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Chemokines have been implicated in the promotion of leucocyte trafficking to diseased muscle. The purpose of this study was to determine whether a subset of inflammatory chemokines are able to directly drive myoblast proliferation, an essential early component of muscle regeneration, in a manner which is entirely independent of leucocytes. Cultured myoblasts (C2C12) were exposed to monocyte chemoattractant protein-1 (MCP-1; CCL2), macrophage inflammatory protein-1α(MIP-1α; CCL3) or MIP-1β (CCL4). All chemokines induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) and greatly increased myoblast proliferative responses. Chemokine-induced myoblast proliferation was abolished by pertussis toxin and the MEK1/2 inhibitor U0126, implicating both Gαi-coupled receptors and ERK1/2-dependent signalling. Myoblasts expressed receptors for all of the chemokines tested, and mitogenic responses were specifically inhibited by antibodies directed against CC family chemokine receptors 2 and 5 (CCR2 and CCR5). Within an in vitro myogenic wound healing assay devoid of leucocytes, all chemokines significantly accelerated the time course of myoblast wound closure after mechanical injury. Injections of MCP-1 into cardiotoxin-injured skeletal muscles in vivo also suppressed expression of the differentiation marker myogenin, consistent with a mitogenic effect. Taken together, our results indicate that CC chemokines have potent and direct effects on myoblast behaviour, thus indicating a novel role in muscle repair beyond leucocyte chemoattraction. Therefore, interventions aimed at modulating the balance between myoblast and leucocyte effects of CC chemokines in injured muscle could represent a novel strategy for the treatment of destructive muscle pathologies.
Skeletal muscle regeneration is an essential compensatory response to both genetic and acquired forms of muscle fibre damage and loss. Damaged muscle regenerates itself by activating a population of undifferentiated muscle precursor cells, commonly referred to as satellite cells (Hawke & Garry, 2001; Charge & Rudnicki, 2004), which contribute to the formation of new healthy fibres. The satellite cells normally reside between the plasma membrane and basal lamina in a relatively quiescent, non-proliferative state. However, once the satellite cells are activated by muscle injury, they undergo intense proliferation as well as migration to sites of muscle fibre damage (Hawke & Garry, 2001). After several rounds of cellular division, the majority of these cells (now considered adult myoblasts) will exit the cell cycle and differentiate into post-mitotic myotubes, which then evolve into mature adult fibres. In experimental models of skeletal muscle injury, major leucocyte accumulation also occurs at sites of muscle regeneration, consisting initially of neutrophils and then primarily of macrophages (Tidball, 2005). It has been shown that interference with macrophage influx will delay subsequent muscle repair (Lescaudron et al. 1999; Summan et al. 2006; Tidball & Wehling-Henricks, 2007). Moreover, a number of cytokines and growth factors with the capacity to induce satellite cell proliferation or migration can be produced by macrophages (Hawke & Garry, 2001; Tidball, 2005), and macrophage-derived soluble factors have been shown to favour the muscle regeneration process (Robertson et al. 1993; Merly et al. 1999; Wehling et al. 2001; Cantini et al. 2002).
Recently, it has been suggested that chemokines are important actors in skeletal muscle regeneration (Warren et al. 2004, 2005; Contreras-Shannon et al. 2006). The chemokines are subdivided into four main families (CXC, CC, C and CX3C), based upon the number and arrangement of their amino-terminal cysteine residues (Zlotnik & Yoshie, 2000). Increased expression levels of several chemokine ligands and their cognate receptors have been found in muscle biopsies obtained from animal models and human patients suffering from muscular dystrophy or inflammatory myopathies. In particular, a predominant up-regulation of the CC chemokines, including MCP-1 (CCL2), MIP-1α (CCL3) and MIP-1β (CCL4), has been reported (Confalonieri et al. 2000; Reyes-Reyna et al. 2002; Porter et al. 2003; Demoule et al. 2005). In addition, skeletal muscle cells constitutively express MCP-1 and MIP-1α (Nagaraju et al. 2000; De Rossi et al. 2000; Reyes-Reyna & Krolick, 2000), and CC chemokines are greatly up-regulated following experimental muscle injury (Hirata et al. 2003; Warren et al. 2004; Contreras-Shannon et al. 2006) or exposure to pro-inflammatory cytokines such as TNF-α (De Rossi et al. 2000; Demoule et al. 2005). Interestingly, the up-regulation of CC chemokines and their receptors observed following muscle damage (Hirata et al. 2003) appears to correspond temporally with the period of satellite cell proliferation found in experimental models of muscle injury (Grounds & McGeachie, 1992). More direct evidence for the importance of CC chemokines in muscle regeneration has been provided by the demonstration that recovery of normal muscle structure and force production after acute injury in vivo is significantly impaired in mice receiving antibodies against MCP-1 or lacking its primary receptor, CCR2 (Warren et al. 2004, 2005; Contreras-Shannon et al. 2006).
As the chemokines are classically associated with the induction of leucocyte trafficking to sites of tissue inflammation, one interpretation of these findings is that the purpose of CC chemokine up-regulation within damaged muscle is to direct leucocytes to sites of muscle injury. This could favour muscle repair in an indirect fashion, such as through the release of macrophage-derived mediators which favour muscle regeneration, as mentioned earlier. However, another possibility is that the CC chemokines expressed within injured skeletal muscle are capable of acting directly on the muscle cells per se, by regulating myoblast behaviour during one or more steps associated with the formation of new muscle fibres. Therefore, the purpose of the present study was to determine whether CC chemokines are capable of playing a role in the muscle regeneration process through mechanisms that are entirely independent of their effects on leucocyte trafficking.
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Skeletal muscle is able to undergo extensive regeneration and repair after injury. Once activated by injury or other stimuli, satellite cells enter the cell cycle and begin to proliferate, at which point they are considered adult myoblasts. Muscle damage also produces factors that trigger the migration of satellite cells to sites of injury (Schultz et al. 1985; Watt et al. 1987). In this study, we employed C2C12 myoblasts as an in vitro model system, since these cells have been shown to proliferate and migrate to form muscle fibres when implanted into mouse muscles in vivo (Watt et al. 1994), and are frequently used to investigate mechanisms of myogenesis (Milasincic et al. 1996; Wu et al. 2000; Tortorella et al. 2001). Here we provide evidence for a chemokine-mediated signal transduction pathway within myoblasts that can be directly linked to their regenerative function in skeletal muscle, starting from the initial interactions with their relevant chemokine receptors and proceeding downstream to signalling events associated with the mitogenic responses subsequently generated.
We focused our investigation upon several CC chemokines (MCP-1, MIP-1α and MIP-1β) that are highly expressed by skeletal muscle in pathological situations (Zlotnik & Yoshie, 2000). Our results show that myoblasts express receptors for these chemokines, and that they trigger potent mitogenic effects on myoblasts which are critically dependent upon the presence of intact signalling through both G proteins (Gαi) and the extracellular signal-regulated kinases (ERK1/2) of the mitogen-activated protein kinase (MAPK) pathway. In addition, using both in vitro and in vivo models, we demonstrate that CC chemokines are able to significantly modify the muscle repair process after the induction of skeletal muscle injury. Taken together, our findings point to a previously unrecognized role for the CC chemokine system in the direct regulation of myoblast behaviour, which could have significant implications for therapeutic strategies aimed at enhancing the muscle regeneration process in muscular dystrophies and other forms of destructive muscle pathology.
The chemokines exert their biological effects by activating seven transmembrane G protein-coupled receptors (Zlotnik & Yoshie, 2000). It is increasingly evident that chemokines can exert multiple functions that extend well beyond their more established effects on leucocyte activation and trafficking. For example, chemokines have been shown to have important effects on non-myeloid cell types as diverse as endothelial cells (Salcedo et al. 2000), synoviocytes (Garcia-Vicuna et al. 2004), neural cells (Klein et al. 1999), and smooth muscle cells (Chandrasekar et al. 2004; Schecter et al. 2004). More recently, interference with SDF-1 (CXCL12) signalling through its cognate receptor, CXCR4, was found to be associated with impaired migration and increased apoptosis of skeletal muscle progenitor cells during embryogenesis (Vasyutina et al. 2005).
Within a given chemokine family (CXC, CC, C or CX3C), several different chemokine ligands are frequently capable of activating the same receptor, and it is not unusual for a single chemokine ligand to target more than one receptor member within the same family (Zlotnik & Yoshie, 2000; Locati et al. 2005). For example, it is well established that MIP-1α signals through both CCR1 and CCR5, whereas MIP-β signalling occurs via CCR5 (Locati et al. 2005). In addition, while it is generally considered that MCP-1 binds more or less exclusively to CCR2, evidence has been presented for an alternative but as yet unidentified receptor (Schecter et al. 2004). In our study, at least one receptor for each of the CC chemokines tested was found to be expressed in myoblasts, and we found that MCP-1, MIP-1α and MIP-β were all able to stimulate myoblasts to enter the S phase of the cell cycle to an equivalent degree. Moreover, the specificity of mitogenic responses to individual chemokines was confirmed by the ability of neutralizing antibodies directed against their known receptors to block these effects, whereas isotypic antibodies against non-cognate receptors had no significant impact.
In other cell types studied to date, the majority of chemokine-induced responses are inhibited by PTX, which is consistent with Gαi protein family members being the primary transduction partners of the chemokine receptors. In keeping with the above, we found that chemokine-induced myoblast proliferative responses were also inhibited by PTX. Gαi proteins are enriched within the t-tubule invaginations of the muscle fibre membrane (Doucet & Tuana, 1991), and several PTX-sensitive mechanisms in skeletal muscle have been described (Vandenburgh et al. 1995; Fedorov et al. 1998; Gosmanov et al. 2002). Furthermore, our results demonstrate that the effects of MCP-1 and MIP-1β on myoblast proliferation are for the most part mediated through CCR2 and CCR5, respectively. On the other hand, the mitogenic effects of MIP-1α were completely unaffected by inhibition of either CCR2 or CCR5, despite the latter being a major receptor for this ligand. This finding suggests (but does not prove) that under these conditions, MIP-1α is capable of acting to a large extent through its alternative receptor, CCR1, which is also expressed in myoblasts. Indeed, we have previously reported that CCR1 is expressed by mouse muscle fibres in vivo, and that CCR1 mRNA transcript levels are significantly up-regulated in the context of pro-inflammatory stimulation by cytokines as well as in dystrophic skeletal muscle (Demoule et al. 2005). Interestingly, it has been reported that human CCR2 and CCR5 can heterodimerize and activate Gq and/or G11 rather than Gαi-dependent signalling pathways (Mellado et al. 2001). Whether such alternative signalling pathways, involving heterodimers of different receptors and alternate G protein subunits, also exist in skeletal muscle cells under certain physiological conditions, remains to be determined.
Several different signalling pathways have been implicated in the proliferative responses of myoblasts to various growth factors, including JAK2-STAT3 (Spangenburg & Booth, 2002), PI3K/Akt (Milasincic et al. 1996), and the MAPKs (Wu et al. 2000; Tortorella et al. 2001; Penn et al. 2001). The ERK1/2 MAPK pathway is most classically triggered by growth factor binding, and is activated in skeletal muscle in response to physical exercise or injury (Nakamura et al. 2005; Sakamoto & Goodyear, 2002). Most studies have found that ERK1/2 activation promotes myoblast proliferation (Bennett & Tonks, 1997; Coolican et al. 1997; Wu et al. 2000), and this effect appears to be dependent upon the timing of ERK1/2 activation relative to induction of the myogenic program (Penn et al. 2001). For example, bFGF (FGF2), a potent myoblast mitogen and inhibitor of differentiation, induces peak activation of ERKs within 2–10 min, and blocking this effect with a MEK inhibitor prevents FGF-induced myoblast proliferation (Milasincic et al. 1996; Tortorella et al. 2001; Jones et al. 2001). Similarly, while IGF-1 and -2 are able to stimulate either proliferation or differentiation of myoblasts depending upon experimental conditions, it appears that ERKs are primarily involved in IGF-mediated proliferative effects on myoblasts (Coolican et al. 1997; Jones et al. 2001; Adi et al. 2002). Our data indicate that ERK1/2 activation is absolutely required to allow for the observed proliferative responses of myoblasts to CC chemokine stimulation. In addition, previous work has implicated the ERK1/2 MAPK pathway in the myoblast migratory response to cell wounding (Yeow et al. 2002). Indeed, the accelerated wound space closure observed in our study after chemokine treatment was probably due in large part to increases in the migratory behaviour of myoblasts, since the differences occurred at a much earlier time point than was observed for chemokine-induced increases in myoblast cell number.
After experimental muscle injury in vivo, there is an early up-regulation of MIP-1α, MIP-β and MCP-1, which corresponds temporally to the proliferative behaviour of myoblasts within the injured muscles (Grounds & McGeachie, 1992; Hirata et al. 2003). However, due to the fact that cardiotoxin-induced injury also triggers the widespread proliferation of several other cell types within the muscle in vivo, we found that this high background noise level precluded accurate quantification of chemokine-induced changes in standard proliferation markers within the specific myoblast population of interest. Therefore, we employed myogenin as an alternative marker, since its expression reflects declining myoblast proliferation and transition toward the post-mitotic differentiated muscle fibre. In this regard, prior studies have shown that other growth factors which induce myoblast proliferation in an ERK-dependent manner also transiently suppress the expression of myogenin (Yablonka-Reuveni & Rivera, 1997), and that the onset of myogenin expression temporally follows the decline in cells positive for proliferating cell nuclear antigen (Yablonka-Reuveni & Rivera, 1994). Accordingly, our in vivo studies are also consistent with a CC chemokine-induced mitogenic effect on myoblasts, since we demonstrate that myogenin expression is suppressed to a greater degree during the early phases of regeneration when the injured muscles are treated by intermittent injections of MCP-1. However, while this experiment demonstrates that MCP-1 can modulate myogenesis in vivo, we cannot rule out the possibility that MCP-1 influenced the interaction between myoblasts and leucocytes under these conditions, since both cell types are present within the in vivo environment after injury.
Hereditary muscle pathologies such as muscular dystrophies, as well as traumatic or exercise-induced muscle injuries, are associated with a significant inflammatory response within the muscle (Tidball, 2005). In models of chronic muscle disease such as the muscular dystrophies and other myopathies, the presence of inflammatory cells and/or their mediators within the muscle has been associated with an aggravation of muscle pathology (Wehling et al. 2001; Gosselin & McCormick, 2004; Acharyya et al. 2007). Both neutrophils and macrophages also have the capacity to kill muscle cells (Nguyen & Tidball, 2003). On the other hand, macrophages recruited to areas of acutely damaged muscle have been shown to promote more effective muscle repair (Lescaudron et al. 1999; Summan et al. 2006; Tidball & Wehling-Henricks, 2007). This dichotomy is probably related to the presence of different subpopulations of macrophages, which express specialized and polarized functions under the influence of different environmental cues (Arnold et al. 2007; Mantovani et al. 2007). Chemokines are excellent candidate molecules for playing a central role in regulating the proportions of different subpopulations of macrophages and other leucocytes within damaged muscles, as well as the chronicity of inflammation and efficacy of the subsequent muscle remodelling response. In principle, the chemokines released from damaged muscles under these conditions could originate from multiple sources including non-muscle cell types (e.g. resident macrophages, endothelial cells, etc.) and infiltrating leucocytes, as well as the muscle cells themselves.
Based upon our findings, a dual role for the CC chemokines expressed by injured muscle is thus suggested (see Fig. 9): one being a direct local effect on myoblasts to stimulate their proliferation, and the other consisting of a more distant effect on leucocytes to induce their trafficking to sites of muscle damage. Achieving the appropriate equilibrium between these two functions of chemokines is likely to be a critically important determinant of the ultimate success of the muscle repair process. Therefore, interventions aimed at modulating the balance between these distinct roles of CC chemokines in injured muscle could represent a novel strategy for the treatment of destructive muscle pathologies.
Figure 9. Proposed mechanisms by which CC chemokines can participate in the muscle repair process Chemokines can potentially be released from damaged muscle fibres, other resident cell types within the muscle, infiltrating leucocytes or myoblasts themselves. After binding to their receptors (CCR1, CCR2, CCR5) on myoblasts, the chemokines induce phosphorylation of ERK1/2 MAPK through a Gαi and MEK-dependent signalling pathway. This leads to myoblast proliferation and most probably enhanced migration, which is later followed by differentiation and eventual reconstitution of the mature muscle fibre.
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