilage collagen fibrils
Human Mutation, 2010Co-Authors: Christiane Otten, Raimund Wagener, Mats Paulsson, Uwe Hansen, Anja Talke, Frank ZauckeAbstract:Mutations in Matrilin-3 have been associated with common skeletal diseases like osteoarthritis as well as with the rare chondrodysplasias MED and SEMD. We have previously shown that the mutations p.R116W and p.C299S, associated with MED and SEMD, respectively, cause retention of Matrilin-3 within the endoplasmic reticulum of primary chondrocytes, while the mutation associated with osteoarthritis, p.T298M, does not hinder secretion. The present study focused on the consequences of the p.T298M mutation on the structure of Matrilin-3 and on the role of Matrilin-3 in the formation of a functional extracellular matrix. Analysis of recombinant full-length Matrilin-3 revealed that the p.T298M mutation does not influence oligomerization of Matrilin-3 or its proteolytic processing by ADAMTS-4 and -5. Nevertheless, structural analyses indicate local conformational changes. These changes do not affect the affinity for collagens II, IX, XI, or COMP, but have a major impact on the in vitro fibrillogenesis of collagen II/IX/XI heterofibrils. Hum Mutat 31:254–263, 2010. © 2010 Wiley-Liss, Inc.
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A Matrilin‐3 mutation associated with osteoarthritis does not affect collagen affinity but promotes the formation of wider cartilage collagen fibrils
Human mutation, 2010Co-Authors: Christiane Otten, Raimund Wagener, Mats Paulsson, Uwe Hansen, Anja Talke, Frank ZauckeAbstract:Mutations in Matrilin-3 have been associated with common skeletal diseases like osteoarthritis as well as with the rare chondrodysplasias MED and SEMD. We have previously shown that the mutations p.R116W and p.C299S, associated with MED and SEMD, respectively, cause retention of Matrilin-3 within the endoplasmic reticulum of primary chondrocytes, while the mutation associated with osteoarthritis, p.T298M, does not hinder secretion. The present study focused on the consequences of the p.T298M mutation on the structure of Matrilin-3 and on the role of Matrilin-3 in the formation of a functional extracellular matrix. Analysis of recombinant full-length Matrilin-3 revealed that the p.T298M mutation does not influence oligomerization of Matrilin-3 or its proteolytic processing by ADAMTS-4 and -5. Nevertheless, structural analyses indicate local conformational changes. These changes do not affect the affinity for collagens II, IX, XI, or COMP, but have a major impact on the in vitro fibrillogenesis of collagen II/IX/XI heterofibrils. Hum Mutat 31:254–263, 2010. © 2010 Wiley-Liss, Inc.
Michael D. Briggs - One of the best experts on this subject based on the ideXlab platform.
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XBP1 signalling is essential for alleviating mutant protein aggregation in ER-stress related skeletal disease.
PLoS genetics, 2019Co-Authors: Katarzyna A. Piróg, Claire L. Hartley, Ray Boot-handford, Ella P. Dennis, Robert M. Jackson, Jamie Soul, Jean-marc Schwartz, John F. Bateman, Michael D. BriggsAbstract: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.
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Loss of Matrilin 1 Does Not Exacerbate the Skeletal Phenotype in a Mouse Model of Multiple Epiphyseal Dysplasia Caused by a
2016Co-Authors: Matn Vd Mutation, David J Thornton, Maryline Fresquet, Peter A. Bell, Katarzyna A. Piróg, Raymond P. Boot-h, Michael D. BriggsAbstract:Objective. Mutations in Matrilin 3 can result in multiple epiphyseal dysplasia (MED), a disease charac-terized by delayed and irregular bone growth and early-onset osteoarthritis. Although intracellular retention of the majority of mutant Matrilin 3 was previously ob-served 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
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RESEARCH ARTICLE Multiple Epiphyseal Dysplasia Mutations in MATN3 Cause Misfolding of the A-Domain and Prevent
2016Co-Authors: William G. Cole, Michael D. Briggs, Communicated Iain McintoshAbstract:Multiple epiphyseal dysplasia (MED) is a relatively common skeletal dysplasia that can present in childhood with a variable phenotype of short stature and pain and stiffness in the large joints, and often progresses to early-onset osteoarthritis in adulthood. Mutations in the Matrilin-3 gene (MATN3) have recently been shown to underlie some forms of autosomal dominant MED. To date all MED mutations in Matrilin-3 cluster in the single A-domain, suggesting that they may disrupt the structure and/or function of this important domain. To determine the effects of MATN3 mutations on the structure and function of Matrilin-3 we expressed both normal and mutant Matrilin-3 in mammalian cells. Wild-type (wt) Matrilin-3 was efficiently secreted into conditioned medium, whereas mutant Matrilin-3 was retained and accumulated within the cell. Furthermore, when the mutant A-domains were examined individually, they existed primarily in an unfolded conformation. Co-immunoprecipitation experiments demonstrated that the mutant A-domains were specifically associated with ERp72, a chaperone protein known to be involved in mediating disulfide bond formation. Light microscopy of cartilage from an MED patient with aMATN3 mutation showed the presence of intracellular material within the chondrocytes, whilst the overall matrix appeared normal. On electron micrographs, the inclusions noted at the light microscopy level appeared to be dilated cisternae of rough endoplasmic reticulum and immunohistochemical analysis confirmed that the retained protein was Matrilin-3. In summary, the dat
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Mild Myopathy Is Associated with COMP but Not MATN3 Mutations in Mouse Models of Genetic Skeletal Diseases
PloS one, 2013Co-Authors: Katarzyna A. Piróg, Yoshihisa Katakura, Alexander A. Mironov, Michael D. BriggsAbstract:Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are skeletal disorders resulting from mutations in COMP, Matrilin-3 or collagen IX and are characterised by short-limbed dwarfism and premature osteoarthritis. Interestingly, recent reports suggest patients can also manifest with muscle weakness. Here we present a detailed analysis of two mouse models of the PSACH/MED disease spectrum; ΔD469 T3-COMP (PSACH) and V194D Matrilin-3 (MED). In grip test experiments T3-COMP mice were weaker than wild-type littermates, whereas V194D mice behaved as controls, confirming that short-limbed dwarfism alone does not contribute to PSACH/MED-related muscle weakness. Muscles from T3-COMP mice showed an increase in centronuclear fibers at the myotendinous junction. T3-COMP tendons became more lax in cyclic testing and showed thicker collagen fibers when compared with wild-type tissue; Matrilin-3 mutant tissues were indistinguishable from controls. This comprehensive study of the myopathy associated with PSACH/MED mutations enables a better understanding of the disease progression, confirms that it is genotype specific and that the limb weakness originates from muscle and tendon pathology rather than short-limbed dwarfism itself. Since some patients are primarily diagnosed with neuromuscular symptoms, this study will facilitate better awareness of the differential diagnoses that might be associated with the PSACH/MED spectrum and subsequent care of PSACH/MED patients.
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Armet/Manf and Creld2 are components of a specialized ER stress response provoked by inappropriate formation of disulphide bonds: implications for genetic skeletal diseases.
Human molecular genetics, 2013Co-Authors: Claire L. Hartley, Maryline Fresquet, Peter A. Bell, Sarah M. Edwards, Lorna Mullan, Ray Boot-handford, Michael D. BriggsAbstract: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.
Mats Paulsson - One of the best experts on this subject based on the ideXlab platform.
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The Matrilin-3 VWA1 domain modulates interleukin-6 release from primary human chondrocytes.
Osteoarthritis and cartilage, 2013Co-Authors: Andreas R. Klatt, Mats Paulsson, Brigitte Paul-klausch, Gabriele Klinger, U. Hillebrand, Gertrud Kühn, Birgit Kobbe, Joerg H. Renno, Wibke Johannis, Raimund WagenerAbstract:Summary Objective We previously demonstrated the ability of Matrilin-3 to modulate the gene expression profile of primary human chondrocytes (PHCs) toward a state favoring cartilage catabolism. The structure within Matrilin-3 responsible for the induction of these catabolic genes is unknown. Here, we investigated the potential of Matrilin-3 (MATN3) and truncated Matrilin-3 proteins, in both monomeric and oligomeric form, to stimulate interleukin (IL)-6 release in PHCs. Methods We expressed full-length Matrilin-3 oligomers, Matrilin-3 von Willebrand factor A (VWA) domain oligomers, Matrilin-3 four epidermal growth factor (EGF) domain oligomers, Matrilin-3 monomers without oligomerization domains, Matrilin-3 VWA domain monomers, and Matrilin-3 4EGF monomers. We then incubated PHCs in the absence or presence of full-length Matrilin-3 or one of the truncated Matrilin-3 proteins and finally determined the release of IL-6 in cell-culture supernatants. Results The addition of full-length Matrilin-3 oligomers, Matrilin-3 VWA domain oligomers, and, less pronounced, Matrilin-3 monomers without oligomerization domains, and Matrilin-3 4EGF-oligomers to the cell-culture medium led to a significant induction of IL-6 in PHCs. Discussion Based on recombinant expression of different Matrilin-3 domains in both monomeric and oligomeric form, this work demonstrated that the VWA1 domain of Matrilin-3 is primarily responsible for the induction of IL-6 release and that the oligomerization of the VWA1 domain markedly promotes its activity.
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Abnormal bone quality in cartilage oligomeric matrix protein and Matrilin 3 double-deficient mice caused by increased tissue inhibitor of metalloproteinases 3 deposition and delayed aggrecan degradation.
Arthritis and rheumatism, 2012Co-Authors: Gergely Groma, Mats Paulsson, Anja Niehoff, Wei Xin, Ivan Grskovic, Bent Brachvogel, Frank ZauckeAbstract:Objective Cartilage oligomeric matrix protein (COMP) and Matrilin 3 are extracellular matrix proteins that are abundant in cartilage. As adaptor molecules, both proteins bridge and stabilize macromolecular networks consisting of fibrillar collagens and proteoglycans. Mutations in the genes coding for COMP and Matrilin 3 have been linked to human chondrodysplasias, while in mice, deficiency in COMP or Matrilin 3 does not cause any pronounced skeletal abnormalities. Given the similar functions of COMP and Matrilin 3 in the assembly and stabilization of the extracellular matrix, our aim was to determine whether these proteins could functionally compensate for each other. Methods To assess this putative redundancy of COMP and Matrilin 3, we generated COMP/Matrilin 3 double-deficient mice and performed an in-depth analysis of their skeletal development. Results At the newborn stage, the overall skeletal morphology of the double mutants was normal, but at 1 month of age, the long bones were shortened and the total body length reduced. Peripheral quantitative computed tomography revealed increased metaphyseal trabecular bone mineral density in the femora. Moreover, the degradation of aggrecan in the cartilage remnants in the metaphyseal trabecular bone was delayed, paralleled by increased deposition of tissue inhibitor of metalloproteinases 3 (TIMP-3). The structure and morphology of the growth plate were grossly normal, but in the center, focal closures were observed, a phenotype very similar to that described in matrix metalloproteinase 13 (MMP-13)–deficient mice. Conclusion We propose that a lack of COMP and Matrilin 3 leads to increased deposition of TIMP-3, which causes partial inactivation of MMPs, including MMP-13, a mechanism that would explain the similarities in phenotype between COMP/Matrilin 3 double-deficient and MMP-13–deficient mice.
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The Matrilins: modulators of extracellular matrix assembly.
The international journal of biochemistry & cell biology, 2010Co-Authors: Andreas R. Klatt, Mats Paulsson, Ann-kathrin A. Becker, Cristian Dan Neacsu, Raimund WagenerAbstract:The Matrilins form a family of oligomeric extracellular adaptor proteins that are most strongly expressed in cartilage but also present in many other extracellular matrices. Matrilins bind to different types of collagen fibrils, to other noncollagenous proteins and to aggrecan. They thereby support matrix assembly by connecting fibrillar components and mediating interactions between these and the aggrecan gel. The binding avidity of a Matrilin can be varied by alternative splicing, proteolytic processing and formation of homo- and heterooligomers. Such changes in Matrilin structure may lead to a modulation of extracellular matrix assembly. Some Matrilins bind weakly to α1β1 integrin and cell surface proteoglycans, but even though Matrilins play a role in mechanotransduction and Matrilin-3 activates the expression of osteoarthritis-associated genes the physiological relevance of Matrilin-cell interactions is unclear. Matrilin knockout mice do not display pronounced phenotypes, which points to a redundancy within the protein family or with functionally related proteins. In man, dominant mutations in the von Willebrand factor A like domain of Matrilin-3 lead to a protein retention in the endoplasmic reticulum that causes multiple epiphyseal dysplasia by initiating a cell stress response. In contrast, a mutation in an EGF domain of Matrilin-3 that is associated with hand osteoarthritis and disc degeneration does not interfere with secretion but instead with extracellular assembly of matrix structures. In this review we summarize such information on Matrilin structure and function that we believe is important for the understanding of extracellular matrix assembly and for deciphering pathophysiological mechanisms in diseases causing skeletal malformations or cartilage degeneration.
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a Matrilin 3 mutation associated with osteoarthritis does not affect collagen affinity but promotes the formation of wider cartilage collagen fibrils
Human Mutation, 2010Co-Authors: Christiane Otten, Raimund Wagener, Mats Paulsson, Uwe Hansen, Anja Talke, Frank ZauckeAbstract:Mutations in Matrilin-3 have been associated with common skeletal diseases like osteoarthritis as well as with the rare chondrodysplasias MED and SEMD. We have previously shown that the mutations p.R116W and p.C299S, associated with MED and SEMD, respectively, cause retention of Matrilin-3 within the endoplasmic reticulum of primary chondrocytes, while the mutation associated with osteoarthritis, p.T298M, does not hinder secretion. The present study focused on the consequences of the p.T298M mutation on the structure of Matrilin-3 and on the role of Matrilin-3 in the formation of a functional extracellular matrix. Analysis of recombinant full-length Matrilin-3 revealed that the p.T298M mutation does not influence oligomerization of Matrilin-3 or its proteolytic processing by ADAMTS-4 and -5. Nevertheless, structural analyses indicate local conformational changes. These changes do not affect the affinity for collagens II, IX, XI, or COMP, but have a major impact on the in vitro fibrillogenesis of collagen II/IX/XI heterofibrils. Hum Mutat 31:254–263, 2010. © 2010 Wiley-Liss, Inc.
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A Matrilin‐3 mutation associated with osteoarthritis does not affect collagen affinity but promotes the formation of wider cartilage collagen fibrils
Human mutation, 2010Co-Authors: Christiane Otten, Raimund Wagener, Mats Paulsson, Uwe Hansen, Anja Talke, Frank ZauckeAbstract:Mutations in Matrilin-3 have been associated with common skeletal diseases like osteoarthritis as well as with the rare chondrodysplasias MED and SEMD. We have previously shown that the mutations p.R116W and p.C299S, associated with MED and SEMD, respectively, cause retention of Matrilin-3 within the endoplasmic reticulum of primary chondrocytes, while the mutation associated with osteoarthritis, p.T298M, does not hinder secretion. The present study focused on the consequences of the p.T298M mutation on the structure of Matrilin-3 and on the role of Matrilin-3 in the formation of a functional extracellular matrix. Analysis of recombinant full-length Matrilin-3 revealed that the p.T298M mutation does not influence oligomerization of Matrilin-3 or its proteolytic processing by ADAMTS-4 and -5. Nevertheless, structural analyses indicate local conformational changes. These changes do not affect the affinity for collagens II, IX, XI, or COMP, but have a major impact on the in vitro fibrillogenesis of collagen II/IX/XI heterofibrils. Hum Mutat 31:254–263, 2010. © 2010 Wiley-Liss, Inc.
Soo-hong Lee - One of the best experts on this subject based on the ideXlab platform.
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Matrilin-3-Primed Adipose-Derived Mesenchymal Stromal Cell Spheroids Prevent Mesenchymal Stromal-Cell-Derived Chondrocyte Hypertrophy.
International journal of molecular sciences, 2020Co-Authors: Manjunatha S. Muttigi, Inbo Han, Hansoo Park, Byoung Ju Kim, Bogyu Choi, Soo-hong LeeAbstract:Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for articular cartilage repair and regeneration. However, the terminal hypertrophic differentiation of Ad-MSC-derived cartilage is a critical barrier during hyaline cartilage regeneration. In this study, we investigated the role of Matrilin-3 in preventing Ad-MSC-derived chondrocyte hypertrophy in vitro and in an osteoarthritis (OA) destabilization of the medial meniscus (DMM) model. Methacrylated hyaluron (MAHA) (1%) was used to encapsulate and make scaffolds containing Ad-MSCs and Matrilin-3. Subsequently, the encapsulated cells in the scaffolds were differentiated in chondrogenic medium (TGF-β, 1-14 days) and thyroid hormone hypertrophic medium (T3, 15-28 days). The presence of Matrilin-3 with Ad-MSCs in the MAHA scaffold significantly increased the chondrogenic marker and decreased the hypertrophy marker mRNA and protein expression. Furthermore, Matrilin-3 significantly modified the expression of TGF-β2, BMP-2, and BMP-4. Next, we prepared the OA model and transplanted Ad-MSCs primed with Matrilin-3, either as a single-cell suspension or in spheroid form. Safranin-O staining and the OA score suggested that the regenerated cartilage morphology in the Matrilin-3-primed Ad-MSC spheroids was similar to the positive control. Furthermore, Matrilin-3-primed Ad-MSC spheroids prevented subchondral bone sclerosis in the mouse model. Here, we show that Matrilin-3 plays a major role in modulating Ad-MSCs' therapeutic effect on cartilage regeneration and hypertrophy suppression.
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efficacy of Matrilin 3 primed adipose derived mesenchymal stem cell spheroids in a rabbit model of disc degeneration
Stem Cell Research & Therapy, 2020Co-Authors: Manjunatha S. Muttigi, Inbo Han, Hansoo Park, Byoung Ju Kim, Hemant Kumar, Sunghyun Park, Un Yong Choi, Soo-hong LeeAbstract:Chronic low back pain is a prevalent disability, often caused by intervertebral disc (IVD) degeneration. Mesenchymal stem cell (MSC) therapy could be a safe and feasible option for repairing the degenerated disc. However, for successful translation to the clinic, various challenges need to be overcome including unwanted adverse effects due to acidic pH, hypoxia, and limited nutrition. Matrilin-3 is an essential extracellular matrix (ECM) component during cartilage development and ossification and exerts chondrocyte protective effects. This study evaluated the effects of Matrilin-3-primed adipose-derived MSCs (Ad-MSCs) on the repair of the degenerated disc in vitro and in vivo. We determined the optimal priming concentration and duration and developed an optimal protocol for Ad-MSC spheroid generation. Priming with 10 ng/ml Matrilin-3 for 5 days resulted in the highest mRNA expression of type 2 collagen and aggrecan in vitro. Furthermore, Ad-MSC spheroids with a density of 250 cells/microwell showed the increased secretion of favorable growth factors such as transforming growth factor beta (TGF-β1), TGF-β2, interleukin-10 (IL-10), granulocyte colony-stimulating factor (G-CSF), and matrix metalloproteinase 1 (MMP1) and decreased secretion of hypertrophic ECM components. In addition, Matrilin-3-primed Ad-MSC spheroid implantation was associated with optimal repair in a rabbit model. Our results suggest that priming MSCs with Matrilin-3 and spheroid formation could be an effective strategy to overcome the challenges associated with the use of MSCs for the treatment of IVD degeneration.
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Efficacy of Matrilin-3-primed adipose-derived mesenchymal stem cell spheroids in a rabbit model of disc degeneration.
Stem cell research & therapy, 2020Co-Authors: Manjunatha S. Muttigi, Inbo Han, Hansoo Park, Byoung Ju Kim, Hemant Kumar, Sunghyun Park, Un Yong Choi, Soo-hong LeeAbstract:Background Chronic low back pain is a prevalent disability, often caused by intervertebral disc (IVD) degeneration. Mesenchymal stem cell (MSC) therapy could be a safe and feasible option for repairing the degenerated disc. However, for successful translation to the clinic, various challenges need to be overcome including unwanted adverse effects due to acidic pH, hypoxia, and limited nutrition. Matrilin-3 is an essential extracellular matrix (ECM) component during cartilage development and ossification and exerts chondrocyte protective effects. Methods This study evaluated the effects of Matrilin-3-primed adipose-derived MSCs (Ad-MSCs) on the repair of the degenerated disc in vitro and in vivo. We determined the optimal priming concentration and duration and developed an optimal protocol for Ad-MSC spheroid generation. Results Priming with 10 ng/ml Matrilin-3 for 5 days resulted in the highest mRNA expression of type 2 collagen and aggrecan in vitro. Furthermore, Ad-MSC spheroids with a density of 250 cells/microwell showed the increased secretion of favorable growth factors such as transforming growth factor beta (TGF-β1), TGF-β2, interleukin-10 (IL-10), granulocyte colony-stimulating factor (G-CSF), and matrix metalloproteinase 1 (MMP1) and decreased secretion of hypertrophic ECM components. In addition, Matrilin-3-primed Ad-MSC spheroid implantation was associated with optimal repair in a rabbit model. Conclusion Our results suggest that priming MSCs with Matrilin-3 and spheroid formation could be an effective strategy to overcome the challenges associated with the use of MSCs for the treatment of IVD degeneration.
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Matrilin 3 codelivery with adipose derived mesenchymal stem cells promotes articular cartilage regeneration in a rat osteochondral defect model
Journal of Tissue Engineering and Regenerative Medicine, 2018Co-Authors: Manjunatha S. Muttigi, Inbo Han, Hansoo Park, Byoung Ju Kim, Bogyu Choi, Arai Yoshie, Hemant Kumar, Soo-hong LeeAbstract:Matrilin-3 is an essential extracellular matrix component present only in cartilaginous tissues. Matrilin-3 exerts chondroprotective effects by regulating an anti-inflammatory function and extracellular matrix components. We hypothesized that the codelivery of Matrilin-3 with infrapatellar adipose-tissue-derived mesenchymal stem cells (Ad-MSCs) may enhance articular cartilage regeneration. Matrilin-3 treatment of Ad-MSCs in serum-free media induced collagen II and aggrecan expression, and Matrilin-3 in chondrogenic media also enhanced in vitro chondrogenic differentiation. Next, the in vivo effect of Matrilin-3 codelivery with Ad-MSCs on cartilage regeneration was assessed in an osteochondral defect model in Sprague Dawley rats: Ad-MSCs and hyaluronic acid were implanted at the defect site with or without Matrilin-3 (140, 280, and 700 ng). Safranin O staining revealed that Matrilin-3 (140 and 280 ng) treatment significantly improved cartilage regeneration and glycosaminoglycan accumulation. In the animals treated with 140-ng Matrilin-3, in particular, the defect site exhibited complete integration with surrounding tissue and a smooth glistening surface. The International Cartilage Repair Society macroscopic and O'Driscoll microscopic scores for regenerated cartilage were furthermore shown to be considerably higher for this group (Matrilin-3; 140 ng) compared with the other groups. Furthermore, the defects treated with 140-ng Matrilin-3 revealed significant hyaline-like cartilage regeneration in the osteochondral defect model; in contrast, the defects treated with 700-ng Matrilin-3 exhibited drastically reduced cartilage regeneration with mixed hyaline-fibrocartilage morphology. Codelivery of Matrilin-3 with Ad-MSCs significantly influenced articular cartilage regeneration, supporting the potential use of this tissue-specific protein for a cartilage-targeted stem cell therapy.
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Matrilin‐3 codelivery with adipose‐derived mesenchymal stem cells promotes articular cartilage regeneration in a rat osteochondral defect model
Journal of tissue engineering and regenerative medicine, 2017Co-Authors: Manjunatha S. Muttigi, Inbo Han, Hansoo Park, Byoung Ju Kim, Bogyu Choi, Arai Yoshie, Hemant Kumar, Soo-hong LeeAbstract:Matrilin-3 is an essential extracellular matrix component present only in cartilaginous tissues. Matrilin-3 exerts chondroprotective effects by regulating an anti-inflammatory function and extracellular matrix components. We hypothesized that the codelivery of Matrilin-3 with infrapatellar adipose-tissue-derived mesenchymal stem cells (Ad-MSCs) may enhance articular cartilage regeneration. Matrilin-3 treatment of Ad-MSCs in serum-free media induced collagen II and aggrecan expression, and Matrilin-3 in chondrogenic media also enhanced in vitro chondrogenic differentiation. Next, the in vivo effect of Matrilin-3 codelivery with Ad-MSCs on cartilage regeneration was assessed in an osteochondral defect model in Sprague Dawley rats: Ad-MSCs and hyaluronic acid were implanted at the defect site with or without Matrilin-3 (140, 280, and 700 ng). Safranin O staining revealed that Matrilin-3 (140 and 280 ng) treatment significantly improved cartilage regeneration and glycosaminoglycan accumulation. In the animals treated with 140-ng Matrilin-3, in particular, the defect site exhibited complete integration with surrounding tissue and a smooth glistening surface. The International Cartilage Repair Society macroscopic and O'Driscoll microscopic scores for regenerated cartilage were furthermore shown to be considerably higher for this group (Matrilin-3; 140 ng) compared with the other groups. Furthermore, the defects treated with 140-ng Matrilin-3 revealed significant hyaline-like cartilage regeneration in the osteochondral defect model; in contrast, the defects treated with 700-ng Matrilin-3 exhibited drastically reduced cartilage regeneration with mixed hyaline-fibrocartilage morphology. Codelivery of Matrilin-3 with Ad-MSCs significantly influenced articular cartilage regeneration, supporting the potential use of this tissue-specific protein for a cartilage-targeted stem cell therapy.