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

Abstract

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-β (TGF-β), also likely play an important role in growth control and muscle differentiation. Decorin, biglycan and betaglycan are proteoglycans that bind TGF-β during skeletal muscle differentiation. In this paper, we show that the binding of TGF-β to the receptors TGF-βRI and-βRII 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-β to TGF-βRI and -βRII increased concomitantly with receptors levels. Both the addition and re-expression of decorin, in these myoblasts, diminished the binding of TGF-β to its transducing receptors. Similar results were obtained when biglycan was added or over-expressed in Dcn null myoblasts. The binding of TGF-β to TGF-βRIII, 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-β to its transducing receptors. Transfection studies with the TGF-β-dependent promoter of the plasminogen activator inhibitor-1, coupled with luciferase, revealed that the addition of each proteoglycan diminished TGF-β-dependent activity, for both TGF-β1 and -β2. The modulation of TGF-β signaling by ECM proteoglycans diminishing the bio-availability of TGF-β for its transducing receptors appears to be a feasible mechanism for the attenuation of this inhibitory growth factor during skeletal muscle formation.

Introduction

In mammals, skeletal muscle formation continues postnatally during the growth of muscle masses and also occurs as a damage-induced regenerative response. Muscle regeneration helps to maintain muscle function during aging and delays the functional impairment caused by progressive neuromuscular diseases, such as Duchenne muscular dystrophy. Satellite cells, mononucleated cells located at the periphery of mature myofibers and beneath the basal lamina, 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.

Growth factors, such as transforming growth factor-β (TGF-β), insulin-like growth factors (IGF), hepatocyte growth factor (HGF) and members of the fibroblast growth factor (FGF) family, are among known extracellular signals for the control of skeletal muscle differentiation. A satellite cell line, derived from regenerating adult mouse skeletal muscle, C2C12, undergoes in vitro terminal myogenic differentiation, after serum removal from the culture medium (Yaffe and Saxel, 1977, Blau et al., 1985). The surrounding extracellular matrix (ECM) also plays an important role in growth control and differentiation. It acts not only as a scaffold for the cells, but also as a reservoir of growth factors and cytokines, regulating their activation status and turnover. In C2C12 cells, disorganization of the ECM, caused by the inhibition of proteoglycan sulfation, affects the proper progression of the myogenic program, independently of myogenin expression (Melo et al., 1996, Osses and Brandan, 2002). In vivo, muscular injuries that destroy the muscle basal lamina generally present a poorer functional recovery than injuries that minimally disrupt its integrity and orientation (Sanes, 2003).

TGF-β is an essential regulator in the processes of development, cell proliferation, and ECM deposition (Massague, 1998). This growth factor presents three structurally very similar mammalian isoforms: TGF-β1, TGF-β2 and TGF-β3, which belong to a superfamily of structurally-related proteins that comprises activins, inhibins, and bone morphogenic proteins (Gray et al., 2002). TGF-β regulates cellular processes by binding to three high-affinity cell surface receptors: TGF-β type I (TGF-βRI), type II (TGF-βRII) and type III receptors (TGF-βRIII, also known as betaglycan). A general mechanism for TGF-β signaling has been established, in which TGF-β is either presented to TGF-βRII receptors, or bound to them directly. Once activated by TGF-β, TGF-βRII recruits, binds and transphosphorylates TGF-βRI, thereby stimulating its protein kinase activity. Active TGF-βRI kinase then phosphorylates Smad-2 or Smad-3, permitting their association with Smad-4, as complexes that migrate to the cell nucleus and mediate the transcriptional regulation of TGF-β target genes. In this signaling pathway, Smad-7 acts as a strong inhibitor (Piek et al., 1999, Massague et al., 2000). Besides Smad-mediated transcription, TGF-β activates and crosstalks with other signaling cascades, including MAPK pathways, some of which regulate Smad activation, and others induce responses not related to transcription (Derynck and Zhang, 2003). Several levels have been described for the regulation of TGF-β signaling components, by measuring receptor turnover (regulated by ligand binding and Smurf/Smad-7 complexes) and the half-life of activated Smads (involving dephosphorylation and degradation) (Koli and Arteaga, 1997, Wells et al., 1997, Lo and Massague, 1999, Kavsak et al., 2000, Ebisawa et al., 2001). In addition, the transcriptional regulation of pathway components, such as Smad-7 and the TGF-β transducing receptors (TGF-βRI and-βRII), has also been reported (Nakao et al., 1997, Ammanamanchi et al., 1998, Ammanamanchi and Brattain, 2004).

Decorin, biglycan and betaglycan are proteoglycans that bind TGF-β, collagens and other ECM proteins (for review see (Ameye and Young, 2002) and (Iozzo, 1997)). In adult skeletal muscle tissue, decorin is the most abundant proteoglycan, and is found mainly in the perimysium (Brandan et al., 1992). The synthesis and expression of decorin are up-regulated during in vitro skeletal muscle differentiation (Brandan et al., 1991). On the other hand, biglycan is expressed by secondary myotubes during fetal muscle formation (Bianco et al., 1990), and in the adult, is preferentially located at the endomysium and certain neuromuscular junctions, probably complexed to the dystrophin-associated protein complex through its binding to α-dystroglycan (Bowe et al., 2000). We have shown that biglycan is transiently expressed in skeletal muscle fibers, after inducing muscle damage (Casar et al., 2004) and in biglycan knock-out mice, the muscular regeneration process is delayed in comparison to wild-type mice (Casar et al., 2004). Both decorin and biglycan are up-regulated in the ECM of the dystrophic muscles of mdx mice (Bowe et al., 2000, Caceres et al., 2000, Porter et al., 2004). Betaglycan is a heparan sulfate proteoglycan of 250–350 kDa, traditionally thought to function by binding TGF-β via its core protein (Esparza-Lopez et al., 2001) and then transferring the growth factor to its signaling receptor, TGF-βRII. This step is particularly important for the TGF-β2 isoform, which cannot bind TGF-βRII independently. Moreover, the expression and incorporation of betaglycan into the ECM are up-regulated during skeletal muscle differentiation (Lopez-Casillas et al., 2003). However, there is increasing evidence that the function of betaglycan is more complex. Normally, membrane-anchored betaglycan undergoes proteolytic processing in vivo, resulting in the release of a soluble ectodomain (Velasco-Loyden et al., 2004). This ectodomain can either enhance or inhibit signaling, depending on the levels of TGF-β (Fukushima et al., 1993), although in most cases, its binding is believed to sequester TGF-β, thereby functioning as a receptor antagonist (Andres et al., 1989, Lopez-Casillas et al., 1991, Philip et al., 1999). We have recently shown that betaglycan can transduce signals in a TGF-β-independent manner (Santander and Brandan, in press). Hence, ECM proteoglycans that bind TGF-β, such as decorin, biglycan and betaglycan, and are expressed in skeletal muscle, could be involved in modulating the amount of TGF-β present in the extracellular milieu, thereby modulating myogenic progression.

In this paper, we show that the binding of TGF-β to TGF-βRI and-βRII diminishes during skeletal muscle differentiation, despite the increase observed in total receptor levels. This is explained by the competition of the proteoglycans decorin, biglycan and betaglycan for TGF-β, modifying the bio-availability of this ligand and directly inhibiting TGF-β receptor-dependent signaling. The modulatory effect of these ECM proteoglycans represents another regulatory mechanism for the TGF-β-dependent signaling pathways, during skeletal muscle formation.