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Mutant cartilage oligomeric matrix protein (COMP) compromises bone integrity, joint function and the balance between adipogenesis and osteogenesis.

ABSTRACT: Mutations in COMP (cartilage oligomeric matrix protein) cause severe long bone shortening in mice and humans. Previously, we showed that massive accumulation of misfolded COMP in the ER of growth plate chondrocytes in our MT-COMP mouse model of pseudoachondroplasia (PSACH) causes premature chondrocyte death and loss of linear growth. Premature chondrocyte death results from activation of oxidative stress and inflammation through the CHOP-ER pathway and is reduced by removing CHOP or by anti-inflammatory or antioxidant therapies. Although the mutant COMP chondrocyte pathologic mechanism is now recognized, the effect of mutant COMP on bone quality and joint health (laxity) is largely unknown. Applying multiple analytic approaches, we describe a novel mechanism by which the deleterious consequences of mutant COMP retention results in upregulation of miR-223 disturbing the adipogenesis - osteogenesis balance. This results in reduction in bone mineral density, bone quality, mechanical strength and subchondral bone thickness. These, in addition to abnormal patterns of ossification at the ends of the femoral bones likely contribute to precocious osteoarthritis (OA) of the hips and knees in the MT-COMP mouse and PSACH. Moreover, joint laxity is compromised by abnormally thin ligaments. Altogether, these novel findings align with the PSACH phenotype of delayed ossification and bone age, extreme joint laxity and joint erosion, and extend our understanding of the underlying processes that affect bone in PSACH. These results introduce a novel finding that miR-223 is involved in the ossification defect in MT-COMP mice making it a therapeutic target.

SUBMITTER: Coustry F

PROVIDER: S-EPMC5910205 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Mutant cartilage oligomeric matrix protein (COMP) compromises bone integrity, joint function and the balance between adipogenesis and osteogenesis.

Coustry Francoise F Posey Karen L KL Maerz Tristan T Baker Kevin K Abraham Annie M AM Ambrose Catherine G CG Nobakhti Sabah S Shefelbine Sandra J SJ Bi Xiaohong X Newton Michael M Gawronski Karissa K Remer Lindsay L Veerisetty Alka C AC Hossain Mohammad G MG Chiu Frankie F Hecht Jacqueline T JT

Matrix biology : journal of the International Society for Matrix Biology 20180105

Mutations in COMP (cartilage oligomeric matrix protein) cause severe long bone shortening in mice and humans. Previously, we showed that massive accumulation of misfolded COMP in the ER of growth plate chondrocytes in our MT-COMP mouse model of pseudoachondroplasia (PSACH) causes premature chondrocyte death and loss of linear growth. Premature chondrocyte death results from activation of oxidative stress and inflammation through the CHOP-ER pathway and is reduced by removing CHOP or by anti-infl ...[more]

PMID: 29309831

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Project description:ObjectiveCOMP (cartilage oligomeric matrix protein) is abundantly expressed in the cardiovascular system, cartilage, and atherosclerotic plaques. We investigated if the total COMP (COMPtotal) and COMP neoepitope (COMPneo) with other cardiovascular markers and clinical parameters could identify symptomatic carotid stenosis. Approach and Results: Blood samples were collected from patients with symptomatic carotid stenosis (stenosis, n=50), patients with stroke without carotid stenosis but small plaques (plaque, n=50), and control subjects (n=50). COMPtotal and COMPneo were measured using an ELISA. Ninety-two cardiovascular disease markers were measured by the Olink CVD kit. The presence of native COMP and COMPneo was determined by immunohistochemistry. The concentration of COMPneo was higher and COMPtotal was lower in the stenosis group. When the concentration was compared between the stenosis and control groups, IL-1ra (interleukin-1 receptor antagonist protein), IL6 (interleukin-6), REN (Renin), MMP1 (matrix metalloproteinase-1), TRAIL-R2 (tumor necrosis factor-related apoptosis-inducing ligand receptor 2), ITGB1BP2 (integrin beta 1 binding protein 2), and COMPneo were predictive of stenosis. Conversely, KLK6 (kallikrein-6), COMPtotal, NEMO (nuclear factor-kappa-B essential modulator), SRC (Proto-oncogene tyrosine-protein kinase Src), SIRT2 (SIR2-like protein), CD40 (cluster of differentiation 40), TF (tissue factor), MP (myoglobin), and RAGE (receptor for advanced glycation end-products) were predictive of the control group. Model reproducibility was good with the receiver operating characteristic plot area under the curve being 0.86. When comparing the plaque group and stenosis group, COMPneo, GAL (galanin), and PTX3 (pentraxin-related protein PTX3) were predictive of stenosis. Model reproducibility was excellent (receiver operating characteristic plot area under the curve 0.92). COMPneo was detected in smooth muscle-, endothelial-, and foam-cells in carotid stenosis.ConclusionsDegradation of COMP may be associated with atherosclerosis progression and generation of a specific COMP fragment-COMPneo. This may represent a novel biomarker that together with COMPtotal and other risk-markers could be used to identify symptomatic carotid stenosis. Graphic Abstract: A graphic abstract is available for this article.

Project description:ObjectiveThe composition of the extracellular matrix (ECM) impacts adipocyte function and might determine adipose tissue (AT) function and distribution. Cartilage oligomeric matrix protein (COMP), a matricellular protein usually studied in bone and cartilage, is highly differentially expressed between subcutaneous abdominal and gluteal AT. This study aimed to explore COMP's role in human subcutaneous abdominal and gluteal AT and preadipocyte biology.MethodsCOMP mRNA levels were measured in whole AT and immortalised preadipocytes via quantitative (q)-PCR. Tissue and cellular COMP protein were measured via Western blot and immunohistochemistry; plasma COMP was measured by ELISA. The effect of COMP on adipogenesis in immortalised preadipocytes was evaluated by qPCR of adipogenic markers and cellular triacylglycerol (TAG) accumulation.ResultsqPCR analysis of paired subcutaneous abdominal and gluteal AT biopsies (n = 190) across a range of BMI (20.7-45.5 kg/m2) indicated ?3-fold higher COMP expression in gluteal AT (P = 1.7 × 10-31); protein levels mirrored this. Immunohistochemistry indicated COMP was abundant in gluteal AT ECM and co-localised with collagen-1. AT COMP mRNA levels and circulating COMP protein levels were positively associated with BMI/adiposity but unrelated to AT distribution. COMP expression changed dynamically during adipogenesis (time × depot, P = 0.01). Supplementation of adipogenic medium with exogenous COMP protein (500 ng/ml) increased PPARG2 expression ?1.5-fold (P = 0.0003) and TAG accumulation ?1.25-fold in abdominal and gluteal preadipocytes (P = 0.02).ConclusionsWe confirmed that COMP is an ECM protein which is differentially expressed between subcutaneous abdominal and gluteal AT. Despite its depot-specific expression pattern, however, AT COMP mRNA levels and plasma COMP concentration correlated positively with overall obesity but not body fat distribution. Exogenous COMP enhanced adipogenesis. These data identify COMP as a novel regulator of AT and highlight the importance of the ECM to AT biology.

Project description:IntroductionSkeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferator-activated receptor gamma (PPAR?) is commonly considered the master transcriptional regulator of adipogenesis, however, it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPAR? shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model.MethodsPPAR? shRNA was delivered by a replication-deficient lentiviral vector, after in vitro testing to confirm purity, concentration, and efficacy for Pparg transcript reduction. Next, control green fluorescent protein lentivirus or PPAR? shRNA expressing lentivirus were delivered by intramedullary injection into the femoral bone marrow of male SCID mice. Analyses included daily monitoring of animal health, and postmortem analysis at 4 weeks. Postmortem analyses included high resolution microcomputed tomography (microCT) reconstructions and analysis, routine histology and histomorphometric analysis, quantitative real time polymerase chain reaction analysis of Pparg transcript levels, and immunohistochemical analysis for markers of adipocytes (PPAR?, fatty acid binding protein 4 [FABP4]), osteoblasts (alkaline phosphatase [ALP], osteocalcin [OCN]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP], Cathepsin K).ResultsIn vitro, PPAR? shRNA delivery significantly reduced Pparg expression in mouse BMSC, accompanied by a significant reduction in lipid droplet accumulation. In vivo, a near total reduction in mature marrow adipocytes was observed at 4 weeks postinjection. This was accompanied by significant reductions in adipocyte-specific markers. Parameters of trabecular bone were significantly increased by both microCT and histomorphometric analysis. By immunohistochemical staining and semi-quantification, a significant increase in OCN+osteoblasts and decrease in TRAP+multinucleated osteoclasts was observed with PPAR? shRNA treatment.DiscussionThese findings suggest that acute loss of PPAR? in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPAR? shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPAR? shRNA on OB and OC cell types, and it may help determine whether PPAR? antagonists are potential therapeutic agents for osteoporotic bone loss.

Project description:COMP (Cartilage Oligomeric Matrix Protein), also named thrombospondin-5, is a member of the thrombospondin family of extracellular matrix proteins. It is of clinical relevance, as in humans mutations in COMP lead to chondrodysplasias. The gene encoding zebrafish Comp is located on chromosome 11 in synteny with its mammalian orthologs. Zebrafish Comp has a domain structure identical to that of tetrapod COMP and shares 74% sequence similarity with murine COMP. Zebrafish comp is expressed from 5 hours post fertilization (hpf) on, while the protein is first detectable in somites of 11 hpf embryos. During development and in adults comp is strongly expressed in myosepta, craniofacial tendon and ligaments, around ribs and vertebra, but not in its name-giving tissue cartilage. As in mammals, zebrafish Comp forms pentamers. It is easily extracted from 5 days post fertilization (dpf) whole zebrafish. The lack of Comp expression in zebrafish cartilage implies that its cartilage function evolved recently in tetrapods. The expression in tendon and myosepta may indicate a more fundamental function, as in evolutionary distant Drosophila muscle-specific adhesion to tendon cells requires thrombospondin. A sequence encoding a calcium binding motif within the first TSP type-3 repeat of zebrafish Comp was targeted by CRISPR-Cas. The heterozygous and homozygous mutant Comp zebrafish displayed a patchy irregular Comp staining in 3 dpf myosepta, indicating a dominant phenotype. Electron microscopy revealed that the endoplasmic reticulum of myosepta fibroblasts is not affected in homozygous fish. The disorganized extracellular matrix may indicate that this mutation rather interferes with extracellular matrix assembly, similar to what is seen in a subgroup of chondrodysplasia patients. The early expression and easy detection of mutant Comp in zebrafish points to the potential of using the zebrafish model for large scale screening of small molecules that can improve secretion or function of disease-associated COMP mutants.

Project description:Primary chondrocytes dedifferentiate in serial monolayer with respect to their morphological and biosynthetic phenotype. They change from a round to a flattened fibroblast-like shape, and collagen I is secreted instead of the cartilage-specific collagen II. We analysed in detail the time course of dedifferentiation of mature bovine articular chondrocytes in monolayer for up to 32 weeks. Assessment of RNA expression by reverse transcription-PCR led to the identification of two novel phenotypical markers, the cartilage oligomeric matrix protein (COMP) and collagen IX, which are down-regulated faster than the widely accepted marker, collagen II. The different kinetics of COMP and collagen expression suggest differential regulation at the level of transcription. Immunostaining and metabolic labelling experiments confirmed the switch in the collagen expression pattern and the rapid down-regulation of de novo synthesis of COMP and collagen IX. Culture of chondrocytes in a three-dimensional matrix is known to stabilize the chondrocytic phenotype. We maintained cells for up to 28 weeks in an alginate bead system, which prevented dedifferentiation and led to a stabilization of collagen and COMP expression. Immunohistochemical analysis of the alginate beads revealed a similar distribution of matrix proteins to that found in vivo. Chondrocytes were transferred after a variable length of monolayer culture into the alginate matrix and the potential for redifferentiation was investigated. The re-expression of COMP and collagen IX was differentially regulated. The expression of COMP was re-induced within days after transfer into the three-dimensional matrix, while the expression of collagen IX was irreversibly down-regulated. In summary, these results demonstrate that the potential for redifferentiation decreases with increasing length of monolayer culture and show that the alginate bead system represents an attractive in vitro model to study the chondrocyte de- and re-differentiation processes, as well as extracellular matrix assembly.

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Mutant cartilage oligomeric matrix protein (COMP) compromises bone integrity, joint function and the balance between adipogenesis and osteogenesis.

Publications

Mutant cartilage oligomeric matrix protein (COMP) compromises bone integrity, joint function and the balance between adipogenesis and osteogenesis.

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