Interactions between the cartilage oligomeric matrix protein and matrilins. Implications for matrix assembly and the pathogenesis of chondrodysplasias

The cartilage oligomeric matrix protein (COMP) and matrilins are abundant non-collagenous proteins in the cartilage extracellular matrix. In the presence of calcium, COMP and matrilin-1 elute together in the gel filtration of cartilage extracts and can be co-immunoprecipitated. In a screen for ligands of matrilin-1, -3, and -4 using an ELISA-style binding assay, COMP was identified as a prominent binding partner for all three, indicating a conservation of the COMP interaction among matrilins. The interaction of COMP and matrilin-4 is saturable, and an apparent K(D) of 1 nm was determined. However, only the full-length COMP and the full-length matrilin-4 proteins showed a strong interaction, indicating that the oligomeric structures markedly increase the affinity. Mutations in COMP or matrilin-3 cause related forms of human chondrodysplasia, and the COMP mutation D469Delta, which is found in patients with pseudoachondroplasia, has been shown to cause a reduced calcium binding. Despite this, the mutation causes only a slight decrease in matrilin-4 binding. This indicates that impaired binding of COMP to matrilins does not cause the pseudoachondroplasia phenotype but rather that matrilins may be coretained in the rough endoplasmatic reticulum where COMP accumulates in the chondrocytes of patients.     

Expression of matrilin-1, -2 and -3 in developing mouse limbs and heart.

The expression of matrilin-1, -2 and -3 was studied in the heart and limb during mouse development. Matrilin-1 is transiently expressed in the heart between days 9.5 and 14.5 p.c. Matrilin-2 expression was detected in the heart from day 10.5 p.c. onwards. In the developing limb bud, both matrilin-1 and -3 were observed first at day 12.5 p.c. Throughout development matrilin-3 expression was strictly limited to cartilage, while matrilin-1 was also found in some other forms of connective tissue. Matrilin-2, albeit present around hypertrophic chondrocytes in the growth plate, was mainly expressed in non-skeletal structures. The complementary, but in part overlapping, expression of matrilins indicates the possibility for both redundant and unique functions among the members of this novel family of extracellular matrix proteins.     

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.     

The matrilins: modulators of extracellular matrix assembly

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.     

Abnormal collagen fibrils in cartilage of matrilin-1/matrilin-3-deficient mice

Matrilins are oligomeric extracellular matrix adaptor proteins mediating interactions between collagen fibrils and other matrix constituents. All four matrilins are expressed in cartilage and mutations in the human gene encoding matrilin-3 (MATN3) are associated with different forms of chondrodysplasia. Surprisingly, however, Matn3-null as well as Matn1- and Matn2-null mice do not show an overt skeletal phenotype, suggesting a dominant negative pathomechanism for the human disorders and redundancy/compensation among the family members in the knock-out situation. Here, we show that mice lacking both matrilin-1 and matrilin-3 develop an apparently normal skeleton, but exhibit biochemical and ultrastructural abnormalities of the knee joint cartilage. At the protein level, an altered SDS-PAGE band pattern and a clear up-regulation of the homotrimeric form of matrilin-4 were evident in newborn Matn1/Matn3 and Matn1 knock-out mice, but not in Matn3-null mice. The ultrastructure of the cartilage matrix after conventional chemical fixation was grossly normal; however, electron microscopy of high pressure frozen and freeze-substituted samples, revealed two consistent observations: 1) moderately increased collagen fibril diameters throughout the epiphysis and the growth plate in both single and double mutants; and 2) increased collagen volume density in Matn1(-/-)/Matn3(-/-) and Matn3(-/-) mice. Taken together, our results demonstrate that matrilin-1 and matrilin-3 modulate collagen fibrillogenesis in cartilage and provide evidence that biochemical compensation might exist between matrilins.     

Altered integration of matrilin-3 into cartilage extracellular matrix in the absence of collagen IX

The matrilins are a family of four noncollagenous oligomeric extracellular matrix proteins with a modular structure. Matrilins can act as adapters which bridge different macromolecular networks. We therefore investigated the effect of collagen IX deficiency on matrilin-3 integration into cartilage tissues. Mice harboring a deleted Col9a1 gene lack synthesis of a functional protein and produce cartilage fibrils completely devoid of collagen IX. Newborn collagen IX knockout mice exhibited significantly decreased matrilin-3 and cartilage oligomeric matrix protein (COMP) signals, particularly in the cartilage primordium of vertebral bodies and ribs. In the absence of collagen IX, a substantial amount of matrilin-3 is released into the medium of cultured chondrocytes instead of being integrated into the cell layer as in wild-type and COMP-deficient cells. Gene expression of matrilin-3 is not affected in the absence of collagen IX, but protein extraction from cartilage is greatly facilitated. Matrilin-3 interacts with collagen IX-containing cartilage fibrils, while fibrils from collagen IX knockout mice lack matrilin-3, and COMP-deficient fibrils exhibit an intermediate integration. In summary, the integration of matrilin-3 into cartilage fibrils occurs both by a direct interaction with collagen IX and indirectly with COMP serving as an adapter. Matrilin-3 can be considered as an interface component, capable of interconnecting macromolecular networks and mediating interactions between cartilage fibrils and the extrafibrillar matrix.     

Matrilins mediate weak cell attachment without promoting focal adhesion formation.

The matrilins form a family of non-collagenous adaptor proteins in the extracellular matrix. The extracellular ligand interactions of matrilins have been studied in some detail, while the potential interplay between matrilins and cells has been largely neglected. Except for matrilin-4, all matrilins mediate cell attachment, but only for matrilin-1 and -3 the binding is clearly dose dependent and seen already at moderate coating concentrations. Even so, much higher concentrations of matrilin-1 or -3 than of fibronectin are required for cell attachment to reach plateau values. Integrins contribute to the matrilin-mediated cell attachment, but the binding does not lead to formation of focal contacts and reorganisation of the actin cytoskeleton. Cells deficient in beta1 integrins are able to adhere, although weaker, and matrilins do not bind the soluble integrin alpha1beta1 and alpha2beta1 ectodomains. Cell surface proteoglycans may promote the attachment, as cells deficient in glycosaminoglycan biosynthesis adhere less well to matrilin-3. Even so, exogenous glycosaminoglycans are not able to compete for the attachment of HaCaT cells to matrilins.     

The matrilins--adaptor proteins in the extracellular matrix

The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage but also in many other forms of extracellular matrix. They participate in the formation of fibrillar or filamentous structures and are often associated with collagens. It appears that they mediate interactions between collagen-containing fibrils and other matrix constituents, such as aggrecan. This adaptor function may be modulated by physiological proteolysis that causes the loss of single subunits and thereby a decrease in binding avidity. Attempts to study matrilin function by gene inactivation in mouse have been frustrating and so far not yielded pronounced phenotypes, presumably because of the extensive redundancy within the family allowing compensation by one family member for another. However, mutations in matrilin-3 in humans cause different forms of chondrodysplasias and perhaps also hand osteoarthritis. As loss of matrilin-3 is not critical in mouse, these phenotypes are likely to be caused by dominant negative effects.     

Ablation of collagen IX and COMP disrupts epiphyseal cartilage architecture.

Chondrodysplasias are a genetically heterogeneous group of skeletal disorders. Mutations in genes coding for cartilage oligomeric matrix protein (COMP), collagen IX and matrilin-3 have been described to cause the autosomal dominantly inherited form of multiple epiphyseal dysplasia (MED). Even though there is clear evidence that these cartilage matrix proteins interact with each other, their exact functions in matrix organisation and bone development still need to be elucidated. We generated a mouse model lacking both collagen IX and COMP to study the potential complementary role of these proteins in skeletal development. Mice deficient in both proteins exhibit shortened and widened long bones as well as an altered bone structure. They display severe growth plate abnormalities with large hypocellular areas in the central parts of the tibia. In addition, chondrocytes in the proliferative and hypertrophic zones do not show their typical columnar arrangement. These phenotypical traits were not observed in mice deficient only in COMP, while mice lacking only collagen IX showed similar growth plate disturbances and shorter and wider tibiae. The contribution of COMP to the phenotype of mice deficient in both collagen IX and COMP appears minor, even though clear differences in the deposition of matrilin-3 were detected.     

The matrilins: a novel family of oligomeric extracellular matrix proteins.

The matrilin family at present has four members that all share a structure made up of von Willebrand factor A domains, epidermal growth factor-like domains and a coiled coil alpha-helical module. The first member of the family, matrilin-1 (previously called cartilage matrix protein or CMP), is expressed mainly in cartilage. Matrilin-3 has a similar tissue distribution, while matrilin-2 and -4 occur in a wide variety of extracellular matrices. Matrilin-1 is associated with cartilage proteoglycans as well as being a component of both collagen-dependent and collagen-independent fibrils and on the basis of the related structures other matrilins may play similar roles. The matrilin genes are strictly and differently regulated and their expression may serve as markers for cellular differentiation.     

Mice lacking the extracellular matrix adaptor protein matrilin-2 develop without obvious abnormalities.

Matrilins are putative adaptor proteins of the extracellular matrix (ECM) which can form both collagen-dependent and collagen-independent filamentous networks. While all known matrilins (matrilin-1, -2, -3, and -4) are expressed in cartilage, only matrilin-2 and matrilin-4 are abundant in non-skeletal tissues. To clarify the biological role of matrilin-2, we have developed a matrilin-2-deficient mouse strain. Matrilin-2 null mice show no gross abnormalities during embryonic or adult development, are fertile, and have a normal lifespan. Histological and ultrastructural analyses indicate apparently normal structure of all organs and tissues where matrilin-2 is expressed. Although matrilin-2 co-localizes with matrilin-4 in many tissues, Northern hybridization, semiquantitative RT-PCR, immunohistochemistry and biochemical analysis reveal no significant alteration in the steady-state level of matrilin-4 expression in homozygous mutant mice. Immunostaining of wild-type and mutant skin samples indicate no detectable differences in the expression and deposition of matrilin-2 binding partners including collagen I, laminin-nidogen complexes, fibrillin-2 and fibronectin. In addition, electron microscopy reveals an intact basement membrane at the epidermal-dermal junction and normal organization of the dermal collagen fibrils in mutant skin. These data suggest that either matrilin-2 and matrilin-2-mediated matrix-matrix interactions are dispensable for proper ECM assembly and function, or that they are efficiently compensated by other matrix components including wild-type levels of matrilin-4.     

Matrilin-3 is dispensable for mouse skeletal growth and development

Matrilin-3 belongs to the matrilin family of extracellular matrix (ECM) proteins and is primarily expressed in cartilage. Mutations in the gene encoding human matrilin-3 (MATN-3) lead to autosomal dominant skeletal disorders, such as multiple epiphyseal dysplasia (MED), which is characterized by short stature and early-onset osteoarthritis, and bilateral hereditary microepiphyseal dysplasia, a variant form of MED characterized by pain in the hip and knee joints. To assess the function of matrilin-3 during skeletal development, we have generated Matn-3 null mice. Homozygous mutant mice appear normal, are fertile, and show no obvious skeletal malformations. Histological and ultrastructural analyses reveal endochondral bone formation indistinguishable from that of wild-type animals. Northern blot, immunohistochemical, and biochemical analyses indicated no compensatory upregulation of any other member of the matrilin family. Altogether, our findings suggest functional redundancy among matrilins and demonstrate that the phenotypes of MED disorders are not caused by the absence of matrilin-3 in cartilage ECM.     

An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia

Multiple epiphyseal dysplasia (MED) can result from mutations in matrilin-3, a structural protein of the cartilage extracellular matrix. We have previously shown that in a mouse model of MED the tibia growth plates were normal at birth but developed a progressive dysplasia characterised by the intracellular retention of mutant matrilin-3 and abnormal chondrocyte morphology. By 3 weeks of age, mutant mice displayed a significant decrease in chondrocyte proliferation and dysregulated apoptosis. The aim of this current study was to identify the initial post-natal stages of the disease. We confirmed that the disease phenotype is seen in rib and xiphoid cartilage and, like tibia growth plate cartilage is characterised by the intracellular retention of mutant matrilin-3. Gene expression profiling showed a significant activation of classical unfolded protein response (UPR) genes in mutant chondrocytes at 5 days of age, which was still maintained by 21 days of age. Interestingly, we also noted the upregulation of arginine-rich, mutated in early stage of tumours (ARMET) and cysteine-rich with EGF-like domain protein 2 (CRELD2) are two genes that have only recently been implicated in the UPR. This endoplasmic reticulum (ER) stress and UPR did not lead to increased chondrocyte apoptosis in mutant cartilage by 5 days of age. In an attempt to alleviate ER stress, mutant mice were fed with a chemical chaperone, 4-sodium phenylbutyrate (SPB). SPB at the dosage used had no effect on chaperone expression at 5 days of age but modestly decreased levels of chaperone proteins at 3 weeks. However, this did not lead to increased secretion of mutant matrilin-3 and in the long term did not improve the disease phenotype. We performed similar studies with a mouse model of Schmid metaphyseal chondrodysplasia, but again this treatment did not improve the phenotype.     

Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms

The matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in matrilin-1 and particularly complex in matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in matrilin-4 by the matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not matrilin-1 subunits present in matrilin-1/-3 hetero-oligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of matrilin-4 detected processed matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo.The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage and many other forms of extracellular matrix (for review, see Ref. 1). They participate in the formation of fibrillar or filamentous structures (2–7) and mediate interactions between collagen-containing fibrils (8, 9) and other matrix constituents like aggrecan (10), small leucine-rich proteoglycans (9), or COMP (11). Matrilins form homo- and hetero-oligomers by their C-terminal coiled-coil domain. In addition, the subunits contain epidermal growth factor-like and von Willebrand factor A (VWA)²-like domains, where the latter are presumably the major ligand binding domains (11). Mutations in matrilin-3 in humans cause different forms of chondrodysplasia (12–14) and are also linked to the development of hand osteoarthritis (15) and intervertebral disc degeneration (16).Proteolytic processing of extracellular matrix proteins plays both physiological and pathophysiological roles. Proteolysis is a major post-translational modification used to modify the function of proteins. Tissue homeostasis requires a well balanced synthesis and degradation of extracellular matrix proteins, specifically mediated by protease families like matrix metalloproteinases (17), ADAMs (18), or ADAMTSs (19). The development of degenerative diseases is often accompanied by an activation of such proteases. In addition, the cleavage sometimes releases protein fragments that have completely new functions (20, 21).Determination of which extracellular proteases cleave which substrates is crucial to understand the physiological function of both (22). Physiological cleavage has been described for most members of the matrilin family (4–6), but was not yet extensively studied. The adapter function of the matrilins may be modulated by physiological proteolysis that causes the loss of single subunits and thereby decreases the binding avidity (5). Interestingly, an earlier identified cleavage site in the hinge region of matrilin-4, N-terminal of the coiled-coil, is conserved throughout the matrilin family (5) and it was recently shown that matrilin-3 is cleaved by ADAMTS4 in vitro at this site (23). Here we studied matrilin processing in some detail and identified another member of the ADAMTS family, ADAMTS5, as being able to cleave matrilin-3 and -4. Such cleavage is likely to alter the cohesion of the extracellular matrix.     

Comparison of age-dependent expression of aggrecan and ADAMTSs in mandibular condylar cartilage, tibial growth plate, and articular cartilage in rats

A disintegrin and metalloproteinase with thrombospondin motif (adamalysin–thrombospondins, ADAMTS) degrades aggrecan, one of the major extracellular matrix (ECM) components in cartilage. Mandibular condylar cartilage differs from primary cartilage, such as articular and growth plate cartilage, in its metabolism of ECM, proliferation, and differentiation. Mandibular condylar cartilage acts as both articular and growth plate cartilage in the growing period, while it remains as articular cartilage after growth. We hypothesized that functional and ECM differences between condylar and primary cartilages give rise to differences in gene expression patterns and levels of aggrecan and ADAMTS-1, -4, and -5 during growth and aging. We employed in situ hybridization and semiquantitative RT-PCR to identify mRNA expression for these molecules in condylar cartilage and primary cartilages during growth and aging. All of the ADAMTSs presented characteristic, age-dependent expression patterns and levels among the cartilages tested in this study. ADAMTS-5 mainly contributed to ECM metabolism in growth plate and condylar cartilage during growth. ADAMTS-1 and ADAMTS-4 may be involved in ECM turn over in articular cartilage. The results of the present study reveal that ECM metabolism and expression of related proteolytic enzymes in primary and secondary cartilages may be differentially regulated during growth and aging.     

COMP mutations, chondrocyte function and cartilage matrix

Cartilage oligomeric matrix protein (COMP) is a large extracellular pentameric glycoprotein found in the territorial matrix surrounding chondrocytes. More than 60 unique COMP mutations have been identified as causing two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1). Recent studies demonstrate that calcium-binding and calcium induced protein folding differ between wild type and mutant COMP proteins and abnormal processing of the mutant COMP protein causes the characteristic large lamellar appearing rough endoplasimic reticulum (rER) cisternae phenotype observed in PSACH and EDMI growth plate chondrocytes. To understand the cellular events leading to this intracellular phenotype, PSACH chondrocytes with a G427E, D469del and D511Y mutations were grown in 3-D culture to produce cartilage nodules. Each nodule was assessed for the appearance and accumulation of cartilage-specific proteins within the rER and for matrix protein synthesis. All three COMP mutations were associated with accumulation of COMP in the rER cisternae by 4 weeks in culture, and by 8 weeks the majority of chondrocytes had the characteristic cellular phenotype. Mutations in COMP also affect the secretion of type IX collagen and matrilin-3 (MATN3) but not the secretion of aggrecan and type II collagen. COMP, type IX collagen and MATN3 were dramatically reduced in the PSACH matrices, and the distribution of these proteins in the matrix was diffuse. Ultrastructural analysis shows that the type II collagen present in the PSACH matrix does not form organized fibril bundles and, overall, the matrix is disorganized. The combined absence of COMP, type IX collagen and MATN3 causes dramatic changes in the matrix and suggests that these proteins play important roles in matrix assembly.     

Distinguishing aggrecan loss from aggrecan proteolysis in ADAMTS-4 and ADAMTS-5 single and double deficient mice

Aggrecan loss from mouse cartilage is predominantly because of ADAMTS-5 activity; however, the relative contribution of other proteolytic and nonproteolytic processes to this loss is not clear. This is the first study to compare aggrecan loss with aggrecan processing in mice with single and double deletions of ADAMTS-4 and -5 activity (Deltacat). Cartilage explants harvested from single and double ADAMTS-4 and -5 Deltacat mice were cultured with or without interleukin (IL)-1alpha or retinoic acid and analyzed for (i) the kinetics of (35)S-labeled aggrecan loss, (ii) the pattern of (35)S-labeled aggrecan fragments released into the media and retained in the matrix, (iii) the pattern of total aggrecan fragments released into the media and retained in the matrix, and (iv) specific cleavage sites within the interglobular and chondroitin sulfate-2 domains. The loss of radiolabeled aggrecan from ADAMTS-4/-5 Deltacat cartilage was less than that from ADAMTS-4, ADAMTS-5, or wild-type cartilage under nonstimulated conditions. IL-1alpha and retinoic acid stimulated radiolabeled aggrecan loss from wild-type and ADAMTS-4 Deltacat cartilage, but there was little effect on ADAMTS-5 cartilage. Proteolysis of aggrecan contributed most to its loss in wild-type, ADAMTS-4, and ADAMTS-5 Deltacat cartilage explants. The pattern of proteolytic processing of aggrecan in these cultures was consistent with that occurring in cartilage pathologies. Retinoic acid, but not IL-1alpha, stimulated radiolabeled aggrecan loss from ADAMTS-4/-5 Deltacat cartilage explants. Even though there was a 300% increase in aggrecan loss from ADAMTS-4/-5 Deltacat cartilage stimulated with retinoic acid, the loss was not associated with aggrecanase cleavage but with the release of predominantly intact aggrecan consistent with the phenotype of the ADAMTS-4/-5 Deltacat mouse. Our results show that chondrocytes have additional mechanism for the turnover of aggrecan and that when proteolytic mechanisms are blocked by ablation of aggrecanase activity, nonproteolytic mechanisms compensate to maintain cartilage homeostasis.     

Identification of an ADAMTS-4 cleavage motif using phage display leads to the development of fluorogenic peptide substrates and reveals matrilin-3 as a novel substrate

ADAMTS-4 and ADAMTS-5 are aggrecanases responsible for the breakdown of cartilage aggrecan in osteoarthritis. Multiple ADAMTS-4 cleavage sites have been described in several matrix proteins including aggrecan, versican, and brevican, but no concise predictive cleavage motif has been identified for this protease. By screening a 13-mer peptide library with a diversity of 10(8), we have identified the ADAMTS-4 cleavage motif E-(AFVLMY)-X(0,1)-(RK)-X(2,3)-(ST)-(VYIFWMLA), with Glu representing P1. Several 13-mer peptides containing this motif, including DVQEFRGVTAVIR and HNEFRQRETYMVF, were shown to be substrates for ADAMTS-4. These peptides were found to be specific substrates for ADAMTS-4 as they were not cleaved by ADAMTS-5. Modification of these peptides with donor (6-FAM) and acceptor (QSY-9) molecules resulted in the development of fluorescence-based substrates with a Km of approximately 35 microM. Furthermore, the role of Glu at P1 and Phe at P1' in binding and catalysis was studied by exploring substitution of these amino acids with the D-isomeric forms. Substitution of P1 with dGlu was tolerable for binding, but not catalysis, whereas substitution of P1' with dPhe precluded both binding and catalysis. Similarly, replacement of Glu with Asp at P1 abolished recognition and cleavage of the peptide. Finally, BLAST results of the ADAMTS-4 cleavage motif identified matrilin-3 as a new substrate for ADAMTS-4. When tested, recombinant ADAMTS-4 effectively cleaved intact matrilin-3 at the predicted motif at Glu435/Ala436 generating two species of 45 and 5 kDa.     

MiR-26a modulates extracellular matrix homeostasis in cartilage

MicroRNAs (miRNAs) may represent new therapeutic targets for bone and joint diseases. We hypothesized that several cartilage-specific proteins are targeted by a single miRNA and used bioinformatics to identify a miRNA that can modulate extracellular matrix (ECM) homeostasis in cartilage.Bioinformatic analysis of miRNA binding sequences in the 3′-untranslated region (3′-UTR) of target genes was performed to identify a miRNA that could bind to the 3′-UTR of cartilage matrix-related genes. MiRNA expression was studied by quantitative PCR of microdissected growth plate cartilage and binding to the 3′-UTR sequences was analyzed by luciferase interaction studies. Levels of proteins encoded by target genes in cultures of miR-26a mimic- or inhibitor-transfected chondrocytes were determined by FACS or immunoblot analysis.The complementary binding sequence of miR-26a and miR-26b was found in the 3′-UTR of the prehypertrophic/hypertrophic-specific genes Cd200, Col10a1 as well as Col9a1 and Ctgf. Both miRNAs were expressed in cartilage and only miR-26a was downregulated in hypertrophic growth plate cartilage. MiR-26a could interact with the 3′-UTR of Cd200 and Col10a1 in luciferase binding studies, but not with Col9a1 and Ctgf. However, protein expression of target genes and the ECM adaptor genes matrilin-3 and COMP was significantly altered in miR-26a mimic- or inhibitor-transfected chondrocytes, whereas the abundance of the cell surface receptor for insulin was not changed. In conclusion, miR-26a suppresses hypertrophic and ECM adaptor protein production. Dysregulation of miR-26a expression could contribute to ECM changes in cartilage diseases and this miRNA may therefore act as a therapeutic target.