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Molecular mechanisms modulating muscle mass David J. Glass Regeneron Pharmaceuticals, 777 Old Saw Mill River Road, Tarrytown, NY 10591-6707, USA

Skeletal muscle atrophy occurs in multiple clinical settings, including cancer, AIDS and sepsis, and is caused in part by an increase in the rate of ATP-dependent ubiquitin-mediated proteolysis. The expression of two recently identified genes encoding ubiquitin–protein ligases, MAFbx/Atrogin-1 and MuRF1, has been shown to increase during muscle atrophy. Mouse knockout studies have demonstrated that MAFbx and MuRF1 are required for muscle atrophy, and thus might be targets for clinical intervention. A second strategy for blocking atrophy involves the stimulation of pathways leading to skeletal muscle hypertrophy. Insulin-like growth factor 1 (IGF-1) is a protein growth factor that can induce skeletal muscle hypertrophy by activating the phosphatidylinositol 3-kinase (PI3K)–Akt pathway. The pathways modulating hypertrophy and atrophy will be further discussed, to highlight potential targets for clinical intervention. In the adult mammal, skeletal muscle hypertrophy is characterized by an increase in the size (as opposed to the number) of individual myofibers. Hypertrophy occurs as an adaptive response to load-bearing exercise, and as a result of an enhanced rate of protein synthesis [1]. This increase in protein synthesis enables new contractile filaments to be added to the pre-existing muscle fiber, which in turn enables the muscle to generate greater force. Increases in muscle load stimulate the expression of a protein growth factor called insulin-like growth factor 1 (IGF-1) [2]. IGF-1 stimulation is sufficient to induce hypertrophy of skeletal muscle [3]. For example, transgenic mice in which IGF-1 is overexpressed in skeletal muscle undergo hypertrophy [4,5]. Recently, it has been shown that IGF-1 induces hypertrophy by stimulating the phosphatidylinositol 3-kinase (PI3K) – Akt pathway, resulting in the downstream activation of proteins that are required for protein synthesis [6,7]. Cancer and AIDS are disease conditions that cause skeletal muscle atrophy [8]. A variety of circulating proteins have been shown to induce atrophy, including the cachectic cytokine interleukin-1 (IL-1) and tumor necrosis factor (TNF) [8]; more recently, it has been shown that transforming growth factor b (TGF-b) family member myostatin can cause atrophy when administered to an adult animal [9]. Conditions that lead to skeletal muscle atrophy cause a decrease in the size of pre-existing muscle Corresponding author: David J. Glass ([email protected]).

fibers, resulting from increases in the rate of ATPdependent ubiquitin-mediated proteolysis [10]. Recent studies have shown that the expression of two musclespecific ubiquitin ligases, MuRF1 [11] and Atrogin-1/MAFbx [11,12], is upregulated in multiple settings of atrophy. Mice in which either the MuRF1 or the Atrogin-1/MAFbx genes were deleted demonstrated sparing of skeletal muscle during atrophy conditions, as assessed by maintenance of skeletal muscle mass [11]. These signaling pathways implicated in hypertrophy and atrophy will be discussed in more detail below. Hypertrophy via IGF-1 signaling Skeletal muscle can be induced to hypertrophy via several laboratory models. In one such model, work-induced hypertrophy, transcription of the gene encoding IGF-1 was shown to increase [2]. Stimulation of muscle cells with IGF-1 is sufficient to induce hypertrophy [3]. Transgenic mice in which IGF-1 expression is increased using a muscle-specific promoter have muscles that are at least twofold greater in mass when compared with wild-type mice [4,5]. The issue of introducing IGF-1 as a treatment for skeletal muscle atrophy is complicated by several factors. First, there are at least six IGF-1 binding proteins (IGFBP1 – 6) [13]. These binding proteins are secreted, and might serve to modulate the paracrine activity of IGF-1 [13]. Further, because IGF-1 can induce proliferation in mitosis-competent cells, the broad application of IGF-1 might negatively affect other tissues. Muscle-specific overexpression of IGF-1 has been shown to ameliorate the dystrophic phenotype in the MDX mouse [14], and has been shown to accelerate regeneration [15], at least establishing the potential benefit of IGF-1 to skeletal muscle if the issues of delivery and tissue-selectivity can be solved. Although it still might be possible to administer IGF-1 directly, for example by targeted molecular delivery, it is of interest to understand the signaling pathways activated by IGF-1, so that one might find downstream molecular targets which could be more easily modulated by pharmacological intervention. IGF-1 binding activates the IGF-1 receptor (IGFR), a receptor tyrosine kinase. The IGFR subsequently recruits the insulin receptor substrate (IRS-1), which results in the activation of two signaling pathways: the Ras– Raf – MEK– ERK pathway [16] and the PI3K– Akt pathway [16] (Fig. 1). The Ras– Raf– MEK– ERK pathway is crucial in mitosis-competent cells for cell proliferation and cell

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Fig. 1. Insulin-like growth factor 1 (IGF-1)-mediated signaling pathways relevant to hypertrophy. Binding of IGF-1 activates the IGF-1 receptor (purple), which then recruits insulin-receptor substrate (IRS-1). This leads to the activation of two signaling pathways: the Ras– Raf– MEK– ERK pathway and the phosphatidylinositol 3-kinase (PI3K) – Akt pathway. The PI3K– Akt pathway recapitulates hypertrophy caused by IGF-1 stimulation. Akt1 activity can be modulated either by directly controlling its phosphorylation state or by altering the levels of the lipid that it binds at the cell membrane, phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] (orange). Signaling molecules that have been shown to have a negative effect on hypertrophy are colored red, and proteins whose activation induces hypertrophy are shown in green. Proteins that have not been assayed for their role in hypertrophy are shown in blue. Abbreviations: eIF-2B, eukaryotic translation initiation factor 2B; ERK, extracellular-signal-regulated kinase; GSK3b, glycogen-synthase kinase 3b; mTOR, mammalian target of rapamycin; p70S6K, p70 S6 kinase; PDK, phosphoinositide-dependent protein kinase; PtdIns(3,4)P2, phosphatidylinositol (3,4)-bisphosphate; PtdIns(4,5)P2, phosphatidylinositol (4,5)-bisphosphate; PHAS-1, phosphorylated heat- and acid-stable protein 1; PP2A, protein phosphatase 2A; PTEN, phosphatase and tensin homologous on chromosome 10; SHIP2, SH2-domain-containing inositol phosphatase; Tsc1/2, tuberous sclerosis complex 1 and 2. Modified from Ref. [87].

survival. However, in adult skeletal muscle, the function of the Ras– Raf –MEK– ERK pathway is less clear; some studies have shown that the pathway downstream of Raf is actually inactivated during hypertrophy [17], as a result of cross-talk from the PI3K– Akt pathway [16 –18]. By contrast, genetic activation of PI3K was shown to be sufficient to induce skeletal muscle hypertrophy [19]. Furthermore, skeletal myotube hypertrophy induced by IGF-1 could be inhibited by a pharmacological inhibitor of PI3K [17]. Therefore, PI3K activity is necessary for IGF-1 to induce hypertrophy, and its activation is sufficient to induce hypertrophy. PI3K is a lipid kinase; it phosphorylates phosphatidylinositol (4,5)-bisphosphate, producing phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] [20,21]. PtdIns(3,4,5)P3 is a membrane-binding site for two kinases: Akt1 (a serine– threonine kinase, also known as protein kinase B) and phosphoinositide-dependent protein kinase (PDK1). Akt1 is phosphorylated by PDK1, and thereby activated upon translocation to the membrane [22,23]. Once activated, Akt1 phosphorylates an everincreasing set of substrates, including proteins that block apoptosis, induce protein synthesis, gene transcription and cell proliferation [20,21]. http://tmm.trends.com

Knockout mice that are Akt1 2/2 are smaller than wildtype littermates, demonstrating that Akt1 is required for normal organ growth [24]. Transgenic mice that express a mutant, constitutively active form of Akt1 in cardiac muscle have hypertrophic hearts [25]. During skeletal muscle hypertrophy, endogenous Akt1 phosphorylation increases, as does the relative amount of Akt1 protein [7]. Expression of a dominant-negative mutant form of Akt1, which inhibits the endogenous activity of this protein, blocks IGF-1-mediated hypertrophy in vitro [6]. Expression of an activated form of Akt1 in skeletal muscle cells is sufficient to induce hypertrophy [6,17,26]. These data suggest that Akt1 activity is required for IGF-1mediated hypertrophy, and expression of activated Akt1 is sufficient to induce muscle hypertrophy. Furthermore, the finding that Akt1 is activated subsequent to PI3K stimulation, and that Akt1 can recapitulate the hypertrophic effects seen with PI3K, suggests that PI3K and Akt1 are members of a linear pathway. There are two other Akt-related genes, Akt2 and Akt3. Although similar in structure to Akt1, the proteins encoded by Akt2 and Akt3 apparently have distinct functions. For example, knockout mice that are Akt2 2/2 are normal in size but have a diabetic phenotype because of

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insulin resistance [27]. The PI3K– Akt2 pathway has been linked to glucose transport via the Glut4 transporter [28]. Insulin sensitivity and energy metabolism as a result of glucose transport are not completely distinct issues from hypertrophy; during diabetes caused by insulin insensitivity, and a resultant downregulation of the Akt pathways, skeletal muscle atrophy is also observed [8]. Furthermore, exercise and hypertrophy help to restore glucose homeostasis, probably by the coordinate regulation of Akt1 and Akt2. Akt1 activity can be modulated either by directly controlling its phosphorylation state or by altering the levels of the lipid that it binds at the cell membrane, PtdIns(3,4,5)P3 [22] (Fig. 1). Akt1 activity depends on phosphorylation at two sites: Ser473 and Thr309 [29]. Protein phosphatase 2A (PP2A) has been shown to dephosphorylate Akt1 [30 – 32]. This phosphatase is composed of three components, which are encoded by separate genes [33]. PP2A has a wide array of substrates and the gene that encodes the catalytic subunit of PP2A is ubiquitously expressed [33]. However, there might be muscle-specific forms of the PP2A regulatory subunit [34], which could provide potential for the skeletal musclespecific regulation of PP2A. The Akt1 lipid-binding site PtdIns(3,4,5)P3 is dephosphorylated by two lipid phosphatases, SHIP (SH2domain-containing inositol phosphatase) (which has two forms, SHIP1 and SHIP2) [35] and PTEN (phosphatase and tensin homologous on chromosome 10) [36] (Fig. 1). Overexpression of SHIP2 blocks hypertrophy in muscle [6,7]. Expression of a dominant-negative mutant form of SHIP2 (dnSHIP2), in which the phosphatase has been rendered inactive, enables Akt1 to remain active by blocking the dephosphorylation of PtdIns(3,4,5)P3 [37]. Expression of dnSHIP2 in myotubes causes hypertrophy, coincident with an increase in the levels of phosphorylated Akt1 [6]. Similarly, overexpression of PTEN inactivates Akt1 [36] and causes a decrease in cell size [38– 40]. Expression of a dominant-negative mutant form of PTEN in the heart induces cardiac hypertrophy [41]. The inhibition of PTEN does not seem to be a promising clinical strategy for inducing muscle hypertrophy, because heterozygote PTEN þ/2 mice have an increased propensity for cancer (probably caused by activation of the Akt1 pathway in cells capable of proliferation [42]). In a published report of a SHIP2 knockout, SHIP2 2/2 animals died soon after birth because of hypoglycemia [43], perhaps as a result of an increase in Akt2 activity. However, these SHIP2 mice are not genetically null for the entire SHIP2 gene; in these animals, the first 18 exons were left intact [43]. Given that the hypoglycemia phenotype was observed in the heterozygote SHIP2 þ/2 animals, it seems possible that some portion of the protein is made, and functions as a ‘dominant-negative’ molecule, or as a disregulated protein fragment; however, northern and western blot data suggested that the SHIP2 mRNA and protein are absent in the knockouts [43]. Whether SHIP2 might serve as a target for modulating hypertrophy depends on experiments that can discriminate between effects on glucose transport via Akt2 and stimulation of protein synthesis because of activation of Akt1. http://tmm.trends.com

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The Akt – mTOR pathways Early experiments in Drosophila helped to define a particular pathway downstream of PI3K and Akt that can control cell size. Genetic loss or inhibition of either IRS-1 [44], PI3K [45], the Drosophila homolog of the mammalian target of rapamycin (mTOR) (also known as FRAP or RAFT-1) [46], or p70 S6 kinase (p70S6K) [47] all resulted in decreases in cell size in the Drosophila wing. It is somewhat controversial whether these molecules represent a linear signaling pathway downstream of IGF-1 stimulation in mammalian cells. Although IGF-1 activates mTOR and p70S6K downstream of PI3K– Akt activation, amino acids can activate mTOR directly, causing a subsequent stimulation of p70S6K activity [48,49]. Thus, mTOR appears to have an important and central function in integrating a variety of growth signals, from simple nutritional stimulation to activation by protein growth factors, resulting in protein synthesis (Fig. 1). Akt phosphorylates mTOR [50], thereby activating it [50,51], and both Akt phosphorylation [7] and mTOR phosphorylation are increased during muscle hypertrophy [52]. Rapamycin is a chemical that binds mTOR and inhibits its function [53]. This reagent has been of value in elucidating the Akt – mTOR – p70S6K pathway. Rapamycin, when complexed with a protein called FK506-binding protein (FKBP12), disrupts activation of mTOR [53]. When applied to myotube cultures in vitro, rapamycin blocks activation of p70S6K downstream of either activated Akt1 or IGF-1 stimulation [6,17,53]. Treatment with rapamycin decreases hypertrophy [17] and muscle growth [53]. However, rapamycin does not completely block IGF-1mediated hypertrophy in vitro, which might suggest that other pathways downstream of Akt1 but independent of mTOR play a role in some settings of hypertrophy. However, in vivo treatment with rapamycin completely blocked compensatory hypertrophy, and inhibited the activation of p70S6K normally observed in this hypertrophy model [7]. Treatment with rapamycin during compensatory hypertrophy did not block activation of Akt1, again demonstrating that Akt1 is upstream of mTOR, and that p70S6K activation requires the activation of mTOR [7] (Fig. 1). This, rapamycin provides pharmacological evidence for the activation of a linear Akt1 – mTOR – p70S6K pathway during hypertrophy. Genetic support for a linear Akt1 – mTOR – p70S6K pathway has recently come from reports demonstrating that the tuberous sclerosis complex 1 and 2 proteins (Tsc1 and Tsc2) can inhibit mTOR (Fig. 1). Akt1 phosphorylates Tsc2, thereby activating mTOR at least in part by disrupting the Tsc1 – Tsc2 complex [54]. Furthermore, in insulin- or serum-stimulated cells, activation of p70S6K is inhibited by expression of the Tsc1 –Tsc2 complex [54,55]. This finding demonstrates that the introduction of a genetic inhibitor of mTOR, downstream of Akt1, inhibits the activation of p70S6K, adding genetic evidence for an Akt1 – mTOR – p70S6K pathway. Because PDK1 has been shown to phosphorylate p70S6 kinase directly [56], one might have assumed that activation of mTOR was dispensable in some settings of p70S6K activation. That might still be the case; alternatively, it might be that

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p70S6K must first be primed by another kinase, such as mTOR, before being activated by PDK1 [57,58] (Fig. 1). In addition to stimulating p70S6 kinase-mediated protein translation, activation of mTOR inhibits PHAS-1 (phosphorylated heat- and acid-stable protein 1) (also known as 4E-BP), which is a negative regulator of the translation initiation factor eIF-4E [59]. It has recently been shown that PHAS-1 can directly bind a protein called ‘Raptor’, which also binds mTOR [60,61] (Fig. 1). Mutations in PHAS-1 that inhibit interaction with Raptor also inhibit mTOR-mediated phosphorylation of PHAS-1 [62]. Finally, overexpression of Raptor can enhance the phosphorylation of PHAS-1 by mTOR in vitro [62,63]. mTOR binds PHAS-1 by a TOR signaling (TOS) motif; this same motif is found in p70S6K [63]. Thus, mTOR can increase protein synthesis by modulating two distinct pathways, the p70S6K pathway and the Raptor– PHAS-1 pathway. Blockade of PHAS-1 might be a potential route to increasing protein synthesis and therefore hypertrophy. Stimulation of p70S6K might be a second route. The same phosphatase that dephosphorylates Akt1, pp2A, has also been shown to dephosphorylate p70S6K [64]. Therefore, pp2A seems to be an important modulator of IGF-1 signaling. PI3K –Akt –GSK3b pathway Glycogen-synthase kinase 3b (GSK3b) is a distinct substrate of Akt1 that has been shown to modulate hypertrophy. GSK3b activity is inhibited by Akt1 phosphorylation [65] (Fig. 1). Expression of a dominantnegative, kinase-inactive form of GSK3b induces dramatic hypertrophy in skeletal myotubes [6]. In cardiac hypertrophy, GSK3b phosphorylation is also evident [66], and expression of a dominant-negative form of GSK3b can induce cardiac hypertrophy [66]. Cardiac hypertrophy was also shown to proceed via a PI3K-dependent process, linking GSK3b to the PI3K –Akt pathway in the heart [67]. GSK3b blocks protein translation initiated by the eIF-2B protein [66]. Therefore GSK3b inhibition might induce hypertrophy by stimulating protein synthesis independent of the mTOR pathway. Atrophy via induction of ubiquitin ligase pathways One might question whether skeletal muscle atrophy is simply the converse of skeletal muscle hypertrophy. However, during skeletal muscle atrophy, an entirely distinct process is stimulated: a dramatic increase in protein degradation and turnover. Furthermore, unique transcriptional pathways are activated, and these are not necessarily the converse of those seen during hypertrophy [68,69]. This stimulation of proteolysis was shown to occur at least in part because of an activation of the ubiquitin– proteasome pathway [68]. Ubiquitin is a short peptide that can be conjugated to specific protein substrates. A chain reaction might then ensue: a second ubiquitin peptide is ligated to the first, and a third to the second; in this way, a chain of polyubiquitin is built onto the substrate, and this ubiquitin chain targets the substrate to a structure called the proteasome, where the substrate is proteolyzed into small peptides [68]. http://tmm.trends.com

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The addition of ubiquitin to a protein substrate has come to be recognized as an exquisitely modulated process. This process requires three distinct enzymatic components, an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin-ligating enzyme. The E3 ubiquitin ligases are the components that confer substrate specificity. Several hundred distinct E3s have already been identified, and it is likely that each modulates the ubiquitination of a distinct set of substrates. Thus, the regulation of ubiquitination appears to be a coordinate signaling pathway, analagous to phosphorylation, in which key pathways might be activated by the enhanced proteolysis of a key inhibitor protein, or in which pathways might be inactivated via the degradation of an activating enzyme. The involvement of the ubiquitin– proteasome pathway in skeletal muscle atrophy had been well-established: rates of protein breakdown increase during atrophy; inhibition of the proteasome blocks these increases [70]; the amount of polyubiquitin conjugation per total protein measured increases during atrophy [71]; mRNA levels of genes that encode distinct components of the ubiquitin pathway increase during atrophy [68]. Genomic experiments designed to identify markers of the atrophy process identified two genes whose expression increased significantly in multiple models of skeletal muscle atrophy: MuRF1 (muscle ring finger 1) [11] and MAFbx (muscle atrophy F-box; also known as Atrogin-1 [12]) [11]. Both of these genes were shown to encode E3 ubiquitin ligases [11]. Expression of MuRF1 and MAFbx is stimulated when the nerve innervating a muscle is cut, thus resulting in paralysis and severe atrophy; these genes are also upregulated by simple immobilization of the muscle, or by treatment with a glucocorticoid, which causes muscle cachexia [11]. More recently, a predictive experiment was performed, in which sepsis-induced atrophy was induced to determine whether this distinct model of atrophy might also increase expression of MuRF1 and MAFbx. Both of these genes were upregulated several-fold during sepsis [72], and this upregulation could be blocked by a pharmacologic inhibitor of glucocorticoids [72]. MuRF1 encodes a protein that contains three domains: a RING-finger domain [73], which is required for ubiquitin-ligase activity [74]; a ‘B-box’, whose function is unclear; and a ‘coil – coil domain’ which might be required for the formation of heterodimers between MuRF1 and a related protein, MuRF2 [75]. Proteins that have these three domains have been called ‘RBCC’ (RING, B-Box, coil – coil domain) proteins [76], or ‘TRIM’ (tripartite motif) proteins [77]. MuRF1 has been demonstrated to have ubiquitin-ligase activity that depends on the presence of the RING domain for that activity [11]. Although particular substrates have not yet been demonstrated, MuRF1 has been shown to bind to the myofibrillar protein titin, at the M line [75,78,79]. Overexpression of MuRF1 results in the disruption of the subdomain of titin that binds MuRF1, suggesting that MuRF1 might play a role in titin turnover [78]. MuRF1 has also been demonstrated to be in the nucleus, and indications that it interacts with transcription-regulating elements such as GMEB-1 suggest a potential role for MuRF1 in modulating

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transcription [78]. However, this has not yet been demonstrated. MAFbx/Atrogin-1 contains an F-box domain, a characteristic motif seen in a family of E3 ubiquitin ligases called SCFs (Skp1, Cullin, F-box) [80]. F-box-containing E3 ligases usually bind a substrate only after that substrate has first been post-translationally modified, for example by phosphorylation [80]. This suggests the possibility of a signaling pathway in which a potential substrate is first phosphorylated as a response to an atrophy-induced stimulus, and then degraded via MAFbx. MuRF1 2/2 and MAFbx 2/2 mice appear phenotypically normal. However, under atrophy conditions, significantly less muscle mass is lost in either MuRF1 2/2 or MAFbx 2/2 animals in comparison with control littermates [11]. This finding demonstrated for the first time that inhibition of discrete ubiquitin ligases could moderate the amount of muscle lost after an atrophy-inducing stimulus. Therefore, MuRF1 or MAFbx might be attractive targets for pharmacological intervention. They might also serve as early markers of skeletal muscle atrophy, aiding in the diagnosis of muscle disease. Myostatin The development of a strain of cows that had excessive amounts of beef led to the isolation of myostatin, a TGF-b family member [81,82]. The ‘double-muscled’ cows were shown to have a mutation in the gene encoding this protein [81,83,84]. Mice engineered to be myostatin null similarly have a large increase in muscle mass relative to wild-type littermates [82]. However, when muscle obtained from myostatin 2/2 animals was analyzed, it was shown to be larger as a result of an increase in the number of muscle fibers, and not as a result of hypertrophy. Therefore, it was thought that myostatin acts in a way that is distinct from the activation of atrophy or the inhibition of hypertrophy; that it simply blocks the proliferation of muscle precursors, thus decreasing muscle mass. However, a more recent experiment has complicated matters: when adult animals are given an inhibitory antibody to myostatin, they undergo what appears to be muscle hypertrophy [85]. Also, mice given myostatin undergo muscle atrophy [9]. Whether myostatin is acting directly on pre-existing muscle fibers, or whether these data uncover a requisite role for satellite cell proliferation and fusion in the maintenance of normal muscle mass, remains to be seen. However, the possibility exists that blockade of the myostatin pathway might now be a distinct route to inhibiting skeletal muscle atrophy. Inhibition of myostatin has already been shown to be beneficial for dystrophic muscle [86]; however, this might be a unique circumstance because of the rapid turnover of muscle and the subsequent need for satellite cell proliferation and differentiation in the dystrophic animal. Conclusion A considerable amount of progress has been made recently in our understanding of the signaling pathways that mediate skeletal muscle hypertrophy and atrophy. These findings provide hope that novel drug targets might be found to block skeletal muscle atrophy, and the gradual http://tmm.trends.com

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loss of strength seen even in normal aging. The lack of approved drugs for skeletal muscle disease highlights the need for continued research in this area. Acknowledgements Thanks to L.S. Schleifer and P.R. Vagelos for enthusiastic support, to G.D. Yancopoulos for critical guidance, and to the Regeneron community. Thanks also to colleagues at Procter and Gamble Pharmaceuticals for their enthusiastic help. Thanks to T.N. Stitt for comments on this manuscript. Sincere apologies to scientific colleagues whose work was omitted from this review because of space constraints.

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