Expression of matrilins during maturation of mouse skeletal tissues.

The matrilins are a recently discovered family of non-collagenous extracellular matrix proteins. During embryogenesis, all matrilins are expressed in skeletal tissues. Additionally, matrilin-2 and -4 are expressed in the dermis and in connective tissues of internal organs, e.g. of the lung and kidney. After birth, the expression of matrilin-1 and -3 remains specific for cartilage and bone whereas matrilin-2 and -4 display a broader tissue distribution and could be detected in epithelial, muscle, and nervous tissue as well as in loose and dense connective tissue. In epiphyseal cartilage of growing long bones, matrilin-1 and -3 are present in all cartilage regions, in contrast to matrilin-2, which is expressed in the proliferative and the upper hypertrophic zones. Similarly matrilin-4 was detected all over the epiphyseal cartilage, with the weakest expression in the hypertrophic zone. Although it was shown that matrilin-1 and -3 can form hetero-oligomers and are often co-localized in tissue, clear differences in their spatial distribution could be demonstrated by double-immunolabelling. During joint development matrilin-2 and matrilin-4 are present at the developing joint surface, while in articular cartilage of 6-week-old mice all matrilins are only weakly expressed.     

Complexes of matrilin-1 and biglycan or decorin connect collagen VI microfibrils to both collagen II and aggrecan

Native supramolecular assemblies containing collagen VI microfibrils and associated extracellular matrix proteins were isolated from Swarm rat chondrosarcoma tissue. Their composition and spatial organization were characterized by electron microscopy and immunological detection of molecular constituents. The small leucine-rich repeat (LRR) proteoglycans biglycan and decorin were bound to the N-terminal region of collagen VI. Chondroadherin, another member of the LRR family, was identified both at the N and C termini of collagen VI. Matrilin-1, -3, and -4 were found in complexes with biglycan or decorin at the N terminus. The interactions between collagen VI, biglycan, decorin, and matrilin-1 were studied in detail and revealed a biglycan/matrilin-1 or decorin/matrilin-1 complex acting as a linkage between collagen VI microfibrils and aggrecan or alternatively collagen II. The complexes between matrilin-1 and biglycan or decorin were also reconstituted in vitro. Colocalization of collagen VI and the different ligands in the pericellular matrix of cultured chondrosarcoma cells supported the physiological relevance of the observed interactions in matrix assembly.     

Molecular structure and tissue distribution of matrilin-3, a filament-forming extracellular matrix protein expressed during skeletal development

Matrilin-3 is a recently identified member of the superfamily of proteins containing von Willebrand factor A-like domains and is able to form hetero-oligomers with matrilin-1 (cartilage matrix protein) via a C-terminal coiled-coil domain. Full-length matrilin-3 and a fragment lacking the assembly domain were expressed in 293-EBNA cells, purified, and subjected to biochemical characterization. Recombinantly expressed full-length matrilin-3 occurs as monomers, dimers, trimers, and tetramers, as detected by electron microscopy and SDS-polyacrylamide gel electrophoresis, whereas matrilin-3, purified from fetal calf cartilage, forms homotetramers as well as hetero-oligomers of variable stoichiometry with matrilin-1. In the matrix formed by cultured chondrosarcoma cells, matrilin-3 is found in a filamentous, collagen-dependent network connecting cells and in a collagen-independent pericellular network. Affinity-purified antibodies detect matrilin-3 expression in a variety of mouse cartilaginous tissues, such as sternum, articular, and epiphyseal cartilage, and in the cartilage anlage of developing bones. It is found both inside the lacunae and in the interterritorial matrix of the resting, proliferating, hypertrophic, and calcified cartilage zones, whereas the expression is lower in the superficial articular cartilage. In trachea and in costal cartilage of adult mice, an expression was seen in the perichondrium. Furthermore, matrilin-3 is found in bone, and its expression is, therefore, not restricted to chondroblasts and chondrocytes.     

Gene expression and immunohistochemical localization of biglycan in association with mineralization in the matrix of epiphyseal cartilage

This study has used in situ hybridization, Northern blot analysis, and immunohistochemistry at the light and electron microscope levels to localize mRNAs and core proteins of biglycan in developing tibial epiphyseal cartilage of 10-day old Wistar rats. The expression of mRNAs and core proteins of biglycan appeared prominent in hypertrophic and degenerative chondrocytes associated with the epiphyseal ossification centre and the growth plate cartilage, but was not seen in the rest of epiphyseal cartilage. Northern blot analysis confirmed biglycan mRNA expression in the epiphyseal cartilage. Ultrastructural immunogold cytochemistry of the growth plate revealed that prominent immunolabelling was confined to the Golgi apparatus and cisternae of rough-surfaced endoplasmic reticulum of the hypertrophic and the degenerating chondrocytes, the early mineralized cartilage matrices of the longitudinal septum of the lower hypertrophic and the calcifying zones, and fully mineralized cartilage matrices, which were present in the metaphyseal bone trabeculae. Furthermore, Western blot analysis of biglycan in extracts of fresh epiphyseal cartilage revealed that an EDTA extract, after chondroitinase ABC digestion, contains core proteins of biglycan, indicating the presence of biglycan in mineralized cartilage matrices. These results indicate that the distribution of biglycan is associated with cartilage matrix mineralization.     

Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissues

The messenger RNAs and core proteins of the two small chondroitin/dermatan sulfate proteoglycans, biglycan and decorin, were localized in developing human bone and other tissues by both 35S-labeled RNA probes and antibodies directed against synthetic peptides corresponding to nonhomologous regions of the two core proteins. Biglycan and decorin expression and localization were substantially divergent and sometimes mutually exclusive. In developing bones, spatially restricted patterns of gene expression and/or matrix localization of the two proteoglycans were identified in articular regions, epiphyseal cartilage, vascular canals, subperichondral regions, and periosteum, and indicated the association of each molecule with specific developmental events at specific sites. Study of non-skeletal tissues revealed that decorin was associated with all major type I (and type II) collagen-rich connective tissues. Conversely, biglycan was expressed and localized in a range of specialized cell types, including connective tissue (skeletal myofibers, endothelial cells) and epithelial cells (differentiating keratinocytes, renal tubular epithelia). Biglycan core protein was localized at the cell surface of certain cell types (e.g., keratinocytes). Whereas the distribution of decorin was consistent with matrix-centered functions, possibly related to regulation of growth of collagen fibers, the distribution of biglycan pointed to other function(s), perhaps related to cell regulation.     

Division and death of cells in developing synovial joints and long bones

Articular chondrocytes are a unique set of cells from the time the cellular condensations that become the anlagen of the long bones develop in the embryo. In the presumptive joint the cells of the opposing bones are packed very closely together, but at cavitation, the central, flattened cells move apart to form the articular surfaces. As the articular cartilage develops the cells are pushed further apart by the cartilaginous matrix. To determine the contributions of cell proliferation and death to cavitation and the subsequent development and growth of articular cartilage, direct observations were made to identify mitotic cells and those with apoptotic bodies in haematoxylin-stained sections of developing joints, and growing and ageing articular cartilage of the rabbit knee. These observations were extended using antibodies to the proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP nick end labelling (TUNEL) on corresponding sections. Low levels of cell division do occur in the articular cartilage up to 6 weeks postnatally, but matrix formation makes the major contribution to the increase in size of the cartilage. Cell death is not observed during cavitation, nor during the development of the articular cartilage proper. Apoptosis is essential, however, for the removal of the epiphyseal cartilage during ossification of the epiphyses and in the growth plate.     

Light and electron microscopical immunohistochemical localization of the small proteoglycan core proteins decorin and biglycan in human knee joint cartilage

Summary The distribution of decorin and biglycan was investigated at the light and electron microscopical level in adult human articular cartilage. In general, the amount of decorin and biglycan was found to decrease with the depth of the layer of the cartilage. Decorin was found in the interterritorial matrix where most of the collagen is located. This fits in well with the assumption that decorin may modulate collagen metabolism. Biglycan was found next to the chondrocytes in the pericellular matrix and is assumed to be responsible for cellular activities. At the ultrastructural level, decorin was localized in the interterritorial matrix and in vesicles in chondrocytes. Biglycan was found, usually though not exclusively in the pericellular matrix. Both small proteoglycans were detected close to and on the collagen fibres and also associated with the more globular structures of the matrix between the fibrils. A double-staining approach revealed that the two molecules could be located along the same collagen fibril. However, staining for biglycan and decorin was not observed simultaneously within the same region of the fibre.     

Regional assessment of articular cartilage gene expression and small proteoglycan metabolism in an animal model of osteoarthritis

Osteoarthritis (OA), the commonest form of arthritis and a major cause of morbidity, is characterized by progressive degeneration of the articular cartilage. Along with increased production and activation of degradative enzymes, altered synthesis of cartilage matrix molecules and growth factors by resident chondrocytes is believed to play a central role in this pathological process. We used an ovine meniscectomy model of OA to evaluate changes in chondrocyte expression of types I, II and III collagen; aggrecan; the small leucine-rich proteoglycans (SLRPs) biglycan, decorin, lumican and fibromodulin; transforming growth factor-β; and connective tissue growth factor. Changes were evaluated separately in the medial and lateral tibial plateaux, and were confirmed for selected molecules using immunohistochemistry and Western blotting. Significant changes in mRNA levels were confined to the lateral compartment, where active cartilage degeneration was observed. In this region there was significant upregulation in expession of types I, II and III collagen, aggrecan, biglycan and lumican, concomitant with downregulation of decorin and connective tissue growth factor. The increases in type I and III collagen mRNA were accompanied by increased immunostaining for these proteins in cartilage. The upregulated lumican expression in degenerative cartilage was associated with increased lumican core protein deficient in keratan sulphate side-chains. Furthermore, there was evidence of significant fragmentation of SLRPs in both normal and arthritic tissue, with specific catabolites of biglycan and fibromodulin identified only in the cartilage from meniscectomized joints. This study highlights the focal nature of the degenerative changes that occur in OA cartilage and suggests that altered synthesis and proteolysis of SLRPs may play an important role in cartilage destruction in arthritis.     

Patterns of uronosyl epimerization and 4-/6-0-sulphation in chondroitin/dermatan sulphate from decorin and biglycan of various bovine tissues

Dermatan sulphate is a co-polymer of two types of disac-chariderepeats: D-glucuronate-N-acetylgalactosamine and L-iduronate-N-acetylgalactosamlne.The former can be O-sulphated at C-4 or C-6 of the galactosamine,whereas the latter contains almost exclusively 4-O-sulphatedgalactosamine. A minor proportion of the L-iduronate may beO-sulphated at C-2. Chondroitin sulphate has no L-iduronate-containing repeats. We have used our recently developed methodsfor sequence analysis of galactos-aminoglycans to investigatethe structure of dermatan/ chondroitin sulphates of the proteoglycansdecorin and biglycan derived from various bovine tissues, likederails, sclera, tendon, aorta, cartilage and bone. The glycanchains, radioiodinated at the reducing end, were partially cleavedwith specific enzymes (chondroitin lyases), and subjected tohigh-resolution polyacrylamide gel electrophoresis, blottingand autoradiography to identify fragments extending from thelabelled reducing end to the point of cleavage. We used chondroitinB lyase to identify the location of L-iduronate, chondroitinAC-I lyase to locate D-glucuronate and chondroitin C lyase tocleave where D-glucuronate residues were succeeded by 6-O-sulphatedN-acetylgalactosamine. We could demonstrate tissue-specific,periodic and wave-like patterns of distribution for the twoepimeric uronic acids, as well as specific patterns of sulphationhi dermatan sulphates derived from either decorin or biglycan.For example, some dermatan sulphates contained D-glucuronate-richdomains that were always 6-sulphated (sclera] decorin), otherswere always 4-sulphated (decorin from bovine dermis, cartilageand bone; biglycan from aorta) or 6-sulphated near the linkageregion, but 4-sulphated hi more distal domains (decorin fromporcine dermis and bovine tendon). Decorin from bone and articularcartilage, as well as biglycan from articular and nasal cartilage,carried largely chondroitin sulphate chains, but also some dermatansulphate, whereas galactosaminoglycan chains derived from aggrecanof nasal cartilage were free of L-iduronate. Decorin and biglycanfrom the same tissue (articular cartilage or sclera) had similarglycan chains. The two side chains in a biglycan molecule areprobably also similar to one another. The portion of the glycanchains nearest to the core protein was substituted with chargedgroups to a variable degree, which may correlate with the structuralfeatures of the main chain. biglycan decorin     

Endocytosis of different members of the small chondroitin/dermatan sulfate proteoglycan family.

The family of small interstitial chondroitin/dermatan sulfate proteoglycans consists of at least three different molecular species: biglycan (proteoglycan I), decorin (proteoglycan II), and proteoglycan-100, which has a glycosylated core protein of about 100 kDa. The core protein of decorin has been shown to be responsible for receptor-mediated endocytosis of this proteoglycan species by a variety of mesenchymal cells. It is now demonstrated that skin fibroblasts and articular chondrocytes endocytose biglycan with an efficiency similar to that of decorin. Uptake of biglycan is also mediated by its core protein and can be inhibited by decorin in a partially competitive manner. In human fibroblasts, endosomal proteins of 51 and 26 kDa, which are known to bind decorin core protein, also interact with biglycan. This interaction can be inhibited by decorin. Bovine articular chondrocytes contained binding proteins of 48 and 25 kDa. Proteoglycan-100 can be distinguished from biglycan and decorin by its low clearance rate, which however, exceeds the rate of fluid phase endocytosis.     

Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues

Introduction

The small leucine-rich proteoglycans (SLRPs) modulate tissue organization, cellular proliferation, matrix adhesion, growth factor and cytokine responses, and sterically protect the surface of collagen type I and II fibrils from proteolysis. Catabolism of SLRPs has important consequences for the integrity of articular cartilage and meniscus by interfering with their tissue homeostatic functions.

Methods

SLRPs were dissociatively extracted from articular cartilage from total knee and hip replacements, menisci from total knee replacements, macroscopically normal and fibrillated knee articular cartilage from mature age-matched donors, and normal young articular cartilage. The tissue extracts were digested with chondroitinase ABC and keratanase-I before identification of SLRP core protein species by Western blotting using antibodies to the carboxyl-termini of the SLRPs.

Results

Multiple core-protein species were detected for all of the SLRPs (except fibromodulin) in the degenerate osteoarthritic articular cartilage and menisci. Fibromodulin had markedly less fragments detected with the carboxyl-terminal antibody compared with other SLRPs. There were fewer SLRP catabolites in osteoarthritic hip than in knee articular cartilage. Fragmentation of all SLRPs in normal age-matched, nonfibrillated knee articular cartilage was less than in fibrillated articular cartilage from the same knee joint or total knee replacement articular cartilage specimens of similar age. There was little fragmentation of SLRPs in normal control knee articular cartilage. Only decorin exhibited a consistent increase in fragmentation in menisci in association with osteoarthritis. There were no fragments of decorin, biglycan, lumican, or keratocan that were unique to any tissue. A single fibromodulin fragment was detected in osteoarthritic articular cartilage but not meniscus. All SLRPs showed a modest age-related increase in fragmentation in knee articular and meniscal cartilage but not in other tissues.

Conclusion

Enhanced fragmentation of SLRPs is evident in degenerate articular cartilage and meniscus. Specific decorin and fibromodulin core protein fragments in degenerate meniscus and/or human articular cartilage may be of value as biomarkers of disease. Once the enzymes responsible for their generation have been identified, further research may identify them as therapeutic targets.     

Distribution of biglycan and decorin in collateral and cruciate ligaments and menisci of the rabbit knee joint.

The small leucine-rich proteoglycans (PGs) biglycan and decorin, and their mRNAs, have been localized during neonatal development and aging (3 weeks to 2 years) of collateral and cruciate ligaments and of menisci of the rabbit knee joint. In the collateral ligaments, biglycan and decorin are found between the bundles of collagen fibers at all ages. In cruciate ligaments the PGs are primarily around the cells. In neonatal ligaments all the cells express the mRNAs for biglycan and decorin, but in the collateral ligaments the number expressing the mRNAs is reduced at 8 months. In 3--week menisci the PGs are uniformly distributed in the matrix, but by 8 months biglycan is present primarily in the central fibrocartilaginous regions, whereas decorin is found peripherally. In neonates, all the cells express the mRNAs but the number is reduced in 8-month menisci. The results illustrate the precise localizations of biglycan and decorin in healthy rabbit ligaments and menisci which, after injury, must be reproduced in the repair tissue for normal strength to be regained. (J Histochem Cytochem 49:877-885, 2001)     

Identification and characterization of asporin. a novel member of the leucine-rich repeat protein family closely related to decorin and biglycan

Asporin, a novel member of the leucine-rich repeat family of proteins, was partially purified from human articular cartilage and meniscus. Cloning of human and mouse asporin cDNAs revealed that the protein is closely related to decorin and biglycan. It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to decorin and biglycan, asporin is not a proteoglycan. Instead, asporin contains a unique stretch of aspartic acid residues in its amino-terminal region. A polymorphism was identified in that the number of consecutive aspartate residues varied from 11 to 15. The 8 exons of the human asporin gene span 26 kilobases on chromosome 9q31.1-32, and the putative promoter region lacks TATA consensus sequences. The asporin mRNA is expressed in a variety of human tissues with higher levels in osteoarthritic articular cartilage, aorta, uterus, heart, and liver. The deduced amino acid sequence of asporin was confirmed by mass spectrometry of the isolated protein resulting in 84% sequence coverage. The protein contains an N-glycosylation site at Asn(281) with a heterogeneous oligosaccharide structure and a potential O-glycosylation site at Ser(54). The name asporin reflects the aspartate-rich amino terminus and the overall similarity to decorin.     

Changes in leucine-rich repeat proteoglycans during maturation of the bovine growth plate

The primary growth plate of the fetal bovine tibia was studied in order to determine whether changes in the structure, abundance and expression of the leucine-rich repeat proteoglycans were occurring during tissue maturation from reserve cartilage to hypertrophic cartilage. The proteoglycans under study were decorin, biglycan, fibromodulin and lumican. Decorin was readily detectable in both the reserve and proliferating zones of the growth plate, but its abundance decreased markedly in the zones of maturation and hypertrophy where it could not be detected under the same conditions of analysis. In contrast to decorin, fibromodulin and biglycan could be detected throughout the growth plate, though their abundance was decreased in the proliferative and hypertrophic zones. Unlike the other proteoglycans, lumican could not be detected throughout the growth plate. At the message level, the expression of decorin shows a similar trend to that of protein abundance in the extracellular matrix, with its expression dropping markedly in the proliferative and hypertrophic zones. In the case of both biglycan and fibromodulin, message expression continued at a similar level throughout the growth plate. Thus, the leucine-rich repeat proteoglycans are different in the way they behave during growth plate maturation.     

The macromolecular characteristics of cartilage proteoglycans do not change when synthesis is up-regulated by link protein peptide.

Previous studies have shown that a synthetic, unglycosylated analogue of the N-terminal peptide from link protein can function as a growth factor and up-regulate proteoglycan biosynthesis in explant cultures of normal human articular cartilage from a wide age range of subjects (McKenna et al., Arthritis Rheum. 41 (1998) 157-162). The present work further shows that link peptide increased proteoglycan synthesis by cartilage cultured in both the presence and absence of serum, suggesting that the mechanism of up-regulation may be different from that of insulin-like growth factors. The proteoglycans synthesised during stimulation with link peptide were of normal hydrodynamic size and the ratio of core protein to glycosaminoglycan side chains and the proportions of the large proteoglycan aggrecan to the small proteoglycans, decorin and biglycan, remained constant. Aggrecan molecules were equally capable of forming aggregates as those from control tissues and the relative proportions of decorin and biglycan were unchanged showing that both were co-ordinately up-regulated. These results confirmed that this novel peptide is a potent stimulator of proteoglycan synthesis by articular cartilage and showed that the newly synthesised proteoglycans were of normal composition.     

The glucuronyl C5-epimerase activity is the limiting factor in the dermatan sulfate biosynthesis.

An early step in the biosynthesis of dermatan sulfate is polymerization to chondroitin, which then is modified by the D-glucuronyl C5-epimerase and mainly 4-O-sulfotransferase. The final structure of the dermatan sulfate side chains varies and our aim was to identify, which of the two enzymes that are crucial to generate dermatan sulfate copolymeric structures in tissues. Dermatan sulfate side chains of biglycan and decorin were prepared from fibroblasts and nasal and articular chondrocytes and characterized regarding detailed structure. Microsomes were prepared from these cells and the activities of D-glucuronyl C5-epimerase and 4-O-sulfotransferase were determined. Chondrocytes from nasal cartilage synthesized biglycan and decorin containing 10%, articular chondrocytes 20--30%, and fibroblast 80% of the uronosyl residues in the l-iduronyl configuration. All three tissues contained high amount of 4-O-sulfotransferase activity. The activity of d-glucuronyl C5-epimerase showed different relationships. Fibroblasts contained a high level of the epimerase activity, articular chondrocytes intermediary activity, and in nasal cartilage it was barely detectable. The data indicate that the activity of the d-glucuronyl C5-epimerase is the main factor for formation of dermatan sulfate in tissues.     

The distribution patterns of COMP and matrilin-3 in septal,alar and triangular cartilages of the human nose

The biomechanical characteristics of septal cartilage depend strongly on the distinct extracellular matrix of cartilage tissue; therefore, it is essential that the components of this matrix are identified and understood. Cartilage oligomeric matrix protein (COMP) and matrilin-3 are localised in articular cartilage. This study was the first to examine all subtypes of mature human nasal cartilages (alar, triangular and septal) with specific attention to the distribution of COMP and matrilin-3. Three whole fresh-frozen noses from human donors were dissected, and exemplary biopsies were examined using histochemical staining (haematoxylin and eosin and Alcian blue) and immunohistochemistry (collagen II, COMP and matrilin-3). The following three zones within the nasal cartilage were identified: superficial, intermediate and central. COMP was detected as highest in the intermediate zones in all three subtypes of nasal cartilage, whereas matrilin-3 was detected with pericellular deposition mainly within septal cartilage predominantly in the superficial zones. The distinct staining patterns of COMP and matrilin-3 underscore the different functional roles of both proteins in nasal cartilage. According to the literature, COMP might be involved with collagen II in the formation of networks, whereas matrilin-3 is reported to prevent ossification or regulate mechanosensitivity. The predominant staining observed in septal cartilage suggests matrilin-3’s modulatory role because of its presence in the osteochondral junctional zone and given that the biomechanical load in septal cartilage is different from that in alar or triangular cartilage. In conclusion, COMP and matrilin-3 were detected in mature human nasal cartilage but displayed different staining patterns that might be explained by the functional roles of the respective matrix protein; however, further research is necessary to identify and define the functional aspects of this morphological difference.     

Systemic hypoxia alters gene expression levels of structural proteins and growth factors in knee joint cartilage

We investigated the effects of short- (8- and 24-h) and long-term (3 weeks) exposure to systemic normobaric hypoxia (13%) on the gene expression level of structural proteins and growth factors in knee joint cartilage of rabbits. Collagen type Ia2, II, and Va1, TGF-beta1, and b-FGF were upregulated after short-term hypoxia in both menisci, but not in articular cartilage. In contrast, long-term hypoxia downregulated gene expression level of collagens, aggrecan, and growth factors in articular cartilage and meniscal fibrocartilage. Interestingly, gene expression levels of non-collagenous proteins biglycan, decorin, and versican were not affected by short-term or by long-term hypoxia in knee joint cartilage. The present study suggests that changes in oxygen level differentially affect gene expression levels of growth factors, collagens, and non-collagenous proteins in normal knee joint cartilage in rabbits.