Browsing by Author "Santander, Cristian"

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    Betaglycan induces TGF-β signaling in a ligand-independent manner, through activation of the p38 pathway
    (2006) Santander, Cristian; Brandan, Enrique
    Betaglycan, a cell surface heparan sulphate proteoglycan, is traditionally thought to function by binding transforming growth factor type (TGF-beta) via its core protein and then transferring the growth factor to its signaling receptor, the type II receptor. However, there is increasing evidence that the function of betaglycan is more complex. Here, we have evaluated the role of betaglycan through adenoviral expression (Adv-BG) in myoblasts and fibroblasts and found that in Adv-BG-infected cells, the activity of p3TP-Lux and pCTGF-Luc reporter after transient transfection, as well as fibronectin synthesis, all of which are target processes for TGF-beta, were highly increased in the absence of TGF-beta. It is known that this cytokine strongly inhibits myogenin induction in myoblasts. In Adv-BG-infected myoblasts, the activity of pMyo-Luc reporter after transient transfection was strongly inhibited in the absence of TGF-beta. These effects were not precluded by applying TGF-beta-blocking antibodies, the soluble TGF-B type II receptor, or soluble betaglycan to sequester TGF-beta present in the cell medium. Furthermore, the data suggest that the cytoplasmic domain of betaglycan is required for this TGF-beta-independent response, giving further support to a ligand-independent signaling effect for betaglycan. The process also seemed independent of Smad-2 phosphorylation, although Adv-BG infection induced p38 phosphorylation, and SB239063, an inhibitor of the p38 pathway, inhibited p3TP-Lux-driven activity. These results suggest a novel signaling mechanism for betaglycan, which is independent of the canonical TGF-beta signal pathway although it involves TGF-beta receptors and takes place through p38 pathways. (c) 2005 Elsevier Inc. All rights reserved.
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    Syndecan-4 and β1 integrin are regulated by electrical activity in skeletal muscle: Implications for cell adhesion
    (2010) Ugarte, Gonzalo; Santander, Cristian; Brandan, Enrique
    Syndecan-4 and integrins are involved in the cell migration and adhesion processes in several cell types. Syndecan-4, a transmembrane heparan sulfate proteoglycan, is associated to focal adhesions in adherent cells and has been described as a marker of satellite cells in skeletal muscle. In this tissue, beta 1 integrin forms heterodimers with alpha 5 and alpha 6 during myoblast differentiation and with alpha 7 in adult muscle. Here, we show that the levels of these two cell surface membrane molecules are regulated by spontaneous electrical activity during the differentiation of rat primary myoblasts. Syndecan-4 and beta 1 integrin protein levels decrease after the inhibition of electrical activity using tetrodotoxin (TTX). Syndecan-4 also decreases substantially in denervated rat tibialis anterior muscle. Indirect immunofluorescence analysis shows that syndecan-4 and beta 1 integrin co-localize with vinculin, a molecular marker of costameres in skeletal muscle myofibers. Co-localization is lost in inactive myotubes adopting a diffuse pattern, suggesting that the costameric organization is disrupted in TTX-treated myotubes. Moreover, the inhibition of spontaneous electrical activity decreases myotube cell adhesion. In summary, this work shows that syndecan-4 and beta 1 integrin protein levels and their localization in costameric structures are regulated by electrical activity and suggests that this regulatory mechanism influences the adhesion properties of skeletal myotubes during differentiation. (C) 2010 Elsevier B.V. All rights reserved.
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    TGF-β receptors, in a Smad-independent manner, are required for terminal skeletal muscle differentiation
    (2010) Droguett, Rebeca; Cabello-Verrugio, Claudio; Santander, Cristian; Brandan, Enrique
    Skeletal muscle differentiation is strongly inhibited by transforming growth factor type beta (TGF-beta), although muscle formation as well as regeneration normally occurs in an environment rich in this growth factor. In this study, we evaluated the role of intracellular regulatory Smads proteins as well as TGF-beta-receptors (TGF-beta-Rs) during skeletal muscle differentiation. We found a decrease of TGF-beta signaling during differentiation. This phenomenon is explained by a decline in the levels of the regulatory proteins Smad-2, -3, and -4, a decrease in the phosphorylation of Smad-2 and lost of nuclear translocation of Smad-3 and -4 in response to TGF-beta. No change in the levels and inhibitory function of Smad-7 was observed. In contrast, we found that TGF-beta-R type I (TGF-beta-RI) and type II (TGF-beta-RII) increased on the cell surface during skeletal muscle differentiation. To analyze the direct role of the serine/threonine kinase activities of TGF-beta-Rs, we used the specific inhibitor SB 431542 and the dominant-negative form of TGF-beta-RII lacking the cytoplasmic domain. The TGF-beta-Rs were important for successful muscle formation, determined by the induction of myogenin, creatine kinase activity, and myosin. Silencing of Smad-2/3 expression by specific siRNA treatments accelerated myogenin, myosin expression, and myotube formation: although when SB 431542 was present inhibition in myosin induction and myotube formation was observed, suggesting that these last steps of skeletal muscle differentiation require active TGF-beta-Rs. These results suggest that both down-regulation of Smad regulatory proteins and cell signaling through the TGF-beta receptors independent of Smad proteins are essential for skeletal muscle differentiation. (C) 2010 Elsevier Inc. All rights reserved.