Role of proteoglycans in the regulation of the skeletal muscle fibrotic response

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(1)REVIEW ARTICLE. Role of proteoglycans in the regulation of the skeletal muscle fibrotic response Enrique Brandan and Jaime Gutierrez n, CARE, Departamento de Biologıa Celular y Molecular, Facultad de Ciencias Biolo gicas, Centro de Envejecimiento y Regeneracio lica de Chile, Santiago, Chile P. Universidad Cato. Keywords connective tissue growth factor (CTGF); fibrosis; muscular diseases; proteoglycans; transforming growth factor-b (TGF-b) Correspondence E. Brandan, Departamento de Biologıa Celular y Molecular, Facultad de Ciencias gicas, P. Universidad Cato lica de Chile, Biolo Casilla 114-D, Santiago, Chile Fax: +56 2 26355395 Tel: +56 2 26862725 E-mail: (Received 31 January 2013, revised 30 March 2013, accepted 3 April 2013). Myogenesis consists of a highly organized and regulated sequence of cellular processes aimed at forming or repairing muscle tissue. Several processes occur during myogenesis, including cell proliferation, migration, and differentiation. Cytokines, proteinases, cell adhesion molecules and growth factors are involved, either activating or inhibiting these events, and are modulated by a group of molecules called proteoglycans (PGs), which play critical roles in skeletal muscle physiology. Particularly interesting are some of the factors responsible for the fibrotic response associated with skeletal muscular dystrophies. Transforming growth factor-b and connective tissue growth factor have gained great attention as factors participating in the fibrotic response in skeletal muscle. This review is focused on the advances achieved in understanding the roles of proteoglycans as modulators of profibrotic growth factors in fibrosis associated with diseases such as skeletal muscle dystrophies.. doi:10.1111/febs.12278. Introduction Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes aimed at forming or repairing muscle tissue. This sequence includes cell proliferation, migration, and differentiation. Proteoglycans (PGs) play critical roles in skeletal muscle physiology. Structurally, they are composed of a core protein to which glycosaminoglycan (GAG) chains are covalently attached (chondroitin/dermatan, keratan, and heparan, among others), giving them various and different specific activities. PGs can be found associated with the plasma membrane and the extracellular matrix (ECM). The skeletal muscles express different heparan sulfate PGs (HSPGs) and the small leucine-rich PGs (SLRPs).. Among the ligands described for PGs are cytokines, proteinases, cell adhesion molecules, and growth factors. Particularly interesting are the factors involved in the fibrotic response associated with skeletal muscular dystrophies. Transforming growth factor-b (TGF-b) and connective tissue growth factor (CTGF) have gained great attention as factors involved in the fibrotic response in skeletal muscle. PGs have been implicated in several diseases. For instance, SLRPs affect several signaling pathways that are directly involved in the control of cell growth, morphogenesis, and immunity [1]. Other PGs, such as brevicam, perlecan, glypicans, and some members of the syndecan family, are involved in several malignancies [2].. Abbreviations CTGF, connective tissue growth factor; DMD, Duchenne muscular dystrophy; ECM, extracellular matrix; ERK, extracellular signal-related kinase; GAG, glycosaminoglycan; HSPG, heparan sulfate proteoglycan; LRP, lipoprotein receptor-related protein; LRR, leucine-rich repeat; PG, proteoglycan; rhBGN, recombinant human biglycan; SLRP, small leucine-rich proteoglycan; TGF-b-RI, transforming growth factor-b receptor type I; TGF-b, transforming growth factor-b.. FEBS Journal 280 (2013) 4109–4117 ª 2013 The Authors Journal compilation ª 2013 FEBS. 4109.

(2) Regulation of fibrosis by proteoglycans. E. Brandan and J. Gutierrez. This review is focused on the advances achieved in understanding the roles of PGs as modulators of profibrotic growth factors in fibrosis associated with diseases such as skeletal muscle dystrophies. Other, more extensive, reviews may serve to fill the gaps found here [3–11].. Skeletal muscular dystrophies and fibrosis Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by the absence of the cytoskeletal protein dystrophin, as in the DMD mouse model and the mdx mouse. This leads to the loss of the anchoring of the myofiber to the basal lamina, eliciting subsequent myofiber necrosis and degeneration, which, in turn, results in a progressive loss of muscle mass, weakness, and increased ECM accumulation or fibrosis [12–15]. Fibrotic scar tissues are observed early in DMD patients, and increase with age [16,17]. Fibrosis corresponds to a process that is characteristic of several chronic diseases, involving the replacement of functional cells by nonfunctional connective tissue enriched in ECM, leading to worsening of the disease [18,19]. Several factors are thought to be involved in the fibrotic process. In the skeletal muscle of DMD patients or mdx models, the levels of TGF-b and CTGF correlate with the development of skeletal muscle fibrosis [20–23]. Increased mRNA levels for these growth factors have been described in a dog model of DMD [24] and in mdx skeletal muscles [25]. TGF-b is an essential regulator during development, inflammation, cell proliferation and ECM deposition processes [26]. In skeletal muscle cells, TGF-b acts as a strong myogenesis inhibitor. It is also known that TGF-b can inhibit myoblast differentiation in vitro, affecting the expression of muscle proteins such as myosin heavy chain and creatine kinase [26,27]. TGF-b regulates the above cellular processes by binding to three high-affinity cell surface receptors: TGF-b receptor type I (TGF-b-RI), TGF-b receptor type II, and TGF-b receptor type III, an HSPG known as b-glycan. A general mechanism for TGF-b signaling has been established, in which TGF-b binds to the cell surface receptors, activating the Smad canonical pathway [28]. Besides the Smad-mediated TGF-b signaling, [29]. TGF-b also can activate the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways [29]. CTGF is one of the factors involved in the induction of scarring, wound healing and fibrosis in several fibrodegeneration-associated diseases [30,31]. Under normal conditions, CTGF is not expressed; however, 4110. several factors, such TGF-b, hepatocyte growth factor, vascular endothelial growth factor, angiotensin II, endothelin, and hypoxia, enhance its expression [19,31]. CTGF levels are strongly correlated with the degree and severity of the fibrotic process in tissues, such as in skin disorders [32], advanced atherosclerotic lesions [33], lung fibrosis [34], renal fibrosis [35], chronic pancreatitis [36], and hepatic fibrosis [37], and the role of CTGF in muscle damage and fibrosis has recently been described [23]. A canonical CTGF-transducing receptor has not been described, but it is known that CTGF interacts with different membrane proteins, which mediate its cellular effects (see below).. PGs in skeletal muscular dystrophic diseases One of the features of several skeletal muscle dystrophies is the accumulation of ECM around skeletal muscle fibers and the interstitial space. This fibrosis is regularly associated with or characterized by a persistent inflammatory process, which is accompanied by the replacement of the functional skeletal muscle fibers by an excess of ECM proteins, persistently and severely affecting skeletal muscle physiology in several dystrophic diseases [11,15,18,38–40]. Among the PGs whose levels are are increased in skeletal muscular dystrophies or cells isolated from skeletal muscle biopsies, the SLRPs decorin and biglycan, composed of chondroitin/dermatan sulfate GAG chains, are the most abundant [12,41]. The same is true in skeletal muscle samples from the mdx mouse [38,40,42,43], and increased levels of HSPGs in mdx skeletal muscles have been described [16]. Remarkably, biglycan regulates the expression of utrophin, the synaptic form of dystrophin, in immature muscle, and recombinant human biglycan (rhBGN) increases utrophin expression in cultured myotubes [44]. Quite extraordinary, systemically delivered rhBGN upregulates utrophin at the sarcolemma and decreases muscle pathology in the mdx mouse model of DMD, suggesting that rhBGN has potential as a treatment for DMD [44]. At least two hypotheses can be considered concerning the relevance of the augmented levels of PGs in dystrophic disease: as part of the exacerbated accumulation of ECM constituents that is characteristic of the fibrotic process; or as a regulatory cell response to increases in the levels of profibrotic growth factors such as TGF-b [45,46] and CTGF (see below), regulating their bioavailability as part of a possible protective or autoregulatory mechanism [47–51]. The exact physiological relevance of this PG accumulation requires further investigation.. FEBS Journal 280 (2013) 4109–4117 ª 2013 The Authors Journal compilation ª 2013 FEBS.

(3) E. Brandan and J. Gutierrez. TGF-b and CTGF profibrotic factors in skeletal muscular dystrophies – a symphony of common elements Fibrosis is a hallmark of skeletal muscular dystrophies. Several inducers of fibrosis have been described for many fibrotic diseases, including hypoxia [52], endothelin [53], angiotensin II [54], and the growth factors TGF-b [55] and CTGF [56].. Regulation of fibrosis by proteoglycans. contains 12 leucine-rich repeats (LRRs), LRR1– LRR12 [1,3,4]. The decorin region responsible for the interaction between TGF-b–decorin and LRP-1 was determined by the use of decorin deletion mutants and peptides derived from internal LRR regions. LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-b [75] (Fig. 1). Remarkably, the LRR6. TGF-b TGF-b levels are increased in DMD [20,46,57–60]. Increased TGF-b activity regulates miR-21 expression and fibrosis in DMD patients and mdx muscles [57]. Mdx macrophages produce elevated levels of TGF-b in response to fibrinogen. This TGF-b acts on fibroblasts, increasing the accumulation of collagen, which is exacerbated by fibrinogen [17]. Several attempts to inhibit TGF-b activity during skeletal muscle damage in muscular dystrophies have been reported. These comprise expression of TGF-bsequestering proteins by gene transfer [61], inhibition of ligand binding to the receptors [62], targeting of TGF-b receptor expression [63], the use of inhibitory plant-derived drugs [64], and the use of decorin to inhibit TGF-b [61,65–68]. Of particular interest is the SLRP decorin, because of its ability to bind TGF-b and regulate the cell response to TGF-b [26,69,70] and other ligands [3,4,8,71]. Decorin is upregulated during skeletal muscle differentiation, and this seems to be necessary to sequester TGF-b and myostatin, two potent myogenic inhibitors [45,72,73]; this removal by decorin seems to be essential for successful skeletal muscle differentiation. However, TGF-b regulation by decorin is more complex; under proliferative conditions, the undifferentiated myoblasts require decorin for a full TGF-b cell response, by a mechanism that is dependent on the giant receptor lipoprotein receptor-related protein (LRP)1 [74,75]. Decorin also seems to be necessary for myogenesis, as antisense inhibition of its expression in myoblasts accelerates skeletal muscle differentiation by decreasing the sensitivity to TGF-b signaling [26]; further analysis is required to clearly establish the role of decorin in each step of muscle differentiation. As mentioned, the full cell response to TGF-b-mediated signaling [74,76] depends on its canonical transducing receptors (TGF-b-RI and TGF-b receptor type II) and the cell surface complex of decorin and LRP-1, which is an endocytic receptor for decorin [74,76]. This novel mechanism of signaling requires the Smad canonical pathway and AKT [74]. Decorin. Fig. 1. The profibrotic growth factors TGF-b and CTGF share several elements, suggesting a common signaling/regulatory complex. Full TGF-b signaling activity depends on binding to the canonical transducing receptors (TGF-b receptors), activating the Smad pathway (red signals), and to the decorin–LRP-1 receptor complex, which, upon endocytosis, activates the phosphoinositide 3-kinase-dependent pathway (purple signals). The role of b-glycan in canonical Smad signaling is omitted for simplicity (see text for details). CTGF binds to several proteins, among them integrins that would activate the fibrotic signaling response (green signals). Furthermore, TGF-b and CTGF bind to decorin, probably stabilizing their interaction and potentiating their signaling. This decorin–TGFb–CTGF complex can be sequestered in the ECM, acting as a reservoir of the ligands, or be associated with other cell surface proteins. In the latter scenario, the decorin–TGF-b–CTGF complex would interact with LRP-1, which also interacts with the HSPG b-glycan through GAG chains. b-Glycan also binds TGF-b via its core protein and CTGF via its GAG chains. All of these proteins might form a regulatory signaling complex that is critical for modulating the biological activity of these two profibrotic growth factors. The top part of the figure shows a magnification of the indicated box indicating in detail the interaction between decorin, CTGF and TGF-b.. FEBS Journal 280 (2013) 4109–4117 ª 2013 The Authors Journal compilation ª 2013 FEBS. 4111.

(4) Regulation of fibrosis by proteoglycans. E. Brandan and J. Gutierrez. internal region, composed of 11 amino acids, is responsible for decorin binding to LRP-1 and subsequent TGF-b-dependent signaling [75]. Furthermore, with an in vivo approach, the LRR6 region of decorin can inhibit TGF-b-mediated action in response to skeletal muscle injury [75]. As already mentioned, decorin localized in the ECM is able to immobilize TGF-b [45] and myostatin [68,77], thus concentrating these growth factors at the ECM and acting as growth factor reservoirs; these factors could be released under pathological conditions. Interestingly, improved muscle healing with reduced fibrosis was observed in myostatin-null mice [78]. Besides the TGF-b receptors and the decorin–LRP-1 complex, TGF-b has another cell surface receptor, the HSPG b-glycan or TGF-b receptor type III (see above). This HSPG has two independent high-affinity binding domains for TGF-b, one in the membrane-distal half and the other in the membrane-proximal half of the b-glycan ectodomain [79]. Thus, b-glycan can present TGF-b to the type II signaling receptor to activate the canonical Smad signaling pathway [80], but also, when it is overexpressed, is able to activate TGF-b signaling by a mechanism that is independent of the ligand but dependent on the kinase activity of TGF-b-RI [81]. CTGF CTGF induces fibrosis in skeletal muscle both in vitro and in vivo. Increased levels of CTGF mRNA have been reported in the skeletal muscle from dystrophic dogs [24] and mdx mice [13]. Increased amounts of CTGF are found in skeletal muscle biopsies from DMD patients [22]. Myoblasts and myotubes produce and respond to CTGF, increasing ECM accumulation and ERK-1/2 phosphorylation [50]. This CTGF-mediated ERK-1/2 phosphorylation is strongly inhibited by heparin, suggesting a role for HSPG in its mode of action [49,50]. Overexpression of CTGF in normal mice, by use of an adenovirus containing the CTGF mouse sequence, induced extensive skeletal muscle damage followed by regeneration, with an increase in the levels of fibrotic markers (fibronectin, collagen, and a-smooth muscle actin) [23]. This overexpressed CTGF also caused a decrease in the specific isometric contractile muscle force. When CTGF overexpression stopped, the entire phenotype proved to be reversible, in parallel with normalization of CTGF levels [23]. Overexpression of CTGF in skeletal muscle, together with infusion of an angiotensin receptor blocker (losartan), decreased the CTGF-mediated increases in the levels of ECM molecules and a-smooth muscle actin, 4112. and ERK-1/2 phosphorylation levels. Notably, losartan was able to prevent the loss of contractile force of muscles overexpressing CTGF [82]. CTGF is a complex protein and, as mentioned, does not have a traditional high-affinity receptor to activate a signal transduction pathway. However, it is able to interact with several proteins present on the cell surface, such the ECM proteins, fibronectin [83], HSPGs [49], and several growth factors, TGF-b among them [84]. Also, CTGF interacts with plasma membranebound proteins and receptors such as TrkA [85], LRP-1 [86], LRP-6 [87], and integrins [88], participating in many mechanisms at the same time [31,48,56,89]. Because of this complex network of interactions, the cell response to CTGF is highly context-dependent and environment-dependent [90]; thus, it has been reported that CTGF induces the expression of fibronectin, but, when the amount of fibronectin reaches a certain level, the cell response to CTGF is notably affected [43,88], apparently by a mechanism that depends on the competition between fibronectin and CTGF for av-subunit-containing integrin [88]. Efforts have been concentrated on inhibiting CTGF profibrotic activity. Inhibition of CTGF by small interfering RNA prevents liver fibrosis in rats [91]. Specific downregulation of CTGF in the kidney with antisense oligonucleotides attenuates the progression of nephropathy in mouse models of type 1 and type 2 diabetes [92], and CTGF antisense inhibition decreases hypertrophic scarring [93]. The use of mAbs (FG-3019) has given positive results in diminishing fibrosis in a model of multiorgan fibrosis induced by repeated intraperitoneal injections of CTGF and TGF-b [94] and in diabetic nephropathy [95]. Dystrophic mice treated with FG-3019 showed decreased fibrosis, less skeletal muscle damage, and an improvement in the capacity to generate skeletal muscle strength. Moreover, if fibrosis is diminished by targeting CTGF, the efficiency of muscle stem cell therapy in the dystrophic mdx mouse is augmented (manuscript under preparation). Decorin-null myoblasts are more sensitive to CTGF, producing more fibronectin and collagen III than wildtype myoblasts, suggesting that decorin is an endogenous inhibitor of the fibrotic effects of CTGF. Furthermore, decorin exogenously added to myoblasts and fibroblasts negatively regulated CTGF profibrotic activity. CTGF interacts with decorin in a saturable manner, with a Kd of 4.4 nM. By the use of decorin deletion mutants, the region where decorin binds to CTGF has been determined; thus, a mutant form of decorin without LRR10–LRR12 is unable to bind CTGF and inhibit its fibrotic effects. Moreover, a. FEBS Journal 280 (2013) 4109–4117 ª 2013 The Authors Journal compilation ª 2013 FEBS.

(5) E. Brandan and J. Gutierrez. peptide derived from LRR12 was able to inhibit CTGF–decorin complex formation and directly inhibit CTGF activity, indicating that decorin binds to CTGF via decorin’s LRR12, and suggesting that this CTGF domain could be somehow involved in its recognition by one of the CTGF receptors related to the induction of the fibrotic response [50]. All of these findings suggest that decorin interacts with CTGF and regulates its biological activity [96].. Common elements The fact that decorin binds CTGF and TGF-b at different decorin LRR domains, modulating their biological activity, opens a new avenue of research, with unexpected results. CTGF is able to interact with receptors located on the cell surface, including integrins [88], LRP-1 [97], and HSPGs [49,50]. In addition, CTGF binds TGF-b, stimulating its biological activity [84]. The role of CTGF in regulating the complex interactions of TGF-b signaling is poorly understood. We speculate that decorin could be a regulatory protein for both profibrotic factors, through the interaction with one of the common receptors, LRP-1 [51,74– 76,86]. b-Glycan and/or other HSPGs could also form part of this complex of common elements. As mentioned, b-glycan binds TGF-b via its core protein, but also can bind CTGF via its heparan sulfate GAG chains [49], and at the same time, can bind to the heparan sulfate-binding domains of LRP-1 [98] (Fig. 1). These complex and interesting interactions among these two profibrotic growth factors, together with several common elements, could be essential for revealing critical steps in signaling followed by triggering of fibrotic responses, and could eventually lead to the development of specific inhibitors against these profibrotic factors with potential use in therapy.. Future perspectives Profibrotic factors are augmented in skeletal muscular dystrophies, and interact in specific ways with different PGs. Their role in skeletal muscle repair associated with skeletal muscular dystrophies is just emerging, offering a basis for future studies designed to provide insights into the molecular interactions among these PGs with versatile growth factors such as TGF-b and CTGF and their common receptors. Future studies focused on understanding the interactions among these molecules will permit the discovery of novel targets for the treatment of fibrosis associated with skeletal muscular dystrophies and the fibrotic response associated with other chronic diseases.. Regulation of fibrosis by proteoglycans. Acknowledgements This study was supported by research grants CARE PFB12/2007, FONDECYT 1110426, CONICYT 79090027, FONDECYT 11110010, and Fundaci on Chilena para Biologıa Celular Proyecto MF-100.. References 1 Iozzo RV & Schaefer L (2010) Proteoglycans in health and disease: novel regulatory signaling mechanisms evoked by the small leucine-rich proteoglycans. FEBS J 277, 3864–3875. 2 Theocharis AD, Skandalis SS, Tzanakakis GN & Karamanos NK (2010) Proteoglycans in health and disease: novel roles for proteoglycans in malignancy and their pharmacological targeting. FEBS J 277, 3904–3923. 3 Brandan E, Cabello-Verrugio C & Vial C (2008) Novel regulatory mechanisms for the proteoglycans decorin and biglycan during muscle formation and muscular dystrophy. Matrix Biol 27, 700–708. 4 Iozzo RV (1999) The biology of the small leucine-rich proteoglycans. Functional network of interactive proteins. J Biol Chem 274, 18843–18846. 5 Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J & Zako M (1999) Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 68, 729–777. 6 Jenniskens GJ, Veerkamp JH & van Kuppevelt TH (2006) Heparan sulfates in skeletal muscle development and physiology. J Cell Physiol 206, 283–294. 7 Filmus J, Capurro M & Rast J (2008) Glypicans. Genome Biol 9, 224, doi:10.1186/gb-2008-9-5-224. 8 Iozzo RV & Schaefer L (2010) Proteoglycans in health and disease: novel regulatory signaling mechanisms evoked by the small leucine-rich proteoglycans. FEBS J 277, 3864–3875. 9 Gillies AR & Lieber RL (2011) Structure and function of the skeletal muscle extracellular matrix. Muscle Nerve 44, 318–331. 10 Iozzo RV & Karamanos N (2010) Proteoglycans in health and disease: emerging concepts and future directions. FEBS J 277, 3863, doi:10.1111/j.1742-4658. 2010.07796.x. 11 Mann CJ, Perdiguero E, Kharraz Y, Aguilar S, Pessina P, Serrano AL & Munoz-Canoves P (2011) Aberrant repair and fibrosis development in skeletal muscle. Skelet Muscle 1, 21, doi:10.1186/2044-5040-1-21. 12 Fadic R, Mezzano V, Alvarez K, Cabrera D, Holmgren J & Brandan E (2006) Increase in decorin and biglycan in Duchenne muscular dystrophy: role of fibroblasts as cell source of these proteoglycans in the disease. J Cell Mol Med 10, 758–769.. FEBS Journal 280 (2013) 4109–4117 ª 2013 The Authors Journal compilation ª 2013 FEBS. 4113.

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Fig. 1. The profibrotic growth factors TGF- b and CTGF share several elements, suggesting a common signaling/regulatory complex
Fig. 1. The profibrotic growth factors TGF- b and CTGF share several elements, suggesting a common signaling/regulatory complex p.3