Fibrillogenesis of collagen types I, II, and III with small leucine-rich proteoglycans decorin and biglycan

Collagen has found use as a scaffold material for tissue engineering as well as a coating material for implants with a view to enhancing osseointegration through mimicry of the bone extracellular matrix in vivo. The aim of this study was to compare the collagen types I, II, and III with regard to their ability to bind the small leucine-rich proteoglycans (SLRPs) decorin and biglycan during fibrillogenesis in vitro in phosphate buffer. In addition, the influence of SLRPs on the proportion of collagen molecules incorporated into fibrils during fibrillogenesis in vitro at high and low ionic strength was investigated, as were their effects on the morphology of collagen fibrils and the speed of fibrillogenesis. Considerably more biglycan than decorin was bound by all three collagen types. Collagen II bound significantly more SLRPs in fibrils than collagen I and III. Decorin and biglycan decreased the proportion of collagen molecules of all three collagen types incorporated into fibrils in similar fashion. Biglycan affected neither fibril diameter nor the speed of fibrillogenesis. Decorin reduced the fibril diameter of all three collagen types. The differences in SLRP-binding ability between collagen types could be of significance when selecting collagen type and/or SLRPs as scaffold materials for tissue engineering or implant coatings.     

Extracellular matrix proteoglycans control the fate of bone marrow stromal cells

Extracellular matrix glycoproteins and proteoglycans bind a variety of growth factors and cytokines thereby regulating matrix assembly as well as bone formation. However, little is known about the mechanisms by which extracellular matrix molecules modulate osteogenic stem cells and bone formation. Using mice deficient in two members of the small leucine-rich proteoglycans, biglycan and decorin, we uncovered a role for these two extracellular matrix proteoglycans in modulating bone formation from bone marrow stromal cells. Our studies showed that the absence of the critical transforming growth factor-beta (TGF-beta)-binding proteoglycans, biglycan and decorin, prevents TGF-beta from proper sequestration within the extracellular matrix. The excess TGF-beta directly binds to its receptors on bone marrow stromal cells and overactivates its signaling transduction pathway. Overall, the predominant effect of the increased TGF-beta signaling in bgn/dcn-deficient bone marrow stromal cells is a "switch in fate" from growth to apoptosis, leading to decreased numbers of osteoprogenitor cells and subsequently reduced bone formation. Thus, biglycan and decorin appear to be essential for maintaining an appropriate number of mature osteoblasts by modulating the proliferation and survival of bone marrow stromal cells. These findings underscore the importance of the micro-environment in controlling the fate of adult stem cells and reveal a novel cellular and molecular basis for the physiological and pathological control of bone mass.     

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.     

Interactions of collagen types I and II with chondroitin sulfates A-C and their effect on osteoblast adhesion

Collagen has found use as a scaffold material for tissue engineering as well as a coating material for implants. The main aim of this study was to compare the ability of the collagen types I and II to bind preparations of the chondroitin sulfate types A-C (CS A, CS B, CS C). In addition, the effect of the three CS preparations on the extent of collagen incorporated into fibrils and the morphology of collagen fibrils was investigated, as was the influence of collagen fibril coatings containing CS A-C on titanium surfaces on the adhesion of primary rat osteoblasts. Fibrils of both collagen types bound a higher mass of CS C than CS B and a greater mass of CS B than CS A per milligram of fibrils formed. Fibrils of collagen type II bound a higher mass of CS B and C than collagen I fibrils. The proportion of collagen incorporated into fibrils decreased with increasing CS A and CS C concentration but not with increasing CS B concentration. All three CS preparations caused collagen I and II fibrils to become thinner. CS A and CS B but not CS C appeared to stimulate the formation of focal adhesions by osteoblasts after incubation for 2 hours. These results could be of importance when selecting collagen type or CS type as materials for implant coatings or tissue engineering scaffolds.     

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.     

Proteoglycans decorin and biglycan differentially modulate TGF-beta-mediated fibrotic responses in the lung

Transforming growth factor (TGF)-beta is a key cytokine in the pathogenesis of pulmonary fibrosis, and pharmacological interference with TGF-beta can ameliorate the fibrotic tissue response. The small proteoglycans decorin and biglycan are able to bind and inhibit TGF-beta activity in vitro. Although decorin has anti-TGF-beta properties in vivo, little is known about the physiological role of biglycan in vivo. Adenoviral gene transfer was used to overexpress active TGF-beta, decorin, and biglycan in cell culture and in murine lungs. Both proteoglycans were able to interfere with TGF-beta bioactivity in vitro in a dose-dependant manner. In vivo, overexpression of TGF-beta resulted in marked lung fibrosis, which was significantly reduced by concomitant overexpression of decorin. Biglycan, however, had no significant effect on lung fibrosis induced by TGF-beta. The data suggest that differences in tissue distribution are responsible for the different effects on TGF-beta bioactivity in vivo, indicating that decorin, but not biglycan, has potential therapeutic value in fibrotic disorders of the lung.     

The effects of transforming growth factor-beta and serum on proteoglycan synthesis by tendon fibrocartilage.

The effects of transforming growth factor-beta (TGF-beta) and serum on proteoglycan synthesis by tissue explants from the fibrocartilaginous region of adult bovine tendon and by cells in culture from this region were assessed. The most characteristic effect of added TGF-beta on both explant tissue and cells in culture was enhanced synthesis of one small proteoglycan-biglycan. Lowered serum concentration diminished incorporation of Na2 35SO4 into proteoglycans. Added TGF-beta (1 ng/ml) stimulated cell proliferation, increased overall proteoglycan synthesis, and increased the length of glycosaminoglycan chains on all secreted proteoglycans. The effect of TGF-beta on cells in culture was highly consistent whereas explants from different animals showed greater variability in the response. It was concluded that TGF-beta did not specifically promote or maintain the cartilaginous nature of this tissue because supplementing medium with TGF-beta did not significantly alter the ratio of large/small proteoglycans synthesized by tissue explants. However, the observation of enhanced biglycan synthesis by TGF-beta suggests that TGF-beta could be involved in differentiation of regions of tendon subjected to compression, because compressed tendon contains both decorin and biglycan small proteoglycans whereas tensional tendon contains primarily decorin. Excess decorin added to cell culture medium did not affect the ability of TGF-beta to enhance synthesis of biglycan.     

The interactions of cartilage proteoglycans with collagens are determined by their structures.

In the present work, the interaction of aggrecan, decorin and biglycan isolated from pig laryngeal cartilage and of the three squid cartilage proteoglycans with collagen type I and II was studied. The interaction was examined under conditions allowing the formation of collagen fibrils. It was found that biglycan interacted strongly with collagen type II and not with type I and the interaction seemed to proceed exclusively through its core proteins. Decorin interacted with collagen type I but not with type II. Aggrecan interacted very poorly with both collagen types. The two squid proteoglycans of large size, D1D1A and D1D2, interacted only with collagen type I through both glycosaminoglycans and core proteins. The third squid proteoglycan of small size, D1D1B, interacted poorly only with collagen type I. The results suggested that the interactions of cartilage proteoglycans with collagen were mainly due to the primary structure of both molecules, and would contribute to the maintenance of the integrity of the tissue. The biochemical significance of these interactions might be more critical in aged vertebrate cartilage, where loss of aggrecan and increase of the small proteoglycans was observed, a large proportion of which is found in the extracellular matrix free of glycosaminoglycan chains.     

Interactions of the extracellular matrix proteoglycans decorin and biglycan with C1q and collectins

Decorin and biglycan are closely related abundant extracellular matrix proteoglycans that have been shown to bind to C1q. Given the overall structural similarities between C1q and mannose-binding lectin (MBL), the two key recognition molecules of the classical and the lectin complement pathways, respectively, we have examined functional consequences of the interaction of C1q and MBL with decorin and biglycan. Recombinant forms of human decorin and biglycan bound C1q via both collagen and globular domains and inhibited the classical pathway. Decorin also bound C1 without activating complement. Furthermore, decorin and biglycan bound efficiently to MBL, but only biglycan could inhibit activation of the lectin pathway. Other members of the collectin family, including human surfactant protein D, bovine collectin-43, and conglutinin also showed binding to decorin and biglycan. Decorin and biglycan strongly inhibited C1q binding to human endothelial cells and U937 cells, and biglycan suppressed C1q-induced MCP-1 and IL-8 production by human endothelial cells. In conclusion, decorin and biglycan act as inhibitors of activation of the complement cascade, cellular interactions, and proinflammatory cytokine production mediated by C1q. These two proteoglycans are likely to down-regulate proinflammatory effects mediated by C1q, and possibly also the collectins, at the tissue level.     

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.     

Decorin regulates assembly of collagen fibrils and acquisition of biomechanical properties during tendon development

Tendon function involves the development of an organized hierarchy of collagen fibrils. Small leucine-rich proteoglycans have been implicated in the regulation of fibrillogenesis and decorin is the prototypic member of this family. Decorin-deficient mice demonstrate altered fibril structure and mechanical function in mature skin and tail tendons. However, the developmental role(s) of decorin needs to be elucidated. To define these role(s) during tendon development, tendons (flexor digitorum longus) were analyzed ultrastructurally from postnatal day 10 to 90. Decorin-deficient tendons developed abnormal, irregularly contoured fibrils. Finite mixture modeling estimated that the mature tendon was a three-subpopulation mixture of fibrils with characteristic diameter ranges. During development, in each subpopulation the mean diameter was consistently larger in mutant mice. Also, diameter distributions and the percentage of fibrils in each subpopulation were altered. Biomechanical analyses demonstrated that mature decorin-deficient tendons had significantly reduced strength and stiffness; however, there was no reduction in immature tendons. Expression of decorin and biglycan, a closely related family member, was analyzed during development. Decorin increased with development while biglycan decreased. Spatially, both had a comparable localization throughout the tendon. Biglycan expression increased substantially in decorin-deficient tendons suggesting a potential functional compensation. The accumulation of structural defects during fibril growth, a period associated with decorin expression and low biglycan expression, may be the cause of compromised mechanical function in the absence of decorin. Our findings indicate that decorin is a key regulatory molecule and that the temporal switch from biglycan to decorin is an important event in the coordinate regulation of fibrillogenesis and tendon development.     

Relationship between actions of transforming growth factor (TGF)-β and cell surface expression of its receptors in clonal osteoblastic cells

Various osteoblastic cell lines were examined for the relationship between the presence of cell-surface transforming growth factor (TGF)-β receptors and the synthesis of matrix proteins with their responsiveness to TGF-β. Treatment with TGF-β1 inhibited proliferation and stimulated proteoglycan and fibronectin synthesis in MC3T3-E1 and MG 63 cells. The major proteoglycans synthesized by these cells were decorin and biglycan, and TGF-β1 markedly stimulated the synthesis of decorin in MC3T3-E1 and of biglycan in MG 63 cells. SaOS 2 and UMR 106 cells synthesized barely detectable amounts of decorin or biglycan, and TGF-β1 did not stimulate the synthesis of these proteoglycans. In SaOS 2 cells, however, TGF-β1 enhanced fibronectin synthesis. TGF-β1 did not show any of these effects in UMR 106 cells. Receptor cross-linking studies revealed that only MC3T3-E1 and MG 63 cells had both types I and II signal-transducing receptors for TGF-β in addition to betaglycan. SaOS 2 cells possessed type I but no type II receptor on the cell surface. In contrast, SaOS 2 as well as MC3T3-E1 and MG 63 cells expressed type II receptor mRNA by Northern blot analysis, and cell lysates contained type II receptor by Western blot analysis. Thus, it appears that type II receptor present in SaOS 2 cells is not able to bind TGF-β1 under these conditions. UMR 106 cells with no response to TGF-β1 had neither of the signal-transducing receptors by any of the analyses. These observations using clonal osteoblastic cell lines demonstrate that the ability of osteoblastic cells to synthesize bone matrix proteoglycans is associated with the responsiveness of these cells to TGF-β1, that the responsiveness of osteoblastic cells to TGF-β1 in cell proliferation and proteoglycan synthesis correlates with the presence of both types I and II receptors, and that the effect of TGF-β1 on fibronectin synthesis can develop with little binding of TGF-β1 to type II receptor if type I receptor is present. It is suggested that the combination of cell-surface receptors for TGF-β determines the responsiveness of osteoblastic cells to TGF-β and that changes in cell-surface TGF-β receptors may play a role in the regulation of matrix protein synthesis and bone formation in osteoblasts. © 1995 Wiley-Liss, Inc.     

Differential regulation of extracellular matrix proteoglycan (PG) gene expression. Transforming growth factor-beta 1 up-regulates biglycan (PGI), and versican (large fibroblast PG) but down-regulates decorin (PGII) mRNA levels in human fibroblasts in culture

Proteoglycans (PGs) comprise a group of extracellular matrix macromolecules which play an important role in matrix biology. In this study, normal human skin and gingival fibroblast cultures were incubated with transforming growth factor-beta 1 (TGF-beta 1), and the expression of three PGs, viz. biglycan (PGI), decorin (PGII), and versican (a large fibroblast proteoglycan) was examined. The results indicate that TGF-beta 1 (5 ng/ml) markedly increased the expression of biglycan (up to 24-fold) and versican (up to 6-fold) mRNAs and the enhancement of biglycan expression was coordinate with elevated type I procollagen gene expression in the same cultures. In contrast, the expression of decorin mRNA was markedly (up to approximately 70%) inhibited by TGF-beta 1. The response to TGF-beta 1 was similar in both skin and gingival fibroblasts, although the gingival cells were clearly more responsive to stimulation by TGF-beta 1 with respect to biglycan gene expression. Analysis of 35S-labeled proteoglycans in the culture media of skin and gingival fibroblasts also revealed stimulation of biglycan and versican production, and reduction in decorin production. Quantitation of both [35S]sulfate and [3H]leucine-labeled decorin in cell culture media by immunoprecipitation revealed a 50% reduction in decorin production in cell cultures treated with TGF-beta 1. This TGF-beta 1-elicited reduction was accompanied by an apparent increase in the size of the decorin molecules, although the size of the core protein was not altered, as judged by Western immunoblotting following chondroitinase ABC digestion. Analysis of the proteoglycans in the matrix and membrane fractions also revealed increased amounts of versican in cultures treated with TGF-beta 1. These results indicate differential regulation of PG gene expression in fibroblasts by TGF-beta 1, and these observations emphasize the role of PGs in the extracellular matrix biology and pathology.     

Decorin-transforming growth factor- interaction regulates matrix organization and mechanical characteristics of three-dimensional collagen matrices

The small leucine-rich proteoglycan decorin has been demonstrated to be a key regulator of collagen fibrillogenesis; decorin deficiencies lead to irregularly shaped collagen fibrils and weakened material behavior in postnatal murine connective tissues. In an in vitro investigation of the contributions of decorin to tissue organization and material behavior, model tissues were engineered by seeding embryonic fibroblasts, harvested from 12.5-13.5 days gestational aged decorin null (Dcn(-/-)) or wild-type mice, within type I collagen gels. The resulting three-dimensional collagen matrices were cultured for 4 weeks under static tension. The collagen matrices seeded with Dcn(-/-) cells exhibited greater contraction, cell density, ultimate tensile strength, and elastic modulus than those seeded with wild-type cells. Ultrastructurally, the matrices seeded with Dcn(-/-) cells contained a greater density of collagen. The decorin-null tissues contained more biglycan than control tissues, suggesting that this related proteoglycan compensated for the absence of decorin. The effect of transforming growth factor-beta (TGF-beta), which is normally sequestered by decorin, was also investigated in this study. The addition of TGF-beta1 to the matrices seeded with wild-type cells improved their contraction and mechanical strength, whereas blocking TGF-beta1 in the Dcn(-/-) cell-seeded matrices significantly reduced the collagen gel contraction. These results indicate that the inhibitory interaction between decorin and TGF-beta1 significantly influenced the matrix organization and material behavior of these in vitro model tissues.     

The small proteoglycan biglycan is associated with thick collagen fibrils in the mouse decidua.

In mice, embryo implantation induces profound changes in the endometrium. These changes include redifferentiation of endometrial fibroblasts and extensive remodeling of extracellular matrix components. We have previously shown that, during this process, there is an impressive increase in the thickness of collagen fibrils present in decidualised areas that surround the implantation site, while collagen fibrils in non-decidualised areas and in the interimplantation site remain thin. In vitro and in vivo experiments have identified small leucine rich proteoglycans (SLRPs) as regulators of collagen fibrillogenesis. In a previous study, we demonstrated a difference between the pre-implantation and the post-implantation expression and distribution of four SLRPs types in uterine tissues. The present study, utilising immunocytochemical electron microscopy, shows that biglycan is associated with the presence of thick collagen fibrils in decidualised regions of the endometrium and that decorin is associated exclusively with thin collagen fibrils in non-decidualised endometrial areas. These results strongly indicate that biglycan plays a role in collagen fibrillogenesis and probably participates in the determination of collagen fibril thickness in the mouse decidua.     

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.     

Molecular interactions of biglycan and decorin with elastic fiber components: biglycan forms a ternary complex with tropoelastin and microfibril-associated glycoprotein 1.

The interactions of the dermatan sulfate proteoglycans biglycan and decorin have been investigated with the elastic fiber components, tropoelastin, fibrillin-containing microfibrils, and microfibril-associated glycoproteins (MAGP) 1 and 2. Both proteoglycans were found to bind tropoelastin and fibrillin-containing microfibrils but not MAGPs 1 and 2 in solid phase binding assays. The specificity of the binding of biglycan and decorin to tropoelastin was confirmed by co-immunoprecipitation experiments and by the blocking of the interactions with elastin-derived peptides. Isolated core proteins from biglycan and decorin bound to tropoelastin more strongly than the intact proteoglycans, and there were no differences in the tropoelastin binding characteristics of distinct glucuronate-rich and iduronate-rich glycoforms of biglycan. These findings indicated that the binding sites were contained in the protein cores of the proteoglycans rather than the glycosaminoglycan side chains. Scatchard analysis showed that biglycan bound more avidly than decorin to tropoelastin with K(d) values estimated as 1.95 x 10(-7) m and 5.3 x 10(-7) m, respectively. In blocking experiments each proteoglycan showed extensive inhibition of binding of the other to tropoelastin but was most effective at blocking its own binding. This result suggested that biglycan and decorin had closely spaced but distinct binding sites on tropoelastin. Addition of the elastin-binding protein MAGP-1 to the assays enhanced the binding of biglycan to tropoelastin but had no effect on the decorin-tropoelastin interaction. Co-immunoprecipitation experiments showed that MAGP-1 interacted with biglycan but not decorin in the solution phase. The results indicated that biglycan specifically formed a ternary complex with tropoelastin and MAGP-1. Overall the study supports the concept that biglycan may have a specific role in the elastinogenic phase of elastic fiber formation.