Extracellular proteoglycans modify TGF-beta bio-availability attenuating its signaling during skeletal muscle differentiation.

The onset and progression of skeletal muscle regeneration are controlled by a complex set of interactions between muscle precursor cells and their environment. Satellite cells constitute the main source of muscle precursor cells for growth and repair. After skeletal muscle injury, cell-derived signals induce their re-entry into the cell cycle and their migration into the damaged zone, where they proliferate and differentiate into mature myofibers. The surrounding extracellular matrix (ECM) together with inhibitory growth factors, such as transforming growth factor-beta (TGF-beta), also likely play an important role in growth control and muscle differentiation. Decorin, biglycan and betaglycan are proteoglycans that bind TGF-beta during skeletal muscle differentiation. In this paper, we show that the binding of TGF-beta to the receptors TGF-betaRI and-betaRII diminished in a satellite cell-derived cell line during differentiation, in spite of an increase expression of both receptors. In contrast, during the differentiation of decorin-null myoblasts (Dcn null), which lack decorin expression, the binding of TGF-beta to TGF-betaRI and -betaRII increased concomitantly with receptors levels. Both the addition and re-expression of decorin, in these myoblasts, diminished the binding of TGF-beta to its transducing receptors. Similar results were obtained when biglycan was added or over-expressed in Dcn null myoblasts. The binding of TGF-beta to TGF-betaRIII, alternatively known as betaglycan, was also augmented in Dcn null myoblasts and diminished by decorin, biglycan and betaglycan. These results suggest that decorin, biglycan and betaglycan compete for the binding of TGF-beta to its transducing receptors. Transfection studies with the TGF-beta-dependent promoter of the plasminogen activator inhibitor-1, coupled with luciferase, revealed that the addition of each proteoglycan diminished TGF-beta-dependent activity, for both TGF-beta1 and -beta2. The modulation of TGF-beta signaling by ECM proteoglycans diminishing the bio-availability of TGF-beta for its transducing receptors appears to be a feasible mechanism for the attenuation of this inhibitory growth factor during skeletal muscle formation.     

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.     

Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components

Transforming growth factor (TGF)-beta-induced chondrogenesis of mesenchymal stem cells derived from bone marrow involves the rapid deposition of a cartilage-specific extracellular matrix. The sequential events in this pathway leading from the undifferentiated stem cell to a mature chondrocyte were investigated by analysis of key matrix elements. Differentiation was rapidly induced in cells cultured in the presence of TGF-beta 3 or -beta 2 and was accompanied by the early expression of fibromodulin and cartilage oligomeric matrix protein. An increase in aggrecan and versican core protein synthesis defined an intermediate stage, which also involved the small leucine-rich proteoglycans decorin and biglycan. This was followed by the appearance of type II collagen and chondroadherin. The pathway was also characterized by the appearance of type X collagen, usually associated with hypertrophic cartilage. There was also a change in the pattern of sulfation of chondroitin sulfate, with a progressive increase in the proportion of 6-sulfated species. The major proportion of newly synthesized glycosaminoglycan was part of an aggregating proteoglycan network. These data allow us to define the phenotype of the differentiated cell and to understand in greater detail the sequential process of matrix assembly.     

Fibrils of different collagen types containing immobilised proteoglycans (PGs) as coatings: characterisation and influence on osteoblast behaviour

Collagen, the main organic component of bone, is used as a coating on titanium implants and as a scaffold material in bone tissue engineering. Surface modifications of titanium which promote osteoblast adhesion, proliferation and synthesis of collagen by osteoblasts are desirable. One biomimetic approach is the coating of titanium with collagen in fibrillar form. Other organic components of bone may be bound to fibrils and exert additional effects. In this study, the collagen types I-III were compared regarding their ability to bind the proteoglycans decorin and biglycan, which are found in bone. More collagen was bound to collagen II fibrils than to those of types I and III. Therefore, titanium surfaces were coated with fibrils of collagen type II containing biglycan or decorin or neither to investigate the effect of the proteoglycans on human primary osteoblast behaviour. In addition, the growth factor TGF-beta1 was adsorbed onto surfaces coated with fibrils of collagen type II containing biglycan or decorin or neither to investigate the influence of decorin and biglycan on the effect of TGF-beta1 on osteoblasts. Fibril-bound biglycan and decorin influence primary osteoblast behaviour by themselves. The presence of substrate-bound biglycan or decorin influences the effect of TGF-beta1. These results may be important when designing collagen-based coatings or scaffolds for tissue engineering, including those loaded with growth factors.     

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.     

Increase in decorin and biglycan in Duchenne Muscular Dystrophy: role of fibroblasts as cell source of these proteoglycans in the disease

Fibrosis is a common pathological feature observed in muscles of patients with Duchenne muscular dystrophy (DMD). Biglycan and decorin are small chondroitin/dermatan sulfate proteoglycans in the muscle extracellular matrix (ECM) that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins. Decorin is considered an anti-fibrotic agent, preventing the process by blocking TGF-beta activity. There is no information about their expression in DMD patients. We found an increased amount of both proteoglycans in the ECM of skeletal muscle biopsies obtained from DMD patients. Both biglycan and decorin were augmented in the perimysium of muscle tissue, but only decorin increased in the endomysium as seen by immunohistochemical analyses. Fibroblasts were isolated from explants obtained from muscle of DMD patients and the incorporation of radioactive sulfate showed an increased synthesis of both decorin and biglycan in cultured fibroblasts compared to controls. The size of decorin and biglycan synthesized by DMD and control fibroblasts seems to be similar in size and anion charge. These findings show that decorin and biglycan are increased in DMD skeletal muscle and suggest that fibroblasts would be, at least, one source for these proteoglycans likely playing a role in the muscle response to dystrophic cell damage.     

Transient up-regulation of biglycan during skeletal muscle regeneration: delayed fiber growth along with decorin increase in biglycan-deficient mice

The onset and progression of skeletal muscle regeneration are controlled by a complex set of interactions between muscle precursor cells and their environment. Decorin is the main proteoglycan present in the extracellular matrix (ECM) of adult muscle while biglycan expression is lower, but both are increased in mdx mice dystrophic muscle. Both of these small leucine-rich proteoglycans (SLRPs) can bind other matrix proteins and to the three TGF-beta isoforms, acting as modulators of their biological activity. We evaluated biglycan and decorin expression in skeletal muscle during barium chloride-induced skeletal muscle regeneration in mice. A transient and dramatic up-regulation of biglycan was associated with newly formed myotubes, whereas decorin presented only minor variations. Studies both in vitro and in intact developing newborn mice showed that biglycan expression is initially high and then decreases during skeletal muscle differentiation and maturation. To further evaluate the role of biglycan during the regenerative process, skeletal muscle regeneration was studied in biglycan-null mice. Skeletal muscle maintains its regenerative capacity in the absence of biglycan, but a delay in regenerated fiber growth and a decreased expression of embryonic myosin were observed despite to normal expression of MyoD and myogenin. Transient up-regulation of decorin during muscle regeneration in these mice may possibly obscure further roles of SLRPs in this process.     

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.     

Temporal regulation of hyaluronan and proteoglycan metabolism by human bone cells in vitro

Osteoblasts elaborate a dynamic extracellular matrix that is constructed and mineralized as bone is formed. This matrix is primarily composed of collagen, along with noncollagenous proteins which include glycoproteins and proteoglycans. After various times in culture, human bone cells were labeled with [35S]sulfate, [3H] leucine/proline, or [3H]glucosamine and the metabolism of hyaluronan and four distinct species of proteoglycans (PGs) was assayed in the medium, cell layer, and intracellular pools. These cells produce hyaluronan (Mr approximately 1,400,000; a chondroitin sulfate PG (CSPG), Mr approximately 600,000; a heparan sulfate PG (HSPG), Mr approximately 400,000; and two dermatan sulfate PGs with Mr approximately 270,000 (biglycan, PG I) and Mr approximately 135,000 (decorin, PG II) that distribute between the medium and cell layer. Two days following subculture, 12 h [35S]sulfate steady-state labeling yielded a composition of 24, 27, 31, and 18% for total CSPG, HSPG, biglycan, and decorin, respectively. While HSPG and decorin levels and distribution between medium and cell layer remained relatively constant during steady-state labeling at different times in culture, CSPG and biglycan levels increased dramatically at late stages of growth, and their distribution changed throughout culture. These results were independent of cell density, media depletion, and labeling pool effects. In contrast, hyaluronan synthesis was uncoupled from PG synthesis and apparently density-dependent. Pulse chase labeling at different stages of culture showed that the CSPG and decorin behaved as secretory PGs. Both HSPG and biglycan underwent catabolism, with HSPG possessing a t1/2 of 8 h and biglycan a t1/2 of 4 h. While the rate of HSPG turnover did not appreciably change between early and late culture, that of biglycan decreased. The mRNA for decorin was constant, while that of biglycan changed during culture. These results suggest that each PG possesses a distinct pattern of cellular and temporal distribution that may reflect specific stages in matrix formation and maturation.     

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.     

Biglycan and decorin differentially regulate signaling in the fetal membranes

Preterm birth is the leading cause of newborn mortality in the United States and about one third of cases are caused by preterm premature rupture of fetal membranes, a complication that is frequently observed in patients with Ehlers–Danlos Syndrome. Notably, a subtype of Ehlers–Danlos Syndrome is caused by expression of abnormal biglycan and decorin proteoglycans. As compound deficiency of these two small leucine-rich proteoglycans is a model of preterm birth, we investigated the fetal membranes of Bgn^−/−; Dcn^−/− double-null and single-null mice. Our results showed that biglycan signaling supported fetal membrane remodeling during early gestation in the absence of concomitant changes in TGFβ levels. In late gestation, biglycan signaling acted in a TGFβ-dependent manner to aid in membrane stabilization. In contrast, decorin signaling supported fetal membrane remodeling at early stages of gestation in a TGFβ-dependent manner, and fetal membrane stabilization at later stages of gestation without changes in TGFβ levels. Furthermore, exogenous soluble decorin was capable of rescuing the TGFβ signaling pathway in fetal membrane mesenchymal cells. Collectively, these findings provide novel targets for manipulation of fetal membrane extracellular matrix stability and could represent novel targets for research on preventive strategies for preterm premature rupture of fetal membranes.     

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 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.     

Proteoglycan and collagen expression during human air conducting system development

The lung is formed from a bud that grows and divides in a dichotomous way. A bud is a new growth center which is determined by epithelial-mesenchymal interactions where proteins of the extracellular matrix (ECM) might be involved. To understand this protein participation during human lung development, we examined the expression and distribution of proteoglycans in relation to the different types of collagens during the period in which the air conducting system is installed. Using light microscopy and immunohistochemistry we evaluate the expression of collagens (I, III and VI) and proteoglycans (decorin, biglycan and lumican) between 8 to 10 weeks post fertilization and 11 to 14 weeks of gestational age of human embryo and fetus lungs. We show that decorin, lumican and all the collagen types investigated were expressed at the epithelium-mesenchymal interface, forming a sleeve around the bronchiolar ducts. In addition, biglycan was expressed in both the endothelial cells and the smooth muscle of the blood vessels. Thus, the similar distribution pattern of collagen and proteoglycans in the early developmental stages of the human lung may be closely related to the process of dichotomous division of the bronchial tree. This study provides a new insight concerning the participation of collagens and proteoglycans in the epithelial-mesenchymal interface during the period in which the air conducting system is installed in the human fetal lung.Key words: human fetal lung, collagens, proteoglycans, extracellular matrix.     

Small leucine-rich proteoglycans orchestrate receptor crosstalk during inflammation

Inflammation is not only a defensive mechanism against microbial invasion, but also frequently represents a critical response to tissue injury under sterile conditions. It is now well established that tissue injury leads to the release of endogenous molecules of intra- and extracellular origin acting as damage-associated molecular patterns (DAMPs). The small leucine-rich proteoglycans (SLRPs) can act as powerful DAMPs following their proteolytical release from the extracellular matrix. Recent investigations of SLRP signaling networks revealed new levels of complexity, showing that SLRPs can cluster different types of receptors and orchestrate a host of downstream signaling events. This review will summarize the evidence for the multifunctional proinflammatory signaling properties of the two archetypal SLRPs, biglycan and decorin. These secreted proteoglycans link the innate to the adaptive immune response and operate in a broad biological context, encompassing microbial defense, tumor growth and autoimmunity.     

Small leucine-rich proteoglycans orchestrate receptor crosstalk during inflammation

Inflammation is not only a defensive mechanism against microbial invasion, but also frequently represents a critical response to tissue injury under sterile conditions. It is now well established that tissue injury leads to the release of endogenous molecules of intra- and extracellular origin acting as damage-associated molecular patterns (DAMPs). The small leucine-rich proteoglycans (SLRPs) can act as powerful DAMPs following their proteolytical release from the extracellular matrix. Recent investigations of SLRP signaling networks revealed new levels of complexity, showing that SLRPs can cluster different types of receptors and orchestrate a host of downstream signaling events. This review will summarize the evidence for the multifunctional proinflammatory signaling properties of the two archetypal SLRPs, biglycan and decorin. These secreted proteoglycans link the innate to the adaptive immune response and operate in a broad biological context, encompassing microbial defense, tumor growth and autoimmunity.     

Biglycan knockout mice: new models for musculoskeletal diseases

Biglycan is a Class I Small Leucine Rich Proteoglycans (SLRP) that is localized on human chromosome Xq28-ter. The conserved nature of its intron-exon structure and protein coding sequence compared to decorin (another Class I SLRP) indicates the two genes may have arisen from gene duplication. Biglycan contains two chondroitin sulfate glycosaminoglycan (GAG) chains attached near its NH₂ terminus making it different from decorin that has only one GAG chain. To determine the functions of biglycan in vivo, transgenic mice were developed that were deficient in the production of the protein (knockout). These mice acquire diminished bone mass progressively with age. Double tetracycline-calcein labeling revealed that the biglycan deficient mice are defective in their capacity to form bone. Based on this observation, we tested the hypothesis that the osteoporosis-like phenotype is due to defects in cells critical to the process of bone formation. Our data shows that biglycan deficient mice have diminished capacity to produce marrow stromal cells, the bone cell precursors, and that this deficiency increases with age. The cells also have reduced response to tranforming growth factor- (TGF-), reduced collagen synthesis and relatively more apoptosis than cells from normal littermates. In addition, calvaria cells isolated from biglycan deficient mice have reduced expression of late differentiation markers such as bone sialoprotein and osteocalcin and diminished ability to accumulate calcium judged by alizerin red staining. We propose that any one of these defects in osteogenic cells alone, or in combination, could contribute to the osteoporosis observed in the biglycan knockout mice. Other data suggests there is a functional relationship between biglycan and bone morphogenic protein-2/4 (BMP 2/4) action in controlling skeletal cell differentiation. In order to test the hypothesis that functional compensation can occur between SLRPs, we created mice deficient in biglycan and decorin. Decorin deficient mice have normal bone mass while the double biglycan/decorin knockout mice have more severe osteopenia than the single biglycan indicating redundancy in SLRP function in bone tissue. To further determine whether compensation could occur between different classes of SLRPs, mice were generated that are deficient in both biglycan (class I) and fibromodulin, a class II SLRP highly expressed in mineralizing tissue. These doubly deficient mice had an impaired gait, ectopic calcification of tendons and premature osteoarthritis. Transmission electron microscopy analysis showed that like the decorin and biglycan knockouts, they have severely disturbed collagen fibril structures. Biomechanical analysis of the affected tendons showed they were weaker compared to control animals leading to the conclusion that instability of the joints could be the primary cause of all the skeletal defects observed in the fibromodulin/biglycan knockout mice. These studies present important new animal models for musculoskeletal diseases and provide the opportunity to characterize the network of signals that control tissue integrity and function through SLRP activity. Published in 2003.