COMP mutations: domain-dependent relationship between abnormal chondrocyte trafficking and clinical PSACH and MED phenotypes

Mutations in cartilage oligomeric matrix protein (COMP) produce clinical phenotypes ranging from the severe end of the spectrum, pseudoachondroplasia (PSACH), which is a dwarfing condition, to a mild condition, multiple epiphyseal dysplasia (MED). Patient chondrocytes have a unique morphology characterized by distended rER cisternae containing lamellar deposits of COMP and other extracellular matrix proteins. It has been difficult to determine why different mutations give rise to variable clinical phenotypes. Using our in vitro cell system, we previously demonstrated that the most common PSACH mutation, D469del, severely impedes trafficking of COMP and type IX collagen in chondrocytic cells, consistent with observations from patient cells. Here, we hypothesize that PSACH and MED mutations variably affect the cellular trafficking behavior of COMP and that the extent of defective trafficking correlates with clinical phenotype. Twelve different recombinant COMP mutations were expressed in rat chondrosarcoma cells and the percent cells with ER-retained COMP was assessed. For mutations in type 3 (T3) repeats, trafficking defects correlated with clinical phenotype; PSACH mutations had more cells retaining mutant COMP, while MED mutations had fewer. In contrast, the cellular trafficking pattern observed for mutations in the C-terminal globular domain (CTD) was not predictive of clinical phenotype. The results demonstrate that different COMP mutations in the T3 repeat domain have variable effects on intracellular transport, which correlate with clinical severity, while CTD mutations do not show such a correlation. These findings suggest that other unidentified factors contribute to the effect of the CTD mutations. J. Cell. Biochem. 103: 778-787, 2008. (c) 2007 Wiley-Liss, Inc.     

Retention of cartilage oligomeric matrix protein (COMP) and cell death in redifferentiated pseudoachondroplasia chondrocytes

Cartilage oligomeric matrix protein (COMP) is a large extracellular glycoprotein that is found in the territorial matrix surrounding chondrocytes. Two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) are caused by mutations in the calcium binding domains of COMP. In this study, we identified two PSACH mutations and assessed the effect of these mutations on redifferentiated chondrocyte structure and function. We confirmed, in vitro, that COMP is retained in enormous cisternae of the rough endoplasmic reticulum (rER) and relatively absent in the PSACH matrix. The rER accumulation may compromise chondrocyte function, leading to chondrocyte death. Moreover, while COMP appears to be deficient in the PSACH matrix, the matrix appeared to be normal but the over-all quantity was reduced. These results suggest that the abnormality in linear growth in PSACH may result from decreased chondrocyte numbers which would also affect the amount of matrix produced.     

Collagen XI chain misassembly in cartilage of the chondrodysplasia (cho) mouse

Molecular mechanisms controlling the assembly of cartilage-specific types II, IX and XI collagens into a heteropolymeric network of uniformly thin, unbanded fibrils are not well understood, but collagen XI has been implicated. The present study on cartilage from the homozygous chondrodysplasia (cho/cho) mouse adds support to this concept. In the absence of alpha1(XI) collagen chains, thick, banded collagen fibrils are formed in the extracellular matrix of cho/cho cartilage. A functional knock-out of the type XI collagen molecule has been assumed. We have re-examined this at the protein level to see if, rather than a complete knock-out, alternative type XI chain assemblies were formed. Mass spectrometry of purified triple-helical collagen from the rib cartilage of cho/cho mice identified alpha1(V) and alpha2(XI) chains. These chains were recovered in roughly equal amounts based on Coomassie Blue staining of SDS-PAGE gels, in addition to alpha1(II)/alpha3(XI) collagen chains. Using telopeptide-specific antibodies and Western blot analysis, it was further shown that type V/XI trimers were present in the matrix cross-linked to each other and to type II collagen molecules to form heteropolymers. Cartilage from heterozygous (cho/+) mice contained a mix of alpha1(V) and alpha1(XI) chains and a mix of thin and thick fibrils on transmission electron microscopy. In summary, the results imply that native type XI collagen molecules containing an alpha1(XI) chain are required to form uniformly thin fibrils and support a role for type XI collagen as the template for the characteristic type II collagen fibril network of developing cartilage.     

Collagen in tissue-engineered cartilage: Types,structure, and crosslinks

The function of articular cartilage as a weight-bearing tissue depends on the specific arrangement of collagen types II and IX into a three-dimensional organized collagen network that can balance the swelling pressure of the proteoglycan/ water gel. To determine whether cartilage engineered in vitro contains a functional collagen network, chondrocyte-polymer constructs were cultured for up to 6 weeks and analyzed with respect to the composition and ultrastructure of collagen by using biochemical and immunochemical methods and scanning electron microscopy. Total collagen content and the concentration of pyridinium crosslinks were significantly (57% and 70%, respectively) lower in tissue-engineered cartilage that in bovine calf articular cartilage. However, the fractions of collagen types II, IX, and X and the collagen network organization, density, and fibril diameter in engineered cartilage were not significantly different from those in natural articular cartilage. The implications of these findings for the field of tissue engineering are that differentiated chondrocytes are capable of forming a complex structure of collagen matrix in vitro, producing a tissue similar to natural articular cartilage on an ultrastructural scale. J. Cell. Biochem. 71:313–327, 1998. © 1998 Wiley-Liss, Inc.     

The chondrocyte,architect of cartilage. Biomechanics,structure, function and molecular biology of cartilage matrix macromolecules

Chondrocytes are specialised cells which produce and maintain the extracellular matrix of cartilage, a tissue that is resilient and pliant. In vivo, it has to withstand very high compressive loads, and that is explicable in terms of the physico-chemical properties of cartilage-specific macromolecules and with the movement of water and ions within the matrix. The functions of the cartilage-specific collagens, aggrecan (a hydrophilic proteoglycan) and hyaluronan are discussed within this context. The structures of cartilage collagens and proteoglycans and their genes are known and a number of informative mutations have been identified. In particular, collagen fibrillogenesis is a complex process which can be altered by mutations whose effects fit what is known about collagen molecular structural functions. In other instances, mutations have indicated new functions for particular molecular domains. As cartilage provides the template for the developing skeleton, mutations in genes for cartilage-specific proteins often produce developmental abnormalities. The search for mutations amongst such genes in heritable disorders is being actively pursued by many groups, although mutation and phenotype are not always well correlated, probably because of compensatory mechanisms. The special nature of the chondrocyte is stressed in connection with its cell involvement in osteoarthritis, the most widespread disease of diarthrodial joints.     

Novel and recurrent COMP (cartilage oligomeric matrix protein) mutations in pseudoachondroplasia and multiple epiphyseal dysplasia

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are common skeletal dysplasias with impaired enchondral ossification and premature degenerative joint disease. The two disorders were in the past considered to be distinct clinical entities; however, recent studies have proven that both diseases can result from mutations of the gene encoding cartilage oligomeric matrix protein (COMP). To characterize further COMP mutations and investigate phenotype-genotype relationships, we screened this gene in 15 patients with PSACH or MED by directly sequencing polymerase chain reaction products from genomic DNA. We identified ten mutations involving conserved residues among the eight calmodulin-like repeats of the gene product: seven were novel missense mutations in exons 9, 10, 11, 13 or 14, and the other three resulted from deletion of one of the five GAC repeats in exon 13. We have found that the GAC repeats in the 7th calmodulin-like repeat in exon 13 represent a hot-spot for mutation, and that mutations in the 7th calmodulin-like repeat produce severe PSACH phenotypes while mutations elsewhere in the gene exhibit mild PSACH or MED phenotypes. These genotype-phenotype correlations may facilitate molecular diagnosis and classification of PSACH and MED, and provide insight into the relationship between structure and function of the COMP gene product. Received: 1 June 1998 / Accepted: 22 October 1998     

Galectin-1 in cartilage: Expression, influence on chondrocyte growth and interaction with ECM components

Galectin-1 is a 14 kDa beta-galactoside binding protein, capable of forming lattice-like structures with glycans of cellular glycoconjugates and inducing intracellular signaling. The expression of Galectin-1 in porcine cartilage is described in this work for the first time. Immunocytochemical methods revealed distinct distribution patterns for both articular and growth plate cartilage. In articular cartilage, the highest reactivity for Galectin-1 was found in all chondrocytes at the superficial zone and in most of those at the lower layer of the middle zone. In the growth plate, marked reactivity was seen in chondrocytes at the proliferative zone and reached a maximum level for the column-forming cells at the hypertrophic zone. In addition, different Galectin-1 distribution patterns were observed at the subcellular level. With regards to the metabolic effects of Galectin-1, the results in vitro seem to indicate an inhibitory effect of Galectin-1 on articular chondrocyte anabolism (i.e. inhibition of cell proliferation and anabolic gene expression) and a stimulation of catabolic processes (i.e. induction of matrix degradation and hypertrophy marker expression). These data represent a starting point for the understanding the molecular mechanisms underlining ECM-Galectin-1 interaction and the subsequent signaling-cell transduction processes involving cartilage formation and maturation.     

Tamoxifen-inducible gene deletion reveals a distinct cell type associated with trabecular bone, and direct regulation of PTHrP expression and chondrocyte morphology by Ihh in growth region cartilage

Indian hedgehog (Ihh) controls multiple aspects of endochondral skeletal development by signaling to both chondrocytes and perichondrial cells. Previous efforts to delineate direct effects of Ihh on chondrocytes by Col2-Cre-mediated ablation of Smoothened (Smo, encoding a transmembrane protein indispensable for Ihh signaling) has been only partially successful, due to the inability to discriminate between chondrocytes and perichondrial cells. Here we report a transgenic line (Col2-Cre) expressing under the control of the Colalpha1(II) promoter an inert form of Cre that is activatable by exogenous tamoxifen (TM); TM administration at proper times during embryogenesis induced Cre activity in chondrocytes but not in the perichondrium. By using this mouse line, we deleted Smo within subsets of chondrocytes without affecting the perichondrium and found that Smo removal led to localized disruption of the expression of parathyroid hormone-related protein (PTHrP) and the morphology of chondrocytes. Unexpectedly, TM invariably induced Cre activity in a subset of cells associated with the trabecular bone surface of long bones. These cells, when genetically marked and cultured in vitro, were capable of producing bone nodules. Expression of the Col2-Cre transgene in these cells likely reflected the endogenous Colalpha1(II) promoter activity as similar cells were found to express the IIA isoform of Colalpha1(II) mRNA endogenously. In summary, the present study has not only provided evidence that Ihh signaling directly controls PTHrP expression and chondrocyte morphology in the growth region cartilage, but has also uncovered a distinct cell type associated with the trabecular bone that appears to possess osteogenic potential.     

Further biochemical and physicochemical characterization of minor disulfide-bonded (type IX) collagen, extracted from foetal calf cartilage

Minor disulfide-bonded collagen (previously termed X1-X7 and now called type IX collagen) was isolated from foetal calf cartilage after pepsin treatment. At least three native fractions, containing, respectively, the X1X2X3, X4, and X5X6X7 chains, were separated; and from further biochemical and physicochemical experiments (differential scanning calorimetry, electrical birefringence, rotary shadowing), we propose a tentative model for their organization within a parent molecule. X1 and X2 are molecules composed of three chains of apparent Mr 62,000 and 50,000 linked by interchain disulfide bonds and containing pepsin-sensitive regions. The cleavage of at least three of these sites, present within X2, gives rise to the X3 and X5X6X7 fractions composed of molecules 80-100 nm and 40-55 nm in length, respectively. The X5X6X7 fraction is not digested by pepsin at 30 degrees C owing to its high thermal stability (certainly explained by its high hydroxyproline + proline content). This organization is in good accordance with that proposed for chicken cartilage type IX collagen; differences could only exist in the number and (or) the location of the pepsin-sensitive sites.     

Regional differences of tibial and femoral cartilage in the chondrocyte gene expression,immunhistochemistry and composite in different stages of osteoarthritis

The function of articular cartilage as an avascular tissue is mainly served by collagen type II and proteoglycan molecules. Within this matrix homeostasis between production and breakdown of the matrix is exceptionally sensitive.The current study was conducted to identify regional differences in specific alterations in cartilage composition during the osteoarthritic process of the human knee joint. Therefor the changes in the expression of the key molecules of the extracellular matrix were measured in dependence of the anatomical side (femoral vs tibial) and associated with immunohistochemistry and quantitative measurement.60 serial osteochondral femoral condyle and the tibial plateau samples of patients undergoing implantation of total knee endoprosthesis of areas showing mild (Group A, macroscopically ICRS grade 1b) respectively advanced (Group B, macroscopically ICRS grade 3a/3b) (30 each) osteoarthritis according to the histological-histochemical grading system (HHGS) were compared with 20 healthy biopsies with immunohistochemistry and histology. We quantified our results on the gene expression of collagen type I and II and aggrecan with the help of real-time (RT)-PCR. Proteoglycan content was measured colorometrically.In group A slightly increased colour intensity was found for collagen II in deeper layers, suggesting a persisting but initially still intact repair process. But especially on the medial tibia plateau the initial Col II increase in gene expression is followed by a decrease leading to the lowest over all Col II expression on the medial plateau, here especially in the central part. There in late stage diseases the collagen type I expression was also more pronounced. Markedly decreased safranin O staining intensity was observed in the radial zone and less reduced intensity in the transitional zone with loss of zonal anatomy in 40% of the specimens in group A and all specimens in group B. Correlation between colorometrically analysed proteoglycan GAG content and aggrecan Real Time PCR is mainly weak.Tibial and femoral cartilage in contrast to patellar cartilage both are preferential exposed to compressive stresses, but presence of menisci affects the load distribution at the tibial side, which creates varying conditions for the different cartilage surfaces in the knee.As directly measured Poissońs ratio in tibial cartilage is higher but Youn?s modulus is lower than in femoral cartilage, different resulting feedback amplification loops interact with proceeding cartilage damage. The initial loss of aggrecan may support Matrix metalloproteinases (Mmps) in the access to the collagen network and the considerably differing mechanical properties at both joint surfaces result in varying increased synthesis and release of matrix degrading enzymes.The present study has identified a selection of events which reflect the response of cartilage structure and composite, chondrocytes itself and their productivity to changes in mechanical stress depending on the anatomical site.     

Secretion pattern, ultrastructural localization and function of extracellular matrix molecules involved in eggshell formation

The chicken eggshell is a composite bioceramic containing organic and inorganic phases. The organic phase contains, among other constituents, type X collagen and proteoglycans (mammillan, a keratan sulfate proteoglycan, and ovoglycan, a dermatan sulfate proteoglycan), whose localization depends on a topographically defined and temporally regulated deposition. Although the distribution of these macromolecules in the eggshell has been well established, little is known about their precise localization within eggshell substructures and oviduct cells or their pattern of production and function during eggshell formation. By using immunofluorescent and immuno-ultrastructural analyses, we examined the distribution of these macromolecules in oviduct cells at different post-oviposition times. To understand the role of proteoglycan sulfation on eggshell formation, we studied the effects of inhibition of proteoglycan sulfation by treatment with sodium chlorate. We showed that these macromolecules are produced by particular oviduct cell populations and at precise post-oviposition times. Based on the precise ultrastructural localization of these macromolecules in eggshell substructures, the timing of the secretion of these macromolecules by oviduct cells and the effects on eggshell formation caused by the inhibition of proteoglycan sulfation, the putative role of mammillan is in the nucleation of the first calcite crystals, while that of ovoglycan is to regulate the growth and orientation of the later forming crystals of the chicken eggshell.     

Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure

The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.     

Combined effects of dynamic tissue shear deformation and insulin-like growth factor I on chondrocyte biosynthesis in cartilage explants

Biophysical forces and biochemical factors play crucial roles in the maintenance of the integrity of articular cartilage. In this study, we explored the effect of dynamic tissue shear deformation and insulin-like growth factor I (IGF-I) on matrix synthesis by chondrocytes within native cartilage explants. Dynamic tissue shear in the range of 0.5-6% strain amplitude at 0.1 Hz was applied to cartilage explants cultured in serum-free medium. Dynamic tissue shear above 1.5% strain amplitude significantly stimulated protein and proteoglycan synthesis, by maximum values of 35 and 25%, respectively, over statically held control specimens. In the absence of tissue shear, IGF-I augmented protein and proteoglycan synthesis up to twofold at IGF-I concentrations in the range of 100-300 ng/ml. When tissue shear and IGF-I stimuli were combined, matrix biosynthesis levels were significantly higher than the maximal effect caused by either stimulus alone. However, there was no significant interaction between tissue shear and IGF-I as determined by two-way ANOVA. We then quantified the effect of dynamic tissue shear on the transport of IGF-I into and within cartilage explants. [125I]IGF-I was added to the medium, and the levels of intratissue [125I]IGF-I were directly measured as a function of time over 48 h in the presence and absence of continuous dynamic shear strain. Dynamic shear did not alter the rate of uptake of [125I]IGF-I into the explants, suggesting that convective diffusion of [125I]IGF-I is negligible under the shear strain conditions used. This is in marked contrast to the enhancement of transport reported in response to uniaxial dynamic compression. Taken together, these data suggest that (1) the stimulatory effect of tissue shear is via mechanotransduction pathways and not by facilitated transport of biochemical factors and (2) chondrocytes may possess complementary signal transduction pathways for biophysical and biochemical factors leading to changes in metabolic activity.     

Analysis of the role of the COL1 domain and its adjacent cysteine-containing sequence in the chain assembly of type IX collagen

The mechanisms of chain selection and assembly of type IX collagen, a heterotrimer 1(IX)2(IX)3(IX), must differ from that of fibrillar collagens since it lacks the characteristic C-propeptide of these latter molecules. We have tested the hypothesis that the information required for this process is contained within the C-terminal triple helical disulfide-bonded region (LMW). The reassociations of the purified LMW fragments of pepsinized bovine type IX collagen were followed by the formation of disulfide-bonded multimers. Our data demonstrate that only three triple helical assemblies form readily, (1)₃, (2)₃, and 123. The information required for chain selection and assembly is thus, at least in part, contained in the studied fragments. Molecular stoichiometries different from the classical heterotrimer may thus also form under certain conditions.     

Novel Cartilage Oligomeric Matrix Protein (COMP) Neoepitopes Identified in Synovial Fluids from Patients with Joint Diseases Using Affinity Chromatography and Mass Spectrometry

To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDS-PAGE followed by in-gel digestion and mass spectrometric identification and characterization. Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser⁷⁷, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser⁷⁷ were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided.     

The effect of TGF-beta1 and beta-estradiol on glycosaminoglycan and type II collagen distribution in articular chondrocyte cultures

Articular cartilage extracellular matrix (ECM) plays a crucial role in regulating chondrocyte functions via cell-matrix interaction, cytoskeletal organization and integrin-mediated signaling. Factors such as interleukins, basic fibroblast growth factor (bFGF), bone morphogenic proteins (BMPs) and insulin-like growth factor (IGF) have been shown to modulate the synthesis of extracellular matrix in vitro. However, the effects of TGF-beta1 and beta-estradiol in ECM regulation require further investigation, although there have been suggestions that these factors do play a positive role. To establish the role of these factors on chondrocytes derived from articular joints, a study was conducted to investigate the effects of TGF-beta1 and beta-estradiol on glycosaminoglycan secretion and type II collagen distribution (two major component of cartilage ECM in vivo). Thus, chondrocyte cultures initiated from rabbit articular cartilage were treated with 10ng/ml of TGF-beta1, 10nM of beta-estradiol or with a combination of both factors. Sulphated glycosaminoglycan (GAG) and type II collagen levels were then measured in both these culture systems. The results revealed that the synthesis of GAG and type II collagen was shown to be enhanced in the TGF-beta1 treated cultures. This increase was also noted when TGF-beta1 and beta-estradiol were both used as culture supplements. However, beta-estradiol alone did not appear to affect GAG or type II collagen deposition. There was also no difference between the amount of collagen type II and GAG being expressed when chondrocyte cultures were treated with TGF-beta1 when compared with cultures treated with combined factors. From this, we conclude that although TGF-beta1 appears to stimulate chondrocyte ECM synthesis, beta-estradiol fails to produce similar effects. The findings of this study confirm that contrary to previous claims, beta-estradiol has little or no effect on chondrocyte ECM synthesis. Furthermore, the use of TGF-beta1 may be useful in future studies looking into biological mechanisms by which ECM synthesis in chondrocyte cultures can be augmented, particularly for clinical application.     

The role of hydrogel structure and dynamic loading on chondrocyte gene expression and matrix formation

Crosslinked poly(ethylene glycol) (PEG) hydrogels are attractive scaffolds for cartilage tissue engineering because of their ability to mimic the aqueous environment and mechanical properties of native cartilage. In this study, hydrogel crosslinking density was varied to study the influence of gel structure and the application of dynamic loading (continuous, 1 Hz, 15% amplitude strain) on chondrocyte gene expression over 1 week culture. Gene expression was quantified using real-time RT-PCR for collagen II and aggrecan, the major cartilage extracellular matrix (ECM) components, and collagen I, an indicator of chondrocyte de-differentiation. When chondrocytes were encapsulated in PEG gels with low or high crosslinking, a high collagen II expression compared to collagen I expression (1000 or 100,000:1, respectively) indicated the native chondrocyte phenotype was retained. In the absence of loading, relative gene expression for collagen II and aggrecan was significantly higher (e.g., 2-fold and 4-fold, respectively, day 7) in the low crosslinked gels compared to gels with higher crosslinking. Dynamic loading, however, showed little effect on ECM gene expression in both crosslinked systems. To better understand the cellular environment, ECM production was qualitatively assessed using an in situ immunofluorescent technique and standard histology. A pericellular matrix (PCM) was observed as early as day 3 post-encapsulation and the degree of formation was dependent on gel crosslinking. These results suggest the PCM may protect the cells from sensing the applied loads. This study demonstrates that gel structure has a profound effect on chondrocyte gene expression, while dynamic loading has much less of an effect at early culture times.