Potential roles for the small leucine-rich proteoglycans biglycan and fibromodulin in ectopic ossification of tendon induced by exercise and in modulating rotarod performance.

We present a detailed comparison of ectopic ossification (EO) found in tendons of biglycan (Bgn), fibromodulin (Fmod) single and double Bgn/Fmod-deficient (DKO) mice with aging. At 3 months, Fmod KO, Bgn KO and DKO displayed torn cruciate ligaments and EO in their quadriceps tendon, menisci and cruciate and patellar ligaments. The phenotype was the least severe in the Fmod KO, intermediate in the Bgn KO and the most severe in the DKO. This condition progressed with age in all three mouse strains and resulted in the development of large supernumerary sesmoid bones. To determine the role of exercise in the extent of EO, we subjected normal and DKO mice to a treadmill exercise 3 days a week for 4 weeks. In contrast to previous findings using more rigorous exercise regimes, the EO in moderately exercised DKO was decreased compared with unexercised DKO mice. Finally, DKO and Bgn KO mice tested using a rotarod showed a reduced ability to maintain their grip on a rotating cylinder compared with wild-type controls. In summary, we show (1) a detailed description of EO formed by Bgn, Fmod or combined depletion, (2) the role of exercise in modulating EO and (3) that Bgn and Fmod are critical in controlling motor function.

Fibromodulin-null murine knee joints display increased incidences of osteoarthritis and alterations in tissue biochemistry.

OBJECTIVE: To elucidate variations in tissue ultrastructure and incidence of pathology between fibromodulin (FM)-null mice and wild-type (WT) animals. DESIGN: FM-null and WT siblings from different age groups were compared. Serial sections were made through paraffin-embedded whole knees and investigated histologically. Additionally, medial femoral condyle peaks from sibling pairs were investigated ultrastructurally using transmission electron microscopy. RESULTS: Histological findings demonstrated a clear and increasing disparity between tissue degeneration in WT and FM-null animal knees with progressing age. Distinct differences were apparent by 36 weeks. Around the 80 week period and onward these differences became profound. However, qualitative ultrastructural investigation did not indicate either any aberrant tissue ultrastructure or any abnormal collagen fibril forms in FM-null articular cartilage compared with WT. Biochemical and immunohistochemical investigation of FM-null articular cartilage showed a significant increase in tissue levels of lumican (LUM). Conversely, the cruciate ligaments of the knee showed both an increase in LUM content and considerable structural abnormalities including the tendency towards rupture. CONCLUSION: This report indicates for the first time that FM-null mice have a higher propensity towards degenerative changes in their knee joints than comparable WT animals. Interestingly, no underlying ultrastructural or fibril abnormalities within the articular cartilage could be identified to explain why FM-null cartilage is more prone to pathological changes than wild-type tissue. We conclude that alterations in ligaments, and possibly other tissues within the knee, are of considerable importance in the pathogenesis of the observed articular cartilage degeneration.

Biglycan and decorin bind close to the n-terminal region of the collagen VI triple helix.

The binding of native biglycan and decorin to pepsin-extracted collagen VI from human placenta was examined by solid phase assay and by measurement of surface plasmon resonance in the BIAcore(TM)2000 system. Both proteoglycans exhibited a strong affinity for collagen VI with dissociation constants (K(D)) of approximately 30 nm. Removal of the glycosaminoglycan chains by chondroitinase ABC digestion did not significantly affect binding. In coprecipitation experiments, biglycan and decorin bound to collagen VI and equally competed with the other, suggesting that biglycan and decorin bind to the same binding site on collagen VI. This was confirmed by electron microscopy after negative staining of complexes between gold-labeled proteoglycans and collagen VI, demonstrating that both biglycan and decorin bound exclusively to a domain close to the interface between the N terminus of the triple helical region and the following globular domain. In solid phase assay using recombinant collagen VI fragments, it was shown that the alpha2(VI) chain probably plays a role in the interaction.

GDF-5 deficiency in mice leads to disruption of tail tendon form and function.

Although the biological factors which regulate tendon homeostasis are poorly understood, recent evidence suggests that Growth and Differentiation Factor-5 (GDF-5) may play a role in this important process. The purpose of this study was to investigate the effect of GDF-5 deficiency on mouse tail tendon using the brachypodism mouse model. We hypothesized that GDF-5 deficient tail tendon would exhibit altered composition, ultrastructure, and biomechanical behavior when compared to heterozygous control littermates. Mutant tail tendons did not display any compositional differences in sulfated glycosaminoglycans (GAG/DNA), collagen (hydroxyproline/DNA), or levels of fibromodulin, decorin, or lumican. However, GDF-5 deficiency did result in a 17% increase in the proportion of medium diameter (100-225 nm) collagen fibrils in tail tendon (at the expense of larger fibrils) when compared to controls (p < 0.05). Also, mutants exhibited a trend toward an increase in irregularly-shaped polymorphic fibrils (33% more, p > 0.05). While GDF-5 deficient tendon fascicles did not demonstrate any significant differences in quasistatic biomechanical properties, mutant fascicles relaxed 11% more slowly than control tendons during time-dependent stress-relaxation tests (p < 0.05). We hypothesize that this subtle alteration in time-dependent mechanical behavior is most-likely due to the increased prevalence of irregularly shaped type I collagen fibrils in the mutant tail tendons. These findings provide additional evidence to support the conclusion that GDF-5 may play a role in tendon homeostasis in mice.

Differential expression of lumican and fibromodulin regulate collagen fibrillogenesis in developing mouse tendons.

Collagen fibrillogenesis is finely regulated during development of tissue-specific extracellular matrices. The role(s) of a leucine-rich repeat protein subfamily in the regulation of fibrillogenesis during tendon development were defined. Lumican-, fibromodulin-, and double-deficient mice demonstrated disruptions in fibrillogenesis. With development, the amount of lumican decreases to barely detectable levels while fibromodulin increases significantly, and these changing patterns may regulate this process. Electron microscopic analysis demonstrated structural abnormalities in the fibrils and alterations in the progression through different assembly steps. In lumican-deficient tendons, alterations were observed early and the mature tendon was nearly normal. Fibromodulin-deficient tendons were comparable with the lumican-null in early developmental periods and acquired a severe phenotype by maturation. The double-deficient mice had a phenotype that was additive early and comparable with the fibromodulin-deficient mice at maturation. Therefore, lumican and fibromodulin both influence initial assembly of intermediates and the entry into fibril growth, while fibromodulin facilitates the progression through growth steps leading to mature fibrils. The observed increased ratio of fibromodulin to lumican and a competition for the same binding site could mediate these transitions. These studies indicate that lumican and fibromodulin have different developmental stage and leucine-rich repeat protein specific functions in the regulation of fibrillogenesis.

Biosynthesis of decorin and glypican.

Decorin and glypican are two examples of exclusively chondroitin/dermatan sulfate and heparan sulfate-substituted proteoglycans, respectively. Decorin is a secretory product, whereas glypican is linked to membrane lipids via a glycosyl-phosphatidyl-inositol (GPI) anchor. The nascent decorin protein enters the lumen of the ER, whereas that of glypican is transferred to the preformed GPI-anchors. Both types of glycosaminoglycuronans are initiated on Ser residues located in special consensus sequences, and the first glycosylation steps constitute a common pathway: the generation of the linkage region GlcA-Gal-Gal-Xyl-Ser<. The nature of the enzymes involved will be reviewed with special emphasis on the recently discovered transient 2-phosphorylation of xylose. The initiation enzymes (betaGalNAc-T1 and alphaGlcNAc-T1) then use these tetrasaccharide primers for either chondroitin or heparan sulfate assembly. The selection mechanism is not yet fully understood. The transferases that form the linkage-region and add the first hexosamine, as well as the uronosyl C-5 epimerases, appear to be products of single genes, but many isoforms of the copolymerases and sulfotransferases forming the repetitive part of the glycan chains are currently being discovered. When these enzymes work together, the fine structure of the glycosaminoglycuronans appears to be generated through the selective expression of isoforms that only operate in certain structural contexts. During heparan sulfate assembly, generation of GlcNH(2) as a permanent feature is now well recognised. Studies on glypican-1 glycoforms that recycle suggest that heparan sulfate chains are degraded by endoheparanase at or near GlcNH(2) residues, followed by deaminative cleavage catalysed by NO-derived nitrite. Chain-truncated glypican-1 can serve as a precursor for the reformation of a proteoglycan with full-size chains. Regulation of biosynthesis can be exercised at several levels, such as expression of the core protein, selection for chondroitin or heparan sulfate assembly, expression of modifying enzymes, and degradation and remodelling. Cytokines, growth factors, NO and polyamines may have regulatory roles.

Fibromodulin and lumican bind to the same region on collagen type I fibrils.

Fibromodulin and lumican are closely related members of the extracellular matrix leucine-rich repeat glycoprotein/proteoglycan family. Similar to decorin, another member of this protein family, they bind to fibrillar collagens and function in the assembly of the collagen network in connective tissues. We have studied the binding of recombinant fibromodulin, lumican and decorin, expressed in mammalian cells, to collagen type I. Using a collagen fibril formation/sedimentation assay we show that fibromodulin inhibits the binding of lumican, and vice versa. Fibromodulin and lumican do not affect the binding of decorin to collagen, nor does decorin inhibit the binding of fibromodulin or lumican. Binding competition experiments and Scatchard plot analysis indicate that fibromodulin binds to collagen type I with higher affinity than lumican.

Initiation of galactosaminoglycan biosynthesis. Separate galactosylation and dephosphorylation pathways for phosphoxylosylated decorin protein and exogenous xyloside.

By using various radiolabelled precursors, glycosylation and phosphorylation of decorin in a rat fibroblast cell line was investigated in the presence of increasing concentrations of p-nitrophenyl-O-beta-d-xylopyranoside. Decorin core protein glycanation was suppressed to approximately 25% of the normal level in the presence of 2 mm and 3 mm xyloside. Glycans/saccharides were released from the core protein and size-separated by gel chromatography. The intracellular decorin obtained from cells treated with 2 mm xyloside was substituted with Xyl and also with Gal-Xyl and Gal-Gal-Xyl, but not with longer saccharides. Only the trisaccharide contained an almost fully phosphorylated Xyl. We conclude that galactosylation of endogenous, xylosylated decorin and exogenous xyloside probably follow separate pathways or that xylosides and early decorin glycoforms are kept separated. At the addition of the first glucuronic acid the two pathways seem to merge and dephosphorylation of decorin takes place. Xyloside-primed and secreted galactosaminoglycan chains produced simultanously retained phosphorylated Xyl. Inadequate dephosphorylation could be due to excess substrate or to a short transit.time. As shown previously [Moses, J., Oldberg, A., Eklund, E. & Fransson, L.-A. (1997) Eur. J. Biochem. 248, 767-774], brefeldin A-arrested decorin is substituted with the linkage-region extended with an undersulphated and incomplete galactosaminoglycan chain. In cells treated with this drug, xylosides were unable to prime galactosaminoglycan synthesis and unable to inhibit glycosylation and phosporylation of decorin.

Fibromodulin-null mice have abnormal collagen fibrils, tissue organization, and altered lumican deposition in tendon.

Fibromodulin is a member of a family of connective tissue glycoproteins/proteoglycans containing leucine-rich repeat motifs. Several members of this gene family bind to fibrillar collagens and are believed to function in the assembly of the collagen network in connective tissues. Here we show that mice lacking a functional fibromodulin gene exhibit an altered morphological phenotype in tail tendon with fewer and abnormal collagen fiber bundles. In fibromodulin-null animals virtually all collagen fiber bundles are disorganized and have an abnormal morphology. Also 10-20% of the bundles in heterozygous mice are similar to the abnormal bundles in fibromodulin-null tail tendon. Ultrastructural analysis of Achilles tendon from fibromodulin-null mice show collagen fibrils with irregular and rough outlines in cross-section. Morphometric analysis show that fibromodulin-null mice have on the average thinner fibrils than wild type animals as a result of a larger preponderance of very thin fibrils in an overall similar range of fibril diameters. Protein and RNA analyses show an approximately 4-fold increase in the content of lumican in fibromodulin-null as compared with wild type tail tendon, despite a decrease in lumican mRNA. These results demonstrate a role for fibromodulin in collagen fibrillogenesis and suggest that the orchestrated action of several leucine-rich repeat glycoproteins/proteoglycans influence the architecture of collagen matrices.

Biosynthesis of the proteoglycan decorin--transient 2-phosphorylation of xylose during formation of the trisaccharide linkage region.

Phosphorylation of decorin was investigated by incubating a rat fibroblast cell line with radiolabelled phosphate and carbohydrate precursors. There was a transient phosphorylation of the linkage-region saccharides in intracellular decorin prior to assembly of the galactosaminoglycan chain. Phosphorylation gradually increased from xylosylated, galactosyl-xylosylated to galactosyl-galactosyl-xylosylated core protein where all trisaccharide stubs were phosphorylated. Addition of the first glucuronate residue was accompanied by rapid dephosphorylation. Brefeldin A treatment resulted in segregation of galactosaminoglycan synthesis and dephosphorylation. Enzymatic degradation of brefeldin-A-arrested immature proteoglycan with incomplete galactosaminoglycan chain [Moses, J., Oldberg, A., Eklund, E. & Fransson, L.-A. (1997) Eur. J. Biochem., in the press] by using chondroitin AC lyase and chondro-glycuronidase, followed by beta-galactosidase treatment, demonstrated the sequence galactosyl-galactosyl-phosphoxylose. The xylose was resistant to direct periodate oxidation, but sensitive after treatment with alkaline phosphatase, showing that the phosphate was located at C2 of xylose. The transient 2-phosphorylation of xylose may be involved in intracellular transport and/or in the control of modifications of the glycan chain.

Biosynthesis of the proteoglycan decorin -- identification of intermediates in galactosaminoglycan assembly.

Biosynthesis of decorin was investigated by incubating a rat fibroblast cell line with various radiolabelled protein and galactosaminoglycan precursors. The following cell-associated and distinct intermediates were isolated and identified: a pool of non-glycosylated core protein, two pools of decorin with incomplete chains, one with three sulphated disaccharide repeats and another with five or more sulphated disaccharide repeats, as well as decorin with mature chains. Results of pulse/chase experiments indicated that these pools represented discrete stages in chain growth. Treatment with brefeldin A, which blocks transport from the endoplasmic reticulum to the Golgi, resulted in accumulation of decorin with an incomplete chain containing six or seven largely unsulphated disaccharide repeats. During recovery from drug treatment, 4-sulfation reappeared earlier than 6-sulfation. The results suggest that the galactosaminoglycan assembly-line consists of separate multienzyme complexes that build only a limited section of the chain. Furthermore, brefeldin A causes segregation of compartments involved in separate stages of the assembly line. In an earlier report [Moses, J., Oldberg. A., Cheng, F. & Fransson, L.-A. (1997) Eur. J. Biochem. 248, 521-526] we took advantage of such segregation to identify and characterize a transient 2-phosphorylation of xylose in the linkage region.

Staphylococcus aureus causing osteomyelitis binds to a nonapeptide sequence in bone sialoprotein.

Bone sialoprotein is a glycoprotein of the bone and dentine extracellular matrix. This protein consists of 320 amino acids, of which 25% are glutamic and aspartic acid residues. Sialic acid, containing oligosaccharides and tyrosine sulphate residues, supplies additional polyanionic properties. Staphylococcal cells, isolated from patients suffering from infection of bone tissue, bind the bone-derived sialoprotein, an interaction which is specifically inhibited by the recombinant bone sialoprotein core protein. We have previously shown that the 150 N-terminal amino acid residues of bone sialoprotein are responsible for the binding to staphylococcal cells. By using recombinant deleted variants of bone sialoprotein and synthetic peptides, we have now localized the staphylococcal binding site to less than 10 residues within the N-terminal part of the protein.

Decorin-binding sites for collagen type I are mainly located in leucine-rich repeats 4-5.

Decorin and biglycan are structurally related interstitial proteoglycans synthesized in connective tissues like skin, tendon, and cartilage. Despite the conspicuous sequence similarities, where about 55% of the amino acid residues in decorin and biglycan are located in identical positions, the two proteoglycans show differences in their interaction with collagen. Decorin binds to collagen type I, whereas biglycan in several assay systems shows no affinity for this collagen type. Here we have made use of these structural similarities and affinity differences in studies of the collagen binding properties of decorin. Recombinant biglycan/decorin chimeras were produced in mammalian cells and analyzed for their capacity to bind collagen. In the chimeras, biglycan contributes sequences crucial for synthesis and export from the mammalian cells, and decorin provides potential collagen-binding properties. By using this approach we show that decorin binds to the collagen primarily via leucine-rich repeats 4-5 composed of some 40 amino acid residues. Proteoglycan chimeras containing decorin sequences from the N terminus to leucine-rich repeat 3 or sequences from leucine-rich repeat 6 to the C terminus do not show any detectable binding to collagen. A proteoglycan chimera containing decorin leucine-rich repeats 4-5 flanked by biglycan sequences binds to collagen. However, this chimera binds to collagen with somewhat lower affinity than wild type decorin, suggesting that additional low affinity binding sites may be located in other parts of decorin. Alternatively, the conformation of the collagen binding leucine-rich repeats 4-5 are different in decorin and in the biglycan/decorin chimera, leading to a lower collagen affinity for the latter.

Distribution and synthesis of bone sialoprotein in metaphyseal bone of young rats show a distinctly different pattern from that of osteopontin.

Bone sialoprotein (BSP) and osteopontin (OPN) are two phosphorylated and highly glycosylated cell-binding proteins in bone. Both proteins bind to hydroxylapatite. The cell binding is mediated via an Arg-Gly-Asp (RGD) sequence and previous work indicates that both proteins can bind to the vitronectin receptor (alpha v beta 3). The present work shows that a prevailing localization of BSP in metaphyseal bone of the young rat is at the interface between calcified cartilage and bone. Thus BSP shows a conspicuous enrichment in the osteoid laid down by the invading osteoblasts immediately next to the calcified cartilage. Furthermore, the most prominent amount of BSP mRNA was detected in cells at the epiphyseal/metaphyseal border. As opposed to OPN, no prominent accumulation of BSP immunoreactivity was observed at bone surfaces that face cells. Also the synthesis OPN was most pronounced at sites very different from those of BSP. Thus, the most prominent amount of OPN mRNA was observed in cells close to the metaphyseal/diaphyseal border, where osteoclastic bone resorption is particularly active. Indeed, message was often found in cells surrounding osteoclasts without any detectable message. The distinctly different patterns of synthesis and expression of the two proteins indicate different roles in bone turnover at this stage of development. Thus, it appears that BSP has a specific role during the initial phases of bone formation at the cartilage/bone interface. On the other hand, the pattern of OPN synthesis and expression support and extend our previous data showing OPN particularly enriched at attachment sites of osteoclasts resorbing bone.

Structure and deduced amino acid sequence of the human fibromodulin gene.

We have determined the structure and partially sequenced the human fibromodulin gene. The translated region of the gene is composed of two exons. An exon in the 5'-non translated region is separated from the next exon by a 1 kb intron. This exon, which encodes the major part of the translated region, is 983 bp and is followed by an approx. 5 kbp intron. The last 50 nucleotides of the translated region as well as the 3'-nontranslated region are located on the last exon. This exon arrangement is different from the arrangement reported for the gene of the structurally related biglycan. The translated region of the gene was sequenced and compared with bovine fibromodulin. The amino acid sequences of human and bovine fibromodulin show an overall homology of 90%.

Altered expression of small proteoglycans, collagen, and transforming growth factor-beta 1 in developing bleomycin-induced pulmonary fibrosis in rats.

The development of bleomycin-induced pulmonary fibrosis in rats was studied over a period of 21 d after an intratracheal instillation of bleomycin. The expression of three small proteoglycans (biglycan, decorin, and fibromodulin), collagen III and TGF-beta 1 was studied by RNA-transfer blot analysis. The proteoglycans were also studied by SDS-polyacrylamide gel electrophoresis and Western blots. TGF-beta 1 mRNA increased threefold already on day 3 and remained elevated until day 10. After the increase of TGF-beta 1 mRNA the messages for biglycan and collagen III steadily increased to reach a maximum 10 d after bleomycin instillation. The mRNA for biglycan increased maximally fourfold and that of collagen III 2.5-fold. Decorin mRNA, in contrast to biglycan decreased and reached 20% of control on day 10. The message for fibromodulin remained constant throughout the study period. The amounts of biglycan and decorin in the tissue changed in accordance with the mRNA levels. The results corroborate and extend previous in vitro studies concerning the effect of TGF-beta 1 on the metabolism of small proteoglycans and show that these macromolecules are regulated differently also in vivo. The marked alterations of biglycan and decorin during the development of fibrosis suggests that these proteoglycans have a regulating role in this process.

COMP (cartilage oligomeric matrix protein) is structurally related to the thrombospondins.

Cloning and sequence analysis of cartilage oligomeric matrix protein (COMP) cDNA, representing a cartilage pentameric protein, revealed a protein of 755 amino acid residues with a calculated molecular mass of 82,700 Da. Expression of the cDNA in COS cells showed that COMP is a homopolymer composed of five identical disulfide-linked subunits. COMP is homologous to the carboxyl-terminal half of thrombospondin, and the homologies include 89% and 54% of the residues in COMP and thrombospondin, respectively. The similarities are most pronounced in the carboxyl-terminal domains and in the calcium binding type 3 repeat domains in which about 60% of the amino acid residues are identical. In the type 2/epidermal growth factor repeat domains the two proteins contain 41% identical residues. The sequence of the amino-terminal 84-amino acid residues is unique for COMP. Comparison of the amino acid sequences in the type 2 and type 3 repeat domains of COMP and the thrombospondins shows that COMP is the product of a unique gene and not the result of an alternatively spliced thrombospondin gene.

Cartilage matrix proteins. An acidic oligomeric protein (COMP) detected only in cartilage.

An Mr = 524,000 oligomeric protein was isolated from bovine cartilage and designated COMP (Cartilage Oligomeric Matrix Protein). The protein is composed of disulfide-bonded subunits with an apparent Mr of 100,000 each. It is markedly anionic, probably due to its high contents of aspartic acid and glutamic acid, as well as to its substitution with negatively charged carbohydrates. COMP was found in all cartilages analyzed, but could not be detected in other tissues by enzyme-linked immunosorbent assay of guanidine HCl extracts. Within a given cartilage, COMP shows a preferential localization to the territorial matrix surrounding the chondrocytes.

Posttranslational modifications of fibromodulin.

Tyrosine sulfate residues were identified in fibromodulin produced by tracheal chondrocytes, by tendon and sclera fibroblasts in primary culture, as well as in Chinese hamster ovary cells transfected with a construct containing fibromodulin cDNA. The tyrosine sulfate residues were located in the N-terminal part of fibromodulin. Thus, Chinese hamster ovary cells expressing a deleted variant of fibromodulin lacking the N-terminal 52 amino acids following the predicted signal peptide did not contain any tyrosine sulfate residues. The substitution with keratan sulfate chains was not restricted to chondrocytes, but was also identified in fibromodulin synthesized by bovine tendon fibroblasts and sclera fibroblasts, as well as in fibromodulin isolated from tendon. Digestion of fibromodulin with N-glycosidase F reduced the apparent size of fibromodulin to that of the core protein, as predicted from sequence analysis (Oldberg, A., Antonsson, P., Lindblom, K., and Heinegård, D. (1989) EMBO J.8, 2601-2604). Thus fibromodulin from cartilage, tendon, and sclera contains N-glycosidically linked oligosaccharides, some of which are extended to keratan sulfate chains.