Wnt Signaling in Skeletal Muscle Dynamics: Myogenesis, Neuromuscular Synapse and Fibrosis

Mol Neurobiol (2014) 49:574–589 DOI 10.1007/s12035-013-8540-5

Pedro Cisternas & Juan P. Henriquez & Enrique Brandan & Nibaldo C. Inestrosa

Received: 13 May 2013 /Accepted: 15 August 2013 /Published online: 7 September 2013 # Springer Science+Business Media New York 2013

Abstract The signaling pathways activated by Wnt ligands are Keywords Wnt signaling . Muscle development . related to a wide range of critical cell functions, such as cell Neuromuscular junction . Fibrosis division, migration, and synaptogenesis. Here, we summarize compelling evidence on the role of Wnt signaling on several features of skeletal muscle physiology. We briefly review the Introduction role of Wnt pathways on the formation of muscle fibers during prenatal and postnatal myogenesis, highlighting its role on the The proper formation of skeletal muscles is crucial for the activation of stem cells of the adult muscles. We also discuss wide variety of movements executed by vertebrate and inver- how Wnt signaling regulates the precise formation of neuromus- tebrate organisms. Skeletal muscles form as a result of coor- cular synapses, by modulating the differentiation of presynaptic dinated steps beginning during embryonic development and and postsynaptic components, particularly regarding the cluster- further consolidated throughout postnatal life. Muscle forma- ing of acetylcholine receptors on the muscle membrane. In tion, or myogenesis, is mediated by different signaling path- addition, based on previous evidence showing that Wnt pathways triggered by extracellular cues, which activate the ex- ways are linked to several diseases, such as Alzheimer's and pression of different myogenic regulator factors (MRFs), such cancer, we address recent studies indicating that Wnt signaling as Myf5, MyoD, and myogenin [1, 2]. Among these signals, plays a key role in skeletal muscle fibrosis, a disease character- studies in prenatal and postnatal muscle development have ized by an increase in the extracellular matrix components described that signaling pathways activated by Wnt ligands leading to failure in muscle regeneration, tissue disorganization modulate the expression of several MRFs, thus regulating the and loss of muscle activity. In this context, we also discuss the formation of functional multinucleated myotubes [3–5]. possible cross-talk between the Wnt/β-catenin pathway with Once differentiated, the function of skeletal muscles relies two other critical profibrotic pathways, transforming growth on the precise innervation of each myofiber by a motor neuron factor β and connective tissue growth factor, which are potent axon. The synapse between the nerve terminal of a motor stimulators of the accumulation of connective tissue, an effect neuron and a muscle fiber is known as the neuromuscular characteristic of the fibrotic condition. As it has emerged in other junction (NMJ) [6, 7]. Like central synapses, the NMJ has a pathological conditions, we suggests that muscle fibrosis may be high degree of specialization and expression of specific mol- a consequence of alterations of Wnt signaling activity. ecules that allows muscle contraction in response to nerve stimulation [1, 8, 9]. Different pathways triggered by Wnt : : ligands have been recently demonstrated to affect, either pos- P. Cisternas E. Brandan N. C. Inestrosa (*) itively and negatively the differentiation of pre- and postsyn- Centro de Envejecimiento y Regeneración (CARE), Departamento aptic specializations to shape the embryonic formation of the de Biología Celular y Molecular, Facultad de Ciencias Biológicas, – Pontificia Universidad Católica de Chile, Alameda 340, NMJ [10 12]. P.O. Box 114-D, Santiago, Chile Several pathological conditions of the skeletal muscle are e-mail: [email protected] associated with fibrosis, an abnormally increased deposition of extracellular matrix (ECM) components, such as fibronectin J. P. Henriquez Departamento de Biología Celular, Facultad de Ciencias Biológicas, and type I or III collagen. Additional factors involve macro- Universidad de Concepción, Concepción, Chile phage infiltration, inflammation and proliferation of fibroblast- Mol Neurobiol (2014) 49:574–589 575 like cells. All of these factors lead to tissue disorganization and β-catenin is accumulated in the cytoplasm and then to a progressive failure in muscle regeneration that culminates translocated to the nucleus where interacts with the T-cell with a decrease in muscle activity [13, 14]. Recent studies specific transcription factor (TCF) and the lymphoid suggest that the activation of Wnt signaling promotes muscle enhancer-binding factor (LEF), inducing the expression of fibrosis [13, 14]. Particularly, the so-called canonical Wnt/β- Wnt target genes (Fig. 1a)[21, 26, 27]. catenin pathway has been described as profibrotic as it relates with changes in the expression of some ECM components. Noncanonical or β-Catenin-Independent Wnt Signaling However, based on the fact that muscle fibers rely in calcium Pathway for most of their functions, we will also consider the potential and still unexplored role of noncanonical Wnt/Ca+2 signaling At least two noncanonical pathways are also activated by Wnt in this process [13–16]. In addition, the suggested profibrotic ligands. In the planar cell polarity pathway (Wnt/PCP), the effect of Wnt signaling could be explained by its interaction Wnt–Fzd dependent recruitment of Dvl leads to activation of with other pathways previously involved in the onset of fibro- small GTPases proteins, such as Rho and Rac, which subse- sis. For instance, the connective tissue growth factor (CTGF) quently activate the c-Jun N-terminal kinase. This protein can and the transforming growth factor β (TGF-β) signaling cas- either signal to the nucleus and it also can modify the cyto- cades have been described in several organs as profibrotic skeleton stability, as it affects the phosphorylation of microtu- including liver, kidney and skeletal muscle [17–19]. However, bule associated proteins and is also able to interact with actin- the molecular effects of Wnt signaling pathway on the activity regulator proteins (Fig. 1b)[28]. In turn, in the Wnt/Ca+2 of TGF-β and CTGF, as well as the effect of these interactions pathway, signaling downstream of Dvl stimulates trimeric G on the onset of muscle fibrosis are not well understood. proteins and the enzyme phospholipase C, which increase the The aim of this review is to describe the role of the Wnt production of inositol triphosphate (IP3), thus triggering an pathway on skeletal muscle dynamics including myogenesis, increase in intracellular Ca+2. As a consequence Ca+2-depen- NMJ formation, and muscle fibrosis. dent proteins, such as protein kinase C (PKC), calcium- calmodulin-dependent protein kinase II (CaMKII), and the phosphatase calcineurin are activated. Some of these enzymes Wnt Signaling Pathways regulate the transcription factor NF-AT, promoting the expression of specific target genes (Fig. 1c)[20, 29, 30]. Wnt ligands belong to a conserved family of cysteine rich glycoproteins, which are essential to a variety of biological processes. In humans, 19 Wnt genes have been described, each Wnt Signaling in Myogenesis one with a different expression pattern and function [20–22]. The Wnt signaling can be divided into two types: canonical or β- Skeletal muscle tissue in vertebrates originates from the first catenin-dependent and noncanonical or β-catenin-independent germination plate of the mesoderm [31, 32]. Normal muscle pathways (Fig. 1)[21, 23]. differentiation involves the expression of several MRFs, such as Myf5, MyoD, and Pax3/7, which participates in the early Canonical or β-Catenin-Dependent Wnt Signaling Pathway stages of the patterning of mesoderm-derived multipotential cells [33–35]. The relationship between the Wnt signaling and The canonical Wnt pathway begins with the binding of the the myogenesis is supported by in vivo studies using knockout Wnt ligand to the widely expressed seven-transmembrane mutants for Fzd receptors as well as for some Wnt ligands (for Frizzled (Fzd) receptors, of which ten members have been a comprehensive review, see von Maltzahn et al. [36, 37]). In described in vertebrates [24]. The Wnt–Fzd interaction re- these studies, the lack of some crucial Wnt effectors led to quires the LDL-receptor-related proteins 5/6 (LRP5/6), which pronounced tissue damage, and poor muscle development act as co-receptors of Fzd [25]. Intracellulary, the canonical leading to mice death, suggesting that the Wnt pathway is Wnt pathway requires the intracellular protein β-catenin. In critical for prenatal myogenic development [36]. For instance, the absence of Wnt ligands, β-catenin levels remain relatively in studies using mutant Wnt10b−/−, the activation of canonical low by the action of a so-called “destruction complex” formed Wnt signaling by GSK-3β inhibition or the overexpression of by the scaffold protein Axin, adenomatous polyposis coli Wnt10b in myoblasts, accelerates myoblast differentiation (APC) and the enzyme glycogen synthase kinase 3β (GSK- and promotes muscle development [38, 39]. 3 β), that phosphorylates β-catenin stimulating its destruction In prenatal myogenesis, activation of Wnt pathways gen- by the proteosomal pathway [25]. Upon Wnt–Fzd interaction, erates diverse effects. For instance, in chicken embryos, the the scaffold protein Dishevelled (Dvl) is recruited, causing the somite myogenesis in presegmented paraxial mesoderm can dissociation of the β-catenin destruction complex by a series of be stimulated by activators of Shh pathway together with phosphorylations that inhibit GSK-3β. Under these conditions, some Wnt ligands, including Wnt1, Wnt3, and Wnt4. Indeed, 576 Mol Neurobiol (2014) 49:574–589

Fig. 1 Wnt signaling pathways. a In the absence of Wnt, GSK-3β protein promotes the phosphorylation and subsequent degradation of β-catenin. In the canonical Wnt signaling, Fzd and LRP 5/6 activation by Wnt ligand binding leads to the accumulation and migration of β-catenin to the nucleus where it could interact with transcription factors LEF/ TCF family and they potentiate transcription of Wnt target genes including Bcl2, axin-2 and ECM components. b Wnt/PCP signaling pathway, the Wnt ligand binding lead the activation of Fzd and Dvl and this complex activated smalls protein G such us Rho and Rac leading to activation of the JNK pathway and its interaction with the cytoskeleton. c Wnt/Ca+2 signaling, the activation of Fzd-Dvl complex lead to the generation of IP3 that activates the IP3 receptor and releases of intracellular Ca+2 ,this activate several proteins including CamKII and calcineurin that regulate the gene expression via the transcription factors NF-AT

at early stages of differentiation, the expression of MyoD is suggesting that Wnt signaling is a key factor for late prenatal dependent on the simultaneous activation of the Wnt and Shh differentiation [40]. The distribution pattern of Fzd and Wnts pathways; in later stages of development, however, Wnt ac- ligands in different regions of chick embryo support a crucial tivity alone is able to stimulate the expression of MyoD, role for Wnt signaling in early myogenesis, by showing a Mol Neurobiol (2014) 49:574–589 577 direct correlation between the expression of some receptors 58–61], whereas agrin null mice fail to maintain AChR clusters and ligands; however, there is a clear overlapping expression [47, 51]. In contrast, and according to its inhibitory role, mice pattern in an embryonic region that could activate both canon- rendered unable to synthesize ACh, display more and wider ical and noncanonical Wnts pathways to regulate myogenesis. postsynaptic densities than wild-type littermates [50, 51]. This includes somites, segmental plate mesoderm, and neural The identity and function of proteins regulating the presyn- folds [41, 42]. In this regard, even though a crucial role has aptic differentiation of motor neurons have not been extensive- been well documented for the Wnt/β-catenin pathway during ly described; however, genetic in vivo studies have identified different steps of myogenesis, cumulative evidence suggest important regulators of the process. For instance, transgenic that other noncanonical pathways, such as the PCP or an mice for members of the ephrin family of bidirectional signal- alternative mTOR-dependent pathway, also play a role in the ing molecules revealed an essential in vivo role for these axonal process [5]. Nevertheless, the precise mechanisms by which guidance regulators on the formation of properly positioned different Wnt ligands and pathways could interplay to regulate NMJs [62]. Regarding neuromuscular synaptogenesis, it has myogenesis require further studies. been shown that whereas FGF signaling induces early presynaptic differentiation, laminin-2 affects the maturation and function of active zones, while collagen IV plays a later mainte- Wnt Signaling in the Function of the Neuromuscular nance role [63]. Remarkably, mice null for Lrp4 or MuSK, key Junction regulators of postsynaptic assembly, also display defects on presynaptic differentiation [53, 57]. According to this observa- The Neuromuscular Junction tion, Lrp4 has been recently shown to bind motor axons to induce key features of presynaptic differentiation, such as the The vertebrate NMJ is a cholinergic synapse that controls clustering of presynaptic and active zone components, through skeletal muscle contraction. Unveiling how this peripheral a mechanism independent of MuSK and agrin [64]. Together, synapse forms does not only represent a potential benefit to the confluence of multiple signals originated at both sides of the recover movement after pathological or traumatic conditions synapse refine the building of functional NMJs. but also provides valuable information to understand central synapses; indeed, the NMJ has been widely used as an arche- Wnt Signaling on Postsynaptic Development at the Vertebrate typical model to identify signaling molecules secreted at both NMJ sides of the synapse that potentially play positive or negative roles to shape functional synapses (for reviews, see [43–45]). Several lines of evidence have shown that Wnt pathways affect An early hallmark of postsynaptic differentiation at the NMJ postsynaptic differentiation at the vertebrate NMJ in vivo. For is the aggregation of several postsynaptic proteins, including instance, chick muscles exposed to the Wnt-binding inhibitor the acetylcholine receptors (AChRs), in discrete domains of the secreted Frizzled related protein (Sfrp1) display impaired sarcolemma [44]. Even though it was originally believed that AChR clustering [65], suggesting that endogenous Wnt li- only nerve-derived molecules induced AChR aggregation, the gands regulate postsynaptic assembly. In support of this no- existence of an early prepattern of AChR clusters before tion, mutant mice for Dvl, a common mediator of several Wnt inervation has been demonstrated to be crucial for the subse- pathways, display abnormal postsynaptic development at the quent positioning of motor axons for NMJ assembly [46–48]. NMJ [65], a phenotype consistent with previous in vitro data Later on, upon nerve–muscle contact, most of these showing that Dvl regulated the function of MuSK to induce prepatterned AChR clusters are disassembled by the inhibitory AChR clustering [66]. In zebrafish, the Wnt11r ligand, effect of acetylcholine (ACh), which signals through a Cdk5- expressed by tissues adjacent to the newly formed muscle dependent pathway [49–51]. In turn, those aggregates located fibers, interacts with the ligand-binding domain of MuSK to in close apposition with the motor axon are stabilized by the induce the prepatterning of AChR clusters and the guidance of anti-inhibitory role of agrin, a motor neuron-derived proteogly- motor axons [46] (Fig. 2a). Accordingly, mutant fish for can originally believed to induce the aggregation of postsyn- Wnt11r or MuSK display similar severe defects in AChR aptic proteins [50–52]. Agrin signals through the muscle- prepatterning and axonal branching [46, 67]. Consistent with specific tyrosine kinase receptor MuSK [53–55]forminga these findings, the mouse muscle-derived ligand Wnt4 binds to membrane complex with a low density lipoprotein receptor- and phosphorylates MuSK [68]. Wnt4 null mice have less related protein 4 (Lrp4) [56–58] and the cytosolic proteins AChR than control littermates just before inervation, whereas rapsyn, which binds to AChRs, as well as to the MuSK- cultured muscle cells exposed to Wnt4 display increased binding proteins Dok-7 and Tid1 [59–61]. In support of their AChR clustering [68]. Similarly, the mouse-derived ligands crucial role, mice deficient in MuSK, Lrp4, rapsyn, Dok-7, or Wnt9a and Wnt11 induce AChR clustering in cultured Tid1 show no signs of postsynaptic differentiation [53, 56, myotubes through a mechanism dependent on MuSK and 578 Mol Neurobiol (2014) 49:574–589

Fig. 2 Wnt signaling in the vertebrate neuromuscular synaptogenesis. a Wnts induce aneural AChR clustering. Several muscle-derived Wnt ligands activate MuSK-dependent AChR clustering in cultured myotubes. In zebrafish, Wnt11r and Wnt4a induce the internalization of MuSK to endosomes located in the middle region of the myofiber. MuSK assemble a complex with the scaffolding proteins diversin, Daam1 and RhoA to position aneural AChR clusters in a central muscle band (gray stripe), which will guide the incoming motor axons for subsequent NMJ assembly. b. Wnts are positive signals for postsynaptic differentiation. Wnt3 and agrin released from the presynaptic terminal collaborate to promote the formation of AChR clusters. Wnt3 induces the formation of AChR microclusters via Rac1, which are aggregated into full- size clusters by the Rho- dependent effect of agrin. c Wnt/ β-catenin pathways inhibit AChR clustering but promote presynaptic behavior. Wnt3a, secreted by muscle cells at the stages of NMJ formation, activates a β-catenin pathway that induces the dispersal of AChR clusters through the inhibition of rapsyn expression. Specific ablation or stabilization of β- catenin in muscles, but not in motor neurons, result in presynaptic defects, suggesting that muscle β-catenin induces the expression of a still unknown retrograde signal for presynaptic differentiation

Lrp4 [69]. Remarkably, recent findings obtained in zebrafish findings reveal a novel key MuSK-dependent mechanism by show that Wnt11r and Wnt4a induce a localized endocytosis of which Wnt ligands induce the aneural clustering of AChRs on MuSK to recycling endosomes which, in turn, accumulates newly formed muscle cells. AChRs at the sites where motor axons will be guided to Wnt ligands can also regulate neuromuscular synaptogenesis assemble functional NMJs [70] (Fig. 2a). Together, these [12, 71, 72]. On the one hand, transplantation of chick wings Mol Neurobiol (2014) 49:574–589 579 with cells secreting the Wnt3 ligand, which is expressed by Wnt mediated signal [83]. Together, these data support the motor neurons at the time of NMJ formation [73], led to participation of Wnt signaling in the axonal guidance with increased AChRs clustering [65]. In cultured myotubes, Wnt3 repulsive and attractive effects depending of the Wnt ligand activates the small GTPase Rac1 to induce the formation of (Table 1). However, the molecular mechanism of the effect and AChR microclusters, which only coalesce into bigger clusters the interaction networks of Wnt signaling are still to be fully after agrin-dependent activation of Rho [65](Fig.2b). These underscored. Regarding presynaptic differention, Wnt7a stim- findings reveal a potential neural-dependent cross-talk of Wnt- ulates the maturation of neuronal connections and the presence and agrin-mediated pathways to induce postsynaptic differen- of the Wnt antagonist sFRP-1 blocks the growth of axons in tiation at the vertebrate NMJ. On the other hand, the highly the cerebellum [84–86]. Similarly, in hippocampal neurons, identical ligand Wnt3a impairs agrin-induced AChR clustering Wnt7a stimulates the clustering of the presynaptic protein and disassemble preformed aggregates [74, 75]. Wnt3a is synaptophysin and increases the mEPSC frequency [87]. Al- expressed by developing skeletal muscles and mediates its together, these data expose the critical role of Wnt signaling on dispersal activity by down-regulating rapsyn expression via a presynaptic assembly at the central nervous system, modulat- β-catenin-dependent, but TCF-independent, pathway [75] ing several processes, which lead to the correct formation of (Fig. 2c). Even though these findings reveal an inhibitory effect synapses (Table 2). However, the advance in the central syn- for β-catenin on AChR clustering, the in vivo role of this apse is not proportional to our knowledge of the differentiation crucial effector of the Wnt canonical pathway at the NMJ is of the presynaptic component at the vertebrate NMJ. rather complex. Thus, even though specific ablation of β- catenin in skeletal muscles, but not in motor neurons, gives rise to enlarged AChR clusters, this effect is primarily related to Wnt Signaling in Muscle Fibrosis presynaptic defects in axonal branching and neurotransmission [74, 76]. In turn, specific stabilization of β-catenin in muscle Fibrosis in Skeletal Muscle but not in neuronal cells, resulted in excessive nerve branching and defasciculation, while NMJ formation or function were Fibrosis is characterized by the aberrant deposition of ECM unaffected [77](Fig.2c). Taken together, these findings reveal components including fibronectin and collagen type I or III. that activation of different Wnt pathways control opposite but This accumulation of ECM components leads to the tissue complementary roles on the assembly of postsynaptic densities disorganization and to the loss of muscle activity and eventu- at nascent neuromuscular synapses. ally to death [15, 88, 89]. Several organs are susceptible to fibrosis including lung, kidney, liver, and skeletal muscle; Wnt Signaling on Presynaptic Differentiation however, all fibrotic reactions share common underlying cellular and molecular mechanisms, including tissue degenera- Even though our current knowledge regarding the potential tion, macrophage infiltration, inflammation, and proliferation role of Wnt signaling on presynaptic differentiation of verte- of fibroblast-like cells [15, 89–93]. The loss of organ archi- brate motor neurons is virtually null, the function of diverse tecture and the activation of leukocytes and fibroblasts in- Wnt pathways on several steps of presynaptic behavior in crease the production of several molecules, such as growth central synapses, from the establishment of neuronal cell factors, proteolytic enzymes, angiogenic factors, and polarity to synaptogenesis, has been well documented. In fibrogenic cytokines. Together, these molecules lead to the primary cultures of hippocampal neurons, it has been de- accumulation of ECM components [16, 94, 95]. scribed that Wnt5a actives axonal differentiation in a β- catenin-independent manner via the activation of the protein Table 1 Effect of Wnt ligands in axonal guidance complex Par6-Par3-atypical protein kinase C (aPKC) [78, 79]. Wnt5a signaling is also involved in regulating polarity via Type of signal References aPKC activation in a Dvl-dependent manner; however, in Drosophila, aPKC mutants do not show failures in neuronal Attractive Repulsive polarity [80]. Other studies indicate that gradients of Wnt1 and Wnt1 + + [81, 155] – Wnt5a expressed in the dorsal spinal cord in an anterior Wnt3 + [49] posterior direction attracts ascending somatosensory axons Wnt4 + [54] projecting from the spinal cord to the brain, while the same Wnt5 + + [56, 155] gradient repels descending corticospinal tracts in the opposite Wnt5a + [83] direction, from the brain to the spinal cord [81, 82]. In addi- Wnt7b + [76] tion, the repulsive signal of Wnt/Ca+2 signaling has been Wnt8b + [61] reported in mutants for CaMKII; indeed, this mutation causes Wnt11r + [46] severe failures in axonal guidance given the lack of a repulsive 580 Mol Neurobiol (2014) 49:574–589

Table 2 Effect of Wnt ligands in synaptogenesis characterized by skeletal muscle wasting, weakness, decrease Wnt Ligand Effect in synaptogenesis References in the fiber size, and muscle necrosis that compromises patient mobility, leading to death [106–108]. One of these diseases is Wnt3a Stimulates AChR clustering [65] the DMD [15, 91, 109]. The clinical manifestation of DMD Wnt4 Stimulates AChR clustering [68] includes an onset during childhood with progressive weakness Wnt5a Stimulates PSD95 clustering [30, 156, 157] anddeathinearlyadulthood[91, 108]. The DMD is caused by Stimulates LTP a failure in the gene coding for the cytoskeletal protein dystro- Decreases the number phin, which is required for the proper interaction between of presynaptic presynaptic terminals theplasmamembraneandtheECM.Theabsenceofdys- Wnt7a Stimulates synaptophysin clustering [84, 87] trophin leads to the loss of function in the normal muscle Stimulates mEPSC frequency regeneration cycle, leading to an increase in the production of Stimulates neuronal connection ECM components [107, 108, 110]. Wnt11, 11r, 9a, Stimulates AChR clustering [69] 10b, 16 In normal conditions, the skeletal muscle have a great capacity to repair itself after injury; however, under chronic injury conditions such as DMD, the skeletal muscle progres- Several models have been described to study the regenerative sively loses its capacity to regenerate, likely by the decrease in capacity of the skeletal muscle, almost all the models use acute the number of satellite cells, the skeletal muscle stem cells, injury induced by injection of a toxin or myofiber destruction. which are unable to repair the damaged tissue. As a conse- These studies have allowed the description of all stages of the quence, the muscle tissue is progressively replaced by adipose regenerative process including the inflammatory response, and especially fibrotic tissue [4, 95, 111, 112]. Currently, no which follows an ordered pattern [96–98]. Early after injury, effective clinical treatment to combat or attenuate muscle the damaged area is infiltratedbyinflammatorycells.Inthemdx fibrosis in DMD patients is available; however, recent studies mice, the model animal for Duchenne's muscle dystrophy using the mdx mouse have focused more attention on eluci- (DMD, see below), these inflammatory cells are critical to dating the cellular and molecular mechanisms underlying promote the survival and proliferation of myogenic precursor skeletal muscle fibrosis associated with dystrophin deficiency. cells, promoting the repair of the skeletal muscle [16, 95, 99, These studies have tested several pharmacological agents that 100]. The innate immune response is activated by the release to target muscle fibrosis and strongly suggest that combating the extracellular space of several factors from the damaged fiber. fibrosis could ameliorate DMD progression and increase the These factors lead to the infiltration of the damage area by success of new cell- and gene-based therapies [113, 114]. monocytes and neutrophils [99, 101]. The neutrophils play a In fibrotic skeletal muscle, several pathways are turning on key role in repair facilitating phagocytosis and the recruitment of by different cells such as fibroblasts, macrophages and leuko- monocytes by the release of cytokines [102]. Proinflammatory cytes. Despite the complex mixture of signaling implicated in macrophages, observed experimentally in the context of muscle the onset of skeletal muscle fibrosis until now two key mol- repair, are phenotypically similar to classically activated M1 ecules have been described as critical in the development of macrophages and are usually found at early stages after muscle the disease: the TGF-β and the CTGF [15, 17, 110, 115–117]. injury. These cells also release proinflammatory cytokines, such TGF-β is a potent inducer of CTGF, and most models postu- as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha late that CTGF acts as a downstream mediator of TGF-β (TNF-α). In a chronic muscle damage, the M1 macrophages activity; by contrast, other studies support the independent release several ECM remodelers like urokinase-type plasmino- action of each molecule [19, 118–121]. gen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), plasmin, matrix metalloproteinases and tissue inhibitors of me- TGF-β Signaling in Fibrosis and its Possible Cross-Talk talloproteinases (TIMPs). The role of these molecules is to with the Wnt Signaling modulate the proliferation of myoblasts and to promote the excessive ECM production/accumulation and the replace- There are three TGF-β isoforms, TGF-β1, TGF-β2, and ment of muscle fiber with fibrotic tissue [103, 104]. After TGF-β3. The activation of these signaling relies on the bind- the activation of M1 macrophages, it has been described the ing of the ligand to a heterodimeric receptor complex which activation of the M2c phenotype, so-called because of their includes one TGF-β type I receptor molecule, termed activin role in deactivating M1 macrophages. These cells release anti- linked kinase 5 (ALK5) [122–124]. In the canonical TGF-β inflammatory factors including the interleukin-10 (IL-10). signaling, ligand binding leads ALK5 to phosphorylate The activity of these cells is important to regulate the end of Smad2/3, which in turn, activates Smad4 and this protein the inflammatory response [105]. complex translocates into the nucleus to activate transcription In skeletal muscle, fibrosis is commonly related with muscle factors and lead to the expression of target genes including dystrophies, a molecularly heterogeneous group of diseases, fibronectin, CTGF and PAI-1 (Fig. 3)[122, 123]. Mol Neurobiol (2014) 49:574–589 581

has been studied, it is possible a therapeutic approach, as the inhibition of this signaling has been reported to decrease the development of fibrosis. The relationship between canonical Wnt and TGF-β pathways has been described in several fibrotic models, and the evidence suggests a collaboration of both pathways in the fibrotic disease; indeed, the inhibition of both pathways pre- vents the development of fibrosis (Fig. 5)[131, 132]. Recent studies in vivo have shown that activation of the canonical Wnt pathway is required for the action of TGF-β, since the presence of dickkopf 1 (DKK1) (a Wnt signaling inhibitor) decreases the activity of TGF-β in a model of muscle fibrosis (Table 3) [123]. In this regard, in studies using chondrocytes as models, it has been reported that the TGF-β signaling inhibits the expression of axin-2, and this leads to an increases in the levels of β-catenin. This could be a negative feedback for the TGF-β signaling through the inhibition of Smad3 protein [133]. These data suggest a direct relationship between the TGF-β and Wnt/ β-catenin pathways in the establishment of the fibrotic disease, and that the effects of TGF-β needs the activation of the canonical Wnt signaling in the progress of the disease.

Cross-Talk Between Wnt Signaling and CTGF in a Model of Muscle Disease

CTGF is a cysteine rich 36–38 kDa secreted protein, which Fig. 3 TGF-β signaling. Active TGF-β binds to its receptor type I/II this leads to the activation, via phosphorylation of Smad2/3 pathways, which belongs to CCN family (acronym for connective tissue growth regulates the transcription of several target genes involved in several factor, cysteine-rich-protein, and nephroblastoma-over- cellular functions like CTGF and ECM proteins. TGF-β can also activate expressed). CTGF is expressed in various cell types including other signaling in Smad-independent manner fibroblasts, chondrocytes, and leukocytes and is poorly expressed in the central nervous system [18, 134, 135]. The The expression of TGF-β has been described in normal CTGF gene contains five exons. Exon 1 encodes a signal skeletal muscle, mdx models and patients with DMD. Also, it peptide (SP) for secretion, while exons 2–5 encode for mod- has been described an over-expression of these ligands after ules 1–4 of the secreted CTGF protein which are homologous muscle injury [125]. In skeletal muscle cells, TGF-β acts as a to other proteins including insulin-like growth factor binding strong myogenic inhibitor. It is also known that TGF-β can protein (IGFBP) domain (module 1), a von Willebrand factor inhibit myoblast differentiation in vitro, affecting the expres- type C (VWC) domain (module 2), a thrombospondin type 1 sion of muscle proteins, such as myosin heavy chain and (TSP1) domain (module 3), and a C-terminal (CT) module creatine kinase [126, 127]. The profibrotic effect of TGF-β (Fig. 4)[134–136]. The modular structure of CTGF allows the could be explained by the stimulation of fibroblasts to produce interaction of CTGF with multiple molecules suggesting a ECM proteins such as collagen and fibronectin. Furthermore, role in several signaling downstream, including Notch1, it has been described that the activation of TGF-β signaling LRPs, and growth factors such as BMP, TGF-β, and vascular leads to a decrease in the expression of metalloprotease like endothelial growth factor. The interaction of CTGF and the collagenase and to an increase in the expression of TIMPs. receptors described previously allows the activation of several Together these effects could lead to the accumulation of ECM pathways downstream including PKC, MEK/ERK, and pro- [121, 123, 124, 128, 129]. tein kinase B (PKB) [19, 137–140]. In fact, the injection of TGF-β in vivo in skeletal muscle In physiological conditions, CTGF is involved in angiogen- stimulates the production of ECM in the injected area and the esis and cellular differentiation, but in pathological conditions, overexpression of TGF-β in myoblasts promotes the forma- it has been described as a profibrotic molecule, stimulating the tion of myofibroblast cells, suggesting a critical role of the proliferation of fibroblasts, their differentiation towards TGF-β in the onset of the fibrotic condition in the skeletal myofibroblasts and ECM production [110, 115, 121, 134, muscle [110, 130]. Since the critical role of these pathways 141]. In fact, in vivo studies have showed that the CTGF 582 Mol Neurobiol (2014) 49:574–589

Table 3 Effect of different signaling components on fibrosis Effect over fibrosis Tissue References

Decrease Increase

Treatment or drug TGF-β + Liver/muscle/kidney/lung [18, 125, 129] CTGF + Liver/muscle/kidney/lung [19, 134] TGF-β+ CTGF ++ Muscle/kidney [17, 142, 143] Wnt3a ++ Muscle [14, 147] Classical Wnt/β-catenin inhibitors DKK1 + Kidney/liver [158, 159] sFRP1 + Mammary epithelial cells [160] sFRP2 + Heart [161] Wif-1 + Chondrocytes [144] GSK-3β inhibitors PKF118-310 + Dermal fibrosis [162] XAV-939 + Dermal fibrosis [163]

injection stimulates the fibrosis and the coinjection of CTGF and TGF-β resulted in a persistent fibrosis [142, 143]. Together, this has led to the notion that CTGF plays an important role in tissue response to injury and fibrosis (Fig. 5). In chondrocytes, CTGF binds the Wnt inhibitory factor 1 (WIF-1), an antagonist of the Wnt pathway, through the CT domain of CTGF. It has been also described a direct relationship in the expression patterns of CTGF and WIF-1, and the presence of WIF-1 inhibits the expression of CTGF; however, CTGF is unable of interfering with the effect of WIF-1 on the canonical Wnt signaling [144]. Furthermore, it has been described that CTGF can also interact with the CT domain of LRP6, so CTGF is also able to affect the interaction of Wnt with their coreceptor [145, 146]. Other studies in human mesangial cells have been described suggesting that CTGF stimulates the canonical Wnt pathway in an LRP6-dependent manner, and this effect is inhibited in the presence of DKK1 (Table 3)[145]. Remarkably, studies in a fibroblast cell line show that the treatment with the Wnt3a ligand, which signals through the Wnt/β-catenin pathway, increases the expression of CTGF and TGF-β mRNAs, reinforcing the view that these signaling pathways may cross- talk in the context of fibrosis [147].

Proposed Model for the Interaction Between Wnt/β-catenin, CTGF, and TGF-β SignalingintheDevelopmentofSkeletal Muscle Fibrosis Fig. 4 CTGF signaling. The CTGF protein contains a signal peptide (SP) as well as four modules: IGFBP, VWC, TSP1, and CT. Through these domains CTGF can interact with several molecules including BMP, TGF- The data presented above support an important role for the β, and possibly some Wnt ligands. CTGF interacts with several mem- Wnt/β-catenin signaling pathway during the onset and devel- brane proteins, including LRP proteins, integrins, TrkA, BMP, and Notch opment of the fibrotic condition in skeletal muscle. The effect 1; these interactions trigger various pathways downstream including of the Wnt pathway could be explained at least in part by the PKC, MEK, p38, and PKB JNK, and the activation of these signaling lead to several effects like proliferation, fibroblast differentiation, and relationship between this signaling cascade with the signaling production of ECM components, leading to the onset of fibrotic condition mediated by TGF-β and CTGF [17, 128, 144]. In addition, Mol Neurobiol (2014) 49:574–589 583

Fig. 5 Cross-talk between Wnt and TGF-β signaling. In fibrotic condi- complex destruction of β-catenin, and the activation of TGF-β leads to tion, the levels of TGF-β are increased, and this promotes the stimulation an increase in cytoplasmic β-catenin and an activation of canonical Wnt of the target genes of this pathway, like CTGF and ECM components signaling and an increase in their target genes including fibronectin, such as type I collagen; this promote the accumulation of connective leading to a major accumulation of ECM components promoting the tissue in the extracellular space, the transcription effect of the TGF−β development of muscle fibrosis signaling, leading a down-regulation of axin-2 a protein part of the

Wnt signaling could modulate early processes related to the some reports suggesting that the CTGF could be a possible regeneration of skeletal muscles after injury, and, in this way, it target gene of the canonical Wnt signaling [153]. Therefore, could trigger a deregulation of the regenerative capacity of the the Wnt/β-catenin signaling pathway could have a key role in muscle, promoting the onset of fibrosis [14, 148]. Therefore, triggering the activation of TGF-β and CTGF in activated the activation of the Wnt/β-catenin signaling could stimulate fibroblasts and maybe in other cells of the fibrotic environ- the formation and activation of fibroblasts into myofibroblast ment. The presence of several Wnt/β-catenin inhibitors, such derived from the satellite cells activated by injury. The activat- as DKK1 and Sfrp1 and 2 inhibit the signaling of TGF-β and ed fibroblasts could increase the expression and release of CTGF, leading to an inhibition of the fibrosis (Table 3 and TGF-β to the environment. In turn, this ligand, in a autocrine Fig. 6). form, could increase the expression of CTGF and together This new role of the Wnt/β-catenin signaling as a profibrotic these ligands could activate downstream signaling leading to factor makes this signaling pathway an interesting candidate for the over-expression of ECM components like fibronectin therapeutic interventions. In fact, there is evidence describing [149–152]. Furthermore, the activation of Wnt/β-catenin could that inhibition of the Wnt/β-catenin pathway decreases the itself stimulate the expression of fibronectin, and also there are progression of the fibrosis [154]. Certainly, further studies are 584 Mol Neurobiol (2014) 49:574–589

Fig. 6 Proposed model for the role of Wnt signaling in Fibrosis. After the differentiation of the fibroblast into myofibroblast. The activated acute muscle injury, the damage area is infiltrated by macrophages and myofibroblast cells increase the production of ECM components like neutrophils; these cells release several cytokines and lead to the activation fibronectin and collagen and also release profibrotic molecules like CTGF of the inflammation process. In normal postinjury muscle regeneration, and TGF-β. In these chronic damage condition has been described an these inflammation is acute and important for the correct replacement of increase in the expression of Wnt ligands, and these lead to the activation the muscle fiber. In a second step, the satellite cells are activated and star of the Wnt/β-catenin pathway in the myofibroblast increasing the pro- the differentiation/proliferation to myoblast, and finally, these cells star duction of the ECM components and the release of profibrotic molecules; the fusion process to form a new muscle fiber; in these normal cycle of together, this lead to aberrant regeneration. Several inhibitors of Wnt/β- regeneration has been described a low expression of Wnt ligands and a catenin signaling like DKK1 and Wif-1 block the TGF-β and CTGF low activity of the Wnt/β-catenin pathway. By contrast, in a chronic profibrotic effect, suggesting the central role of the Wnt signaling in the muscle injury like DMD, the inflammation process is deregulated, and progress of fibrosis these lead to the deregulation of the regeneration processes stimulating necessary to better define the effect of Wnt signaling inhibition formation of the motoneuron-muscle synapse. In myogenesis, on skeletal muscle fibrosis. the effect of Wnt signaling leads to the progression of the differentation at early developmental stages and inhibition of this signaling leads to a poor skeletal muscle formation. In the General Conclusions maturation of the NMJ, the Wnt signaling modulates the local- ization of several proteins critical for the patterning of the The Wnt signaling pathway plays a critical role in several synapse and for the precise control of the muscle contraction. processes including the development of muscle tissue and the In muscle fibrosis, apparently the Wnt signaling plays a critical Mol Neurobiol (2014) 49:574–589 585 role, as it is able to functionally interact with other profibrotic 19. Abreu JG, Ketpura NI, Reversade B, De Robertis EM (2002) molecules, such as CTGF and TGF-β. Understanding these Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nat Cell Biol 4(8):599–604 interactions could be critical in the discovery of future treat- 20. Nusse R, Varmus H (2012) Three decades of Wnts: a personal ments for this pathological condition. perspective on how a scientific field developed. EMBO J 31(12): 2670–2684 Acknowledgments This work was supported by grants from the Basal 21. Clevers H, Nusse R (2012) Wnt/beta-catenin signaling and disease. – Center of Excellence in Aging and Regeneration (CONICYT-PFB 12/ Cell 149(6):1192 1205 2007) and FONDECYT (no. 1120156 to N.C.I. and no. 1130321 to 22. Gordon MD, Nusse R (2006) Wnt signaling: multiple pathways, J.P.H.). PC was a postdoctoral fellow from the PFB (12/2007) grant to multiple receptors, and multiple transcription factors. J Biol Chem – NCI and EB. Graphic work was carried out by Graphique-Science (http:// 281(32):22429 22433 graphique-science.blogspot.com). 23. Toledo EM, Colombres M, Inestrosa NC (2008) Wnt signaling in neuroprotection and stem cell differentiation. 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