Kollias HD, McDermott JC. Transforming growth factor-␤ and myostatin signaling in skeletal muscle.

Skeletal muscle fibrosis is a major pathological hallmark of chronic myopathies in which myofibers are replaced by progressive deposition of collagen and other extracellular matrix proteins produced by muscle fibroblasts. Recent studies have shown that in the absence of the endogenous muscle growth regulator myostatin, regeneration of muscle is enhanced, and muscle fibrosis is correspondingly reduced. We now demonstrate that myostatin not only regulates the growth of myocytes but also directly regulates muscle fibroblasts. Our results show that myostatin stimulates the proliferation of muscle fibroblasts and the production of extracellular matrix proteins both in vitro and in vivo. Further, muscle fibroblasts express myostatin and its putative receptor activin receptor IIB. Proliferation of muscle fibroblasts, induced by myostatin, involves the activation of Smad, p38 MAPK and Akt pathways. These results expand our understanding of the function of myostatin in muscle tissue and provide a potential target for anti-fibrotic therapies.Fibroblasts play an important role in the repair response of tissues to injury by secreting extracellular matrix proteins including collagen and growth factors. However, in a variety of disease states, continued fibrosis contributes to the pathological process. In chronic myopathies and muscular dystrophies, fibrosis is considered to be associated with decreased strength and elasticity of muscle and may inhibit the diffusion of nutrients to myofibers (1-3). Identification of factors that regulate fibrosis is an important goal not only in understanding the pathogenesis of muscular dystrophies but also in developing novel therapies to treat these disorders. Several potential future therapies for the muscular dystrophies, including those involving gene and myoblast transfer, may be hampered by significant fibrosis.Myostatin is a highly conserved transforming growth factor-␤ (TGF-␤) 2 family member that is expressed in skeletal muscle, which is also the primary target tissue (4). Deletion of the myostatin gene (MSTN) in mice leads to muscle hypertrophy and hyperplasia with an approximate doubling of muscle mass (4). This function of myostatin, as an endogenous inhibitor of muscle growth, is also conserved in humans, as demonstrated by the identification of a hypermuscular child with a loss-of-function mutation in the myostatin gene (5). One mechanism by which myostatin regulates muscle growth in adult animals appears to be direct inhibition of the proliferation and differentiation of resident muscle precursor cells (6 -8).The potential effect of myostatin inhibition on muscle degenerative diseases has been explored in various animal models. In the absence of myostatin, muscle regenerates more quickly and completely following acute and chronic injury (8, 9). In the mdx mouse, a model of Duchenne and Becker muscular dystrophy, myostatin deletion, or postnatal inhibition increases muscle mass and strength (10 -12). An early observation of mdx/mstn null mice was that the diaphragm muscle of t...

Transforming growth factor ␤1 (TGF-␤1) and myostatin signaling, mediated by the same Smad downstream effectors, potently repress skeletal muscle cell differentiation. Smad7 inhibits these cytokine signaling pathways. The role of Smad7 during skeletal muscle cell differentiation was assessed. In these studies, we document that increased expression of Smad7 abrogates myostatin-but not TGF-␤1-mediated repression of myogenesis. Further, constitutive expression of exogenous Smad7 potently enhanced skeletal muscle differentiation and cellular hypertrophy. Conversely, targeting of endogenous Smad7 by small interfering RNA inhibited C2C12 muscle cell differentiation, indicating an essential role for Smad7 during myogenesis. Congruent with a role for Smad7 in myogenesis, we observed that the muscle regulatory factor (MyoD) binds to and transactivates the Smad7 proximal promoter region. Finally, we document that Smad7 directly interacts with MyoD and enhances MyoD transcriptional activity. Thus, Smad7 cooperates with MyoD, creating a positive loop to induce Smad7 expression and to promote MyoD driven myogenesis. Taken together, these data implicate Smad7 as a fundamental regulator of differentiation in skeletal muscle cells.

Key pointsr Smad7 is an intracellular antagonist of transforming growth factor-β signalling pathways and modulates muscle growth in vivo.r Loss of Smad7 results in decreased muscle mass, reduced force generation, fibre type switching from glycolytic towards oxidative type and delayed recovery from injury.r Upregulated Smad2/3 signalling in Smad7 −/− muscle results in reduced myoblast proliferation and differentiation.r Smad7 is an important regulator of muscle growth and may be a potential intracellular therapeutic target for muscle disorders. AbstractThe transforming growth factor-β (TGF-β) family of growth factors plays an essential role in mediating cellular growth and differentiation. Myostatin is a muscle-specific member of the TGF-β superfamily and a negative regulator of muscle growth. Myostatin inhibitors are currently being pursued as therapeutic options for muscle disorders. Smad7 inhibits intracellular myostatin signalling via Smad2/3, and thus presents a means of regulating myostatin and potentiating muscle growth. We investigated the functional loss of Smad7 on muscle in vivo by examining muscle growth and differentiation in mice deficient in Smad7 (Smad7 −/− ). Smad7 −/− mice showed reduced muscle mass, hypotrophy and hypoplasia of muscle fibres, as well as an increase in oxidative fibre types. Examination of muscle strength showed reduced force generation in vivo and ex vivo compared to wild-type controls. Analysis of muscle regeneration showed a delay in recovery, probably as a result of decreased activation, proliferation and differentiation of satellite cells, as confirmed in vitro. Additionally, myostatin expression was upregulated in Smad7 −/− muscle. Our findings suggest that increased Smad2/3 signalling in the absence of Smad7 inhibition impedes muscle growth and regeneration. Taken together, our experiments demonstrate that Smad7 is an important mediator of muscle growth in vivo. Our studies enhance our understanding of in vivo TGF-β pathway modulation and suggest that Smad7 may be an important therapeutic target for muscle disorders. AbbreviationsActRIIB, activin receptor type IIB; CK, creatine kinase; CSA, cross-sectional area; DAPI, 4' ,6-diamidino-2-phenylindole; ECL, electrochemiluminescence; EDL, extensor digitorum longus; EdU, 5-ethynyl-2'-deoxyuridine; eMyHC, embryonic myosin heavy chain; GLS, generalized least squares; H&E, haematoxylin and eosin; MRF, myogenic regulatory factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MyHC, myosin heavy chain; PBS-T, phosphate-buffered saline and 0.1% Tween 20; TA, tibialis anterior; TGF-β, transforming growth factor-β; WT, wild-type.