Mònica Suelves

E47 phosphorylation by p38 MAPK promotes MyoD/E47 association and muscle‐specific gene transcription

Selective recognition of the E‐box sequences on muscle gene promoters by heterodimers of myogenic basic helix–loop–helix (bHLH) transcription factors, such as MyoD, with the ubiquitous bHLH proteins E12 and E47 is a key event in skeletal myogenesis. However, homodimers of MyoD or E47 are unable of binding to and activating muscle chromatin targets, suggesting that formation of functional MyoD/E47 heterodimers is pivotal in controlling muscle transcription. Here we show that p38 MAPK, whose activity is essential for myogenesis, regulates MyoD/E47 heterodimerization. Phosphorylation of E47 at Ser140 by p38 induces MyoD/E47 association and activation of muscle‐specific transcription, while the nonphosphorylatable E47 mutant Ser140Ala fails to heterodimerize with MyoD and displays impaired myogenic potential. Moreover, inhibition of p38 activity in myocytes precludes E47 phosphorylation at Ser140, which results in reduced MyoD/E47 heterodimerization and inefficient muscle differentiation, as a consequence of the impaired binding of the transcription factors to the E regulatory regions of muscle genes. These findings identify a novel pro‐myogenic role of p38 in regulating the formation of functional MyoD/E47 heterodimers that are essential for myogenesis.

Fibrinogen drives dystrophic muscle fibrosis via a TGFβ/alternative macrophage activation pathway

In the fatal degenerative Duchenne muscular dystrophy (DMD), skeletal muscle is progressively replaced by fibrotic tissue. Here, we show that fibrinogen accumulates in dystrophic muscles of DMD patients and mdx mice. Genetic loss or pharmacological depletion of fibrinogen in these mice reduced fibrosis and dystrophy progression. Our results demonstrate that fibrinogen-Mac-1 receptor binding, through induction of IL-1beta, drives the synthesis of transforming growth factor-beta (TGFbeta) by mdx macrophages, which in turn induces collagen production in mdx fibroblasts. Fibrinogen-produced TGFbeta further amplifies collagen accumulation through activation of profibrotic alternatively activated macrophages. Fibrinogen, by engaging its alphavbeta3 receptor on fibroblasts, also directly promotes collagen synthesis. These data unveil a profibrotic role of fibrinogen deposition in muscle dystrophy.

uPA deficiency exacerbates muscular dystrophy in MDX mice.

Duchenne muscular dystrophy (DMD) is a fatal and incurable muscle degenerative disorder. We identify a function of the protease urokinase plasminogen activator (uPA) in mdx mice, a mouse model of DMD. The expression of uPA is induced in mdx dystrophic muscle, and the genetic loss of uPA in mdx mice exacerbated muscle dystrophy and reduced muscular function. Bone marrow (BM) transplantation experiments revealed a critical function for BM-derived uPA in mdx muscle repair via three mechanisms: (1) by promoting the infiltration of BM-derived inflammatory cells; (2) by preventing the excessive deposition of fibrin; and (3) by promoting myoblast migration. Interestingly, genetic loss of the uPA receptor in mdx mice did not exacerbate muscular dystrophy in mdx mice, suggesting that uPA exerts its effects independently of its receptor. These findings underscore the importance of uPA in muscular dystrophy.

Phosphorylation of MRF4 transactivation domain by p38 mediates repression of specific myogenic genes

Skeletal myogenesis is associated with the activation of four muscle regulatory factors (MRFs): Myf5, MyoD, Myogenin and MRF4. Here we report that p38 mitogen‐activated protein kinase represses the transcriptional activity of MRF4 (involved in late stages of myogenesis), resulting in downregulation of specific muscle genes. MRF4 is phosphorylated in vitro and in vivo by p38 on two serines (Ser31 and Ser42) located in the N‐terminal transactivation domain, resulting in reduced MRF4‐mediated transcriptional activity. In contrast, nonphosphorylatable MRF4 mutants display increased transcriptional activity and are able to advance both myoblast fusion and differentiation. We also show that expression of desmin and α‐actin, but not muscle creatin kinase, decreased at late stages of muscle differentiation, correlating with the induction of MRF4 and p38 activation. Accordingly, inhibition of p38 during late myogenesis results in the upregulation of both desmin and α‐actin. We propose that repression of MRF4 activity by p38 phosphorylation may represent a new mechanism for the silencing of specific muscle genes at the terminal stages of muscle differentiation.

Plasmin generation dependent on α-enolase-type plasminogen receptor is required for myogenesis

Plasmin is a potent extracellular protease specialized in the degradation of fibrin (fibrinolysis). Active plasmin is generated by proteolytic activation of the zymogen plasminogen (Plg) by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). a -Enolase constitutes a receptor for plasminogen on several leukocyte cell types, serving to localize and promote plasminogen activation pericellularly. However, a role for a -enolase-type plasminogen receptor (PlgR) in myogenesis has never been demonstrated. In this study, we show that C2C12 mouse myoblasts express PlgR, being its expression greatly induced during the differentiation process. A monoclonal antibody against PIgR MAb 11G1, with cell surface-generated plasmin inhibitory abilities, was able to fully abrogate C2C12 myoblast fusion and differentiation in vitro. Moreover, both plasmin activity and PlgR expression were significantly induced in regenerating skeletal muscle in vivo, either in experimentally-injured muscle or in the dystrophic muscle of mdx mouse (an animal model of human Duchenne muscular dystrophy, DMD). The mdx muscle presents better regeneration capacities and less fibrosis than the human DMD muscle; therefore, the increase in PlgR/plasmin activity in mdx muscle suggests an important contribution of the fibrinolytic system in mdx regeneration. This study constitutes the first indication of α-enolase-type plasminogen receptor as an impor-tant component of skeletal myogenesis, by concentrating and enhancing plasmin generation on the cell surface.

Lineage of origin in rhabdomyosarcoma informs pharmacological response

Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.

The plasminogen activation system in skeletal muscle regeneration: antagonistic roles of urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1).

The plasminogen activation (PA) system is an extensively used mechanism for the generation of proteolytic activity in the extracellular matrix, where it contributes to tissue remodeling in a wide range of physiopathological processes. Despite the limited information available at present on plasminogen activators, their inhibitors and cognate receptors in skeletal muscle, increasing evidence is accumulating on their important roles in the homeostasis of muscle fibers and their surrounding extracellular matrix. The development of mice deficient for the individual components of the PA system has provided an incisive approach to test the proposed muscle functions in vivo. Skeletal muscle regeneration induced by injury has been analyzed in urokinase-type plasminogen activator (uPA)-, tissue-type plasminogen activator (tPA)-, plasminogen (Plg)- and plasminogen activator inhibitor-1 (PAI-1)-deficient mice and has demonstrated profound effects of these molecules on the fibrotic state and the inflammatory response, which contribute to muscle repair. In particular, the opposite roles of uPA and its inhibitor PAI-1 in this process are highlighted. Delineating the mechanisms by which the different plasminogen activation system components regulate tissue repair will be of potential therapeutic value for severe muscle disorders.

ALPHA-ENOLASE PLASMINOGEN RECEPTOR IN MYOGENESIS

Plasmin is a potent extracellular protease specialized in the degradation of fibrin (fibrinolysis). Active plasmin is generated by proteolytic activation of the zymogen plasminogen (Plg) by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Alpha-enolase, although traditionally considered a glycolytic enzyme, constitutes a receptor for plasminogen on several cell types, serving to localize and promote plasminogen activation pericellularly. Localization of plasmin activity on the cell surface plays a critical role in fibrinolysis and in physiopathological processes involving extracellular matrix remodelling. Previous studies have unambiguously demonstrated that uPA-dependent plasmin generation is necessary for myogenesis in vitro and for muscle regeneration in vivo. However, the implication of alpha-enolase plasminogen receptor in myogenesis had never been investigated. This review focuses on the recently reported expression and function of alpha-enolase plasminogen receptor during myogenesis. Skeletal myoblasts express alpha-enolase plasminogen receptor, being its expression greatly induced during the differentiation process in vitro. MAb 11G1, a monoclonal antibody against anti-alpha-enolase plasminogen receptor, that inhibits plasmin generation, was able to fully abrogate myoblast fusion and differentiation. Moreover, both plasmin activity and alpha-enolase plasminogen receptor expression were significantly augmented in injury-induced regenerating muscle of wild type mice and in the dystrophic muscle of mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Altogether, these results indicate that the plasminogen activation (PA) system is an important component of skeletal myogenesis in vitro and in vivo. In particular, the expression of alpha-enolase plasminogen receptor may serve to concentrate and enhance plasmin generation on the cell surface of migratory myoblasts contributing to efficient muscle repair.

Amelioration of Duchenne muscular dystrophy in mdx mice by elimination of matrix-associated fibrin-driven inflammation coupled to the αMβ2 leukocyte integrin receptor

In Duchenne muscular dystrophy (DMD), a persistently altered and reorganizing extracellular matrix (ECM) within inflamed muscle promotes damage and dysfunction. However, the molecular determinants of the ECM that mediate inflammatory changes and faulty tissue reorganization remain poorly defined. Here, we show that fibrin deposition is a conspicuous consequence of muscle-vascular damage in dystrophic muscles of DMD patients and mdx mice and that elimination of fibrin(ogen) attenuated dystrophy progression in mdx mice. These benefits appear to be tied to: (i) a decrease in leukocyte integrin α(M)β(2)-mediated proinflammatory programs, thereby attenuating counterproductive inflammation and muscle degeneration; and (ii) a release of satellite cells from persistent inhibitory signals, thereby promoting regeneration. Remarkably, Fib-gamma(390-396A) (Fibγ(390-396A)) mice expressing a mutant form of fibrinogen with normal clotting function, but lacking the α(M)β(2) binding motif, ameliorated dystrophic pathology. Delivery of a fibrinogen/α(M)β(2) blocking peptide was similarly beneficial. Conversely, intramuscular fibrinogen delivery sufficed to induce inflammation and degeneration in fibrinogen-null mice. Thus, local fibrin(ogen) deposition drives dystrophic muscle inflammation and dysfunction, and disruption of fibrin(ogen)-α(M)β(2) interactions may provide a novel strategy for DMD treatment.

DNA methylation dynamics in muscle development and disease

DNA methylation is an essential epigenetic modification for mammalian development and is crucial for the establishment and maintenance of cellular identity. Traditionally, DNA methylation has been considered as a permanent repressive epigenetic mark. However, the application of genome-wide approaches has allowed the analysis of DNA methylation in different genomic contexts revealing a more dynamic regulation than originally thought, since active DNA methylation and demethylation occur during cellular differentiation and tissue specification. Satellite cells are the primary stem cells in adult skeletal muscle and are responsible for postnatal muscle growth, hypertrophy, and muscle regeneration. This review outlines the published data regarding DNA methylation changes along the skeletal muscle program, in both physiological and pathological conditions, to better understand the epigenetic mechanisms that control myogenesis.