Browsing by Author "Brandan, E"

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    Antisense inhibition of decorin expression in myoblasts decreases cell responsiveness to transforming growth factor β and accelerates skeletal muscle differentiation
    (2001) Riquelme, C; Larraín, J; Schönherr, E; Henriquez, JP; Kresse, H; Brandan, E
    Decorin is a member of the family of the small leucine-rich proteoglycans. In addition to its function as an extracellular matrix organizer, it has the ability to activate the epidermal growth factor receptor, and it forms complexes with various isoforms of transforming growth factor beta (TGF-beta), Decorin is expressed during skeletal muscle differentiation and is up-regulated in dystrophic muscle. In this study we investigated the role of decorin in TGF-beta -dependent inhibition of myogenesis, To probe the function of decorin during myogenesis, C2C12 myoblasts were stably transfected with a plasmid expressing antisense decorin mRNA. The re; suiting inhibition of decorin expression led to the expression of myogenin, a master transcription factor for muscle differentiation, under growth conditions and accelerated skeletal muscle differentiation as determined by the expression of creatine kinase, In contrast myogenin expression was inhibited by adenovirally induced decorin expression or by adding exogenous decorin, Reduced synthesis of decorin resulted in a 7-fold decreased sensitivity to TGF-beta -mediated inhibition of myogenin expression. In contrast, adenovirally induced decorin expression in wild type cells resulted in a 5-fold increased sensitivity to TGF-beta -mediated inhibition of myogenin expression. Transfection studies with the TGF-beta -dependent promoter of the plasminogen activator inhibitor-1 coupled with luciferase revealed that the transducing receptors for TGF-beta1 and TGF-beta2 were involved in the different responses of wild type and anti-sense decorin myoblasts, These results demonstrate that a reduction of decorin expression or of decorin availability results in a decreased responsiveness to TGF-beta. These findings strongly suggest a new role for decorin during skeletal muscle terminal differentiation by activating TGF-beta -dependent signaling pathways.
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    Decorin core protein fragment Leu155-Val260 interacts with TGF-β but does not compete for decorin binding to type I collagen
    (1998) Schönherr, E; Broszat, M; Brandan, E; Bruckner, P; Kresse, H
    It has been shown that small proteoglycans containing leucine-rich repeats in their core proteins can form complexes with TGF-beta. Decorin, a ubiquitously found molecule of the extracellular matrix, is the best-studied example. Therefore, binding domains on its core protein were investigated using recombinant decorin fragments generated as fusion proteins in prokaryotes. The peptide Leu155-Val260 immobilized by the polyhistidine tag on a nickel chelate column bound TGF-beta 1 and -beta 2 almost as effectively as the largest fragment (Asp45-Lys359) studied. Other peptides were less effective. For the two peptides Asp45-Lys359 and Leu155-Val260 dissociation constants in the nanomolar range for high-affinity binding sites were calculated in a solid-phase assay with immobilized TGF-beta 2. Peptide Asp45-Lys359 also contained a lower affinity binding site. Domains with lower affinity were also found in peptides Asp45-Leu155 and Arg63-Gly190. Peptide Leu155-Val260 also formed complexes with TGF-beta in the liquid phase as determined by equilibrium gel filtration. Furthermore, F(ab') fragments of polyclonal antibodies against peptide Leu155-Val260 interfered with TGF-beta binding to peptide Asp45-Lys359 in a dose-dependent manner. Peptide Leu155-Val260, however, is only a weak competitor of the binding of wild-type decorin to reconstituted type I collagen fibrils. Therefore, independent binding sites of decorin for TGF-beta and type I collagen should exist. In support of this hypothesis saturable binding of TGF-beta 1 and TGF-beta 2 to collagen-bound native decorin could be demonstrated. The bound cytokine could be released in a biologically active form by collagenase treatment. Thus, decorin may play a biological role in storing this cytokine temporarily in the extracellular matrix and in thereby modulating an interaction of TGF-beta with its signaling receptors. (C) 1998 Academic Press.
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    Extracellular matrix is required for skeletal muscle differentiation rut not myogenin expression
    (1996) Melo, F; Carey, DJ; Brandan, E
    Skeletal muscle cells are a useful model for studying cell differentiation. Muscle cell differentiation is marked by myoblast proliferation followed by progressive fusion to form large multinucleated myotubes that synthesize muscle-specific proteins and contract spontaneously. The molecular analysis of myogenesis has advanced with the identification of several myogenic regulatory factors, including myod1, myd, and myogenin. These factors regulate each other's expression and that of muscle-specific proteins such as the acetylcholine receptor and acetylcholinesterase (AChE). In order to investigate the role of extracellular matrix (ECM) in myogenesis we have cultured myoblasts (C2C12) in the presence or absence of an exogenous ECM (Matrigel). In addition, we have induced differentiation of myoblasts in the presence or absence of Matrigel and/or chlorate, a specific inhibitor of proteoglycan sulfation. Our results indicated that the formation of fused myotubes and expression of AChE was stimulated by Matrigel. Treatment of myoblasts induced to differentiate with chlorate resulted in an inhibition of cell fusion and AChE activity. Chlorate treatment was also found to inhibit the deposition and assembly of ECM components such fibronectin and laminin. The expression of myogenin mRNA was observed when myoblasts were induced to differentiate, but was unaffected by the presence of Matrigel or by culture of the cells in the presence of chlorate. These results suggest that the expression of myogenin is independent of the presence of ECM, but that the presence of ECM is essential for the formation of myotubes and the expression of later muscle-specific gene products. (C) 1996 Wiley-Liss, Inc.
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    Inhibition of myoblast migration via decorin expression is critical for normal skeletal muscle differentiation
    (2003) Olguin, HC; Santander, C; Brandan, E
    During limb skeletal muscle formation, committed muscle cells proliferate and differentiate in the presence of extracellular signals that stimulate or repress each process. Proteoglyeans are extracellular matrix organizers and modulators of growth factor activities, regulating muscle differentiation in vitro. Previously, we characterized proteoglycan expression during early limb muscle formation and showed a spatiotemporal relation between the onset of myogenesis and the expression of decorin, an important muscle extracellular matrix component and potent regulator of TGF-beta activity. To evaluate decorin's role during in vivo differentiation in committed muscle cells, we grafted wild type and decorin-null myoblasts onto chick limb buds. The absence of decorin enhanced the migration and distribution of myoblasts in the limb, correlating with the inhibition of skeletal muscle differentiation. Both phenotypes were reverted by de novo decorin expression. In vitro, we determined that both decorin core protein and its glycosaminoglycan chain were required to reverse the migration phenotype. Results presented here suggest that the enhanced migration observed in decorin-null myoblasts may not be dependent on chemotactic growth factor signaling nor the differentiation status of the cells. Decorin may be involved in the establishment and/or coordination of a critical myoblast density, through inhibition of migration, that permits normal muscle differentiation during embryonic myogenesis. (C) 2003 Elsevier Science (USA). All rights reserved.
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    Interaction between Alzheimer's disease beta A4 precursor protein (APP) and the extracellular matrix: Evidence for the participation of heparan sulfate proteoglycans
    (1997) Caceres, J; Brandan, E
    The interaction between the Alzheimer amyloid precursor protein (APP) and an intact extracellular matrix (ECM), matrigel, obtained from Engelbreth-Holm-Swarm tumors was evaluated. Based on quantitative analyses of the binding data obtained from solid phase binding assays, two binding sites on the ECM were identified for [I-125]-APP (with apparent Kd(1) of 1.0 x 10(-11) M and Kd(2) of 1.6 x 10(-9) M respectively). Over 70% of [I-125]-APP was displaced by heparin and N-desulfated heparin but not by chondroitin sulfate. Pretreatment of matrigel with heparitinase decreased the binding of [I-125]-APP by 80%. beta-amyloid peptides (residues 1-40, 1-28, and 1-16) containing a heparin binding domain also displaced 80% of bound [I-125]-APP, which was totally displaced by intact APP. The binding of [I-125]-APP to matrigel increased by 210% with a decrease in the pH. These observations suggest that [I-125]-APP interacts mainly with heparan sulfate proteoglycan present in the ECM. The binding of [I-125]-APP to individual ECM components was also analyzed. [I-125]-APP was found to bind laminin and collagen type IV but not fibronectin. However, when these ECM constituents were combined, the extent of APP-binding decreased significantly, to levels comparable to those obtained with intact matrigel, suggesting that multiple interactions may occur between ECM constituents and [I-125]-APP. The results are discussed in terms of APP function and amyloidogenesis. (C) 1997 Wiley-Liss, Inc.
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    Syndecan-1 expression is down-regulated during myoblast terminal differentiation - Modulation by growth factors and retinoic acid
    (1997) Larrain, J; CizmeciSmith, G; Troncoso, V; Stahl, RC; Carey, DJ; Brandan, E
    Syndecan-1 is an integral membrane proteoglycan involved in the interaction of cells with extracellular matrix proteins and growth factors. It is transiently expressed in several condensing mesenchymal tissues after epithelial induction. In this study we evaluated the expression of syndecan-1 during skeletal muscle differentiation. The expression of syndecan-1 as determined by Northern blot analyses and immunofluorescence microscopy is down-regulated during differentiation. The transcriptional activity of a syndecan-1 promoter construct is also down-regulated in differentiating muscle cells. The decrease in syndecan-1 gene expression is not dependent on the presence of E-boxes, binding sites for the MyoD family of transcription factors in the promoter region, or myogenin expression. Deletion of the region containing the E-boxes or treatment of differentiating cells with sodium butyrate, an inhibitor of myogenin expression, had no effect on syndecan-1 expression. Basic fibroblast growth factor and transforming growth factor type beta, which are inhibitors of myogenesis, had little effect on syndecan-1 expression. When added together, however, they induced syndecan-1 expression. Retinoic acid, an inducer of myogenesis, inhibited syndecan-1 expression and abolished the effect of the growth factors. These results indicate that syndecan-1 expression is down-regulated during myogenesis and that growth factors and retinoic acid modulate syndecan-1 expression by a mechanism that is independent of myogenin.
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    Synthesis of proteoglycans is augmented in dystrophic mdx mouse skeletal muscle
    (2000) Cáceres, S; Cuellar, C; Casar, JC; Garrido, J; Schaefer, L; Kresse, H; Brandan, E
    Mdx mice uniquely recover from degenerative dystrophic lesions through an intense myoproliferative response. The onset and progression of this process are controlled by a complex set of interactions between myoblasts and their environment. The presence of the extracellular matrix is essential for normal myogenesis. Proteoglycans are abundant components of the extracellular matrix. The synthesis of proteoglycans in mdx mice during skeletal muscle regeneration was evaluated. Incorporation of radioactive sulfate demonstrated a significant increase in the synthesis of several types of proteoglycans in mdx animals compared to age-matched controls. The size and charge of proteoglycans synthesized by the mdx mice remained unchanged. In particular, one of the up-regulated proteoglycans, the small chondroitin/dermatan sulfate proteoglycan decorin which is known to bind and to sequester transforming growth factor-beta, was investigated. Immunocytolocalization and in situ hybridization studies showed that decorin mainly accumulated in the endomysium, i.e. around individual skeletal muscle fibers from M. titialis anterior and diaphragm.