SMAD2 promotes myogenin expression and terminal myogenic differentiation.

SMAD2 is a transcription factor, the activity of which is regulated by members of the transforming growth factor ß (TGFß) superfamily. Although activation of SMAD2 and SMAD3 downstream of TGFß or myostatin signaling is known to inhibit myogenesis, we found that SMAD2 in the absence of TGFß signaling promotes terminal myogenic differentiation. We found that, during myogenic differentiation, SMAD2 expression is induced. Knockout of SMAD2 expression in primary myoblasts did not affect the efficiency of myogenic differentiation but produced smaller myotubes with reduced expression of the terminal differentiation marker myogenin. Conversely, overexpression of SMAD2 stimulated myogenin expression, and enhanced both differentiation and fusion, and these effects were independent of classical activation by the TGFß receptor complex. Loss of Smad2 in muscle satellite cells in vivo resulted in decreased muscle fiber caliber and impaired regeneration after acute injury. Taken together, we demonstrate that SMAD2 is an important positive regulator of myogenic differentiation, in part through the regulation of Myog.

Angiotensin 1-7 increases fiber cross-sectional area and force in juvenile mouse skeletal muscle.

Recent studies reported that in skeletal muscle angiotensin 1-7 (Ang 1-7), via its receptor Mas (MasR), prevents the atrophy induced by angiotensin II and by cast immobilization; it also improves muscle integrity and function in the mdx mouse, a muscular dystrophy model. The objectives of this study were to document 1) the extent of the Ang 1-7's hypertrophic effect in terms of muscle mass and muscle fiber cross-sectional area (CSA), 2) how Ang 1-7 affects muscle contractile function in terms of twitch and tetanic force, force-frequency relationship, and 3) whether the effect involves MasR. Wild-type and MasR-deficient [Mas receptor knockout mouse model (MasR-/-)] mice were treated with Ang 1-7 (100 ng/kg body wt·min using an osmotic pump) for 4 or 16 wk. Ang 1-7 significantly increased skeletal muscle/body weight ratio of soleus, tibialis, and gastrocnemius, but not of extensor digitorum longus (EDL). It significantly increased fiber cross-sectional area in the order of type I > IIA > IIB. In EDL and soleus muscles, it significantly increased twitch and tetanic force while causing a shift in the force-frequency relationship toward lower stimulation frequencies. It had no effect on fiber type composition. None of the Ang 1-7 effects observed in wild-type mice were observed in MasR-/- muscles. It caused a transient increase in phosphorylated protein kinase B (Akt) and 4EBP proteins while having no effect on S6 phosphorylation, MuRF-1, and atrogin-1 and a decrease in PAX7 expression in satellite cells. This is the first study demonstrating the hypertrophic effects of Ang 1-7 in normal muscle acting via its MasR.

The role of L-type amino acid transporter 1 (Slc7a5) during in vitro myogenesis.

Satellite cells are required for muscle regeneration, remodeling, and repair through their activation, proliferation, and differentiation; however, how dietary factors regulate this process remains poorly understood. The L-type amino acid transporter 1 (LAT1) transports amino acids, such as leucine, into mature myofibers, which then stimulate protein synthesis and anabolic signaling. However, whether LAT1 is expressed on myoblasts and is involved in regulating myogenesis is unknown. The aim of this study was to characterize the expressional and functional relevance of LAT1 during different stages of myogenesis and in response to growth and atrophic conditions in vitro. We determined that LAT1 is expressed by C2C12 and human primary myoblasts, and its gene expression is lower during differentiation (P < 0.05). Pharmacological inhibition and genetic knockdown of LAT1 impaired myoblast viability, differentiation, and fusion (all P < 0.05). LAT1 protein content in C2C12 myoblasts was not significantly altered in response to different leucine concentrations in cell culture media or in two in vitro atrophy models. However, LAT1 content was decreased in myotubes under atrophic conditions in vitro (P < 0.05). These findings indicate that LAT1 is stable throughout myogenesis and in response to several in vitro conditions that induce muscle remodeling. Further, amino acid transport through LAT1 is required for normal myogenesis in vitro.

CCAAT/enhancer-binding protein beta promotes muscle stem cell quiescence through regulation of quiescence-associated genes.

Regeneration of skeletal muscle depends on resident muscle stem cells called satellite cells that in healthy, uninjured muscle remain quiescent (noncycling). After activation and expansion of satellite cells postinjury, satellite cell numbers return to uninjured levels and return to mitotic quiescence. Here, we show that the transcription factor CCAAT/enhancer-binding protein beta (C/EBPß) is required to maintain quiescence of satellite cells in uninjured muscle. We show that C/EBPß is expressed in quiescent satellite cells in vivo and upregulated in noncycling myoblasts in vitro. Loss of C/EBPß in satellite cells promotes their premature exit from quiescence resulting in spontaneous activation and differentiation of the stem cell pool. Forced expression of C/EBPß in myoblasts inhibits proliferation by upregulation of 28 quiescence-associated genes. Furthermore, we find that caveolin-1 is a direct transcriptional target of C/EBPß and is required for cell cycle exit in muscle satellite cells expressing C/EBPß. The induction of mitotic quiescence is considered necessary for the long-term maintenance of adult stem cell populations with dysregulation driving increased differentiation of progenitors and depletion of the stem cell pool. Our findings place C/EBPß as an important transcriptional regulator of muscle satellite cell quiescence.

Mdm2 promotes myogenesis through the ubiquitination and degradation of CCAAT/enhancer-binding protein ß.

Myogenesis is a tightly regulated differentiation process during which precursor cells express in a coordinated fashion the myogenic regulatory factors, while down-regulating the satellite cell marker Pax7. CCAAT/Enhancer-binding protein ß (C/EBPß) is also expressed in satellite cells and acts to maintain the undifferentiated state by stimulating Pax7 expression and by triggering a decrease in MyoD protein expression. Herein, we show that C/EBPß protein is rapidly down-regulated upon induction of myogenesis and this is not due to changes in Cebpb mRNA expression. Rather, loss of C/EBPß protein is accompanied by an increase in Mdm2 expression, an E3 ubiquitin ligase. We demonstrate that Mdm2 interacts with, ubiquitinates and targets C/EBPß for degradation by the 26 S proteasome, leading to increased MyoD expression. Knockdown of Mdm2 expression in myoblasts using a shRNA resulted in high C/EBPß levels and a blockade of myogenesis, indicating that Mdm2 is necessary for myogenic differentiation. Primary myoblasts expressing the shMdm2 construct were unable to contribute to muscle regeneration when grafted into cardiotoxin-injured muscle. The differentiation defect imposed by loss of Mdm2 could be partially rescued by loss of C/EBPß, suggesting that the regulation of C/EBPß turnover is a major role for Mdm2 in myoblasts. Taken together, we provide evidence that Mdm2 regulates entry into myogenesis by targeting C/EBPß for degradation by the 26 S proteasome.

Skeletal myosin light chain kinase regulates skeletal myogenesis by phosphorylation of MEF2C.

The MEF2 factors regulate transcription during cardiac and skeletal myogenesis. MEF2 factors establish skeletal muscle commitment by amplifying and synergizing with MyoD. While phosphorylation is known to regulate MEF2 function, lineage-specific regulation is unknown. Here, we show that phosphorylation of MEF2C on T(80) by skeletal myosin light chain kinase (skMLCK) enhances skeletal and not cardiac myogenesis. A phosphorylation-deficient MEF2C mutant (MEFT80A) enhanced cardiac, but not skeletal myogenesis in P19 stem cells. Further, MEFT80A was deficient in recruitment of p300 to skeletal but not cardiac muscle promoters. In gain-of-function studies, skMLCK upregulated myogenic regulatory factor (MRF) expression, leading to enhanced skeletal myogenesis in P19 cells and more efficient myogenic conversion. In loss-of-function studies, MLCK was essential for efficient MRF expression and subsequent myogenesis in embryonic stem (ES) and P19 cells as well as for proper activation of quiescent satellite cells. Thus, skMLCK regulates MRF expression by controlling the MEF2C-dependent recruitment of histone acetyltransferases to skeletal muscle promoters. This work identifies the first kinase that regulates MyoD and Myf5 expression in ES or satellite cells.

Hedgehog signaling regulates MyoD expression and activity.

The inhibition of MyoD expression is important for obtaining muscle progenitors that can replenish the satellite cell niche during muscle repair. Progenitors could be derived from either embryonic stem cells or satellite cells. Hedgehog (Hh) signaling is important for MyoD expression during embryogenesis and adult muscle regeneration. To date, the mechanistic understanding of MyoD regulation by Hh signaling is unclear. Here, we demonstrate that the Hh effector, Gli2, regulates MyoD expression and associates with MyoD gene elements. Gain- and loss-of-function experiments in pluripotent P19 cells show that Gli2 activity is sufficient and required for efficient MyoD expression during skeletal myogenesis. Inhibition of Hh signaling reduces MyoD expression during satellite cell activation in vitro. In addition to regulating MyoD expression, Hh signaling regulates MyoD transcriptional activity, and MyoD activates Hh signaling in myogenic conversion assays. Finally, Gli2, MyoD, and MEF2C form a protein complex, which enhances MyoD activity on skeletal muscle-related promoters. We therefore link Hh signaling to the function and expression of MyoD protein during myogenesis in stem cells.

Extending social accountability mandates to biomedical research in Canadian faculties of medicine.

Background: Social accountability (SA), as defined by Boelen and Heck, is the obligation of medical schools to address the needs of communities through education, research and service activities. While SA is embedded within health profession education frameworks in medicine, they are rarely taught within graduate-level (MSc/PhD) education. Methods: As these programs train future medical researchers, we invited first-year graduate students enrolled in a mandatory professionalism class at our institution (n = 111) to complete a survey on their perceptions of the importance of SA in their research, training, and future careers. Results: Over 80% (n = 87) of respondents agreed that SA is relevant and felt committed to integrating it into their future research activities, only a limited number of students felt confident and/or supported in their abilities to integrate SA into their research. Conclusions: Specific SA training in graduate education is necessary for students to effectively incorporate elements of SA into their research, and as such support the SA mandates of their training institutions. We posit that awareness of SA principles formalizes the professional standards for biomedical researchers and is thus foundational for developing a professionalism curriculum in graduate education programs in medicine. We propose an expansion of the World Health Organization (WHO) partnership pentagon to include partners within the research ecosystem (funding partners, certification bodies) that collaborate with biomedical researchers to make research socially accountable.

Contexte: La responsabilité sociale (RS), telle que définie par Boelen et Heck, est l'obligation pour les facultés de médecine de répondre aux besoins des communautés par l'entremise de l'éducation, de la recherche et des activités de service. Bien que la responsabilité sociale soit intégrée dans les cadres de formation des professionnels de santé en médecine, elle est rarement enseignée au niveau des études supérieures (MSc/PhD). Méthodes: Étant donné que ces programmes forment les futurs chercheurs médicaux, nous avons invité les étudiants de première année inscrits à un cours obligatoire sur le professionnalisme dans notre établissement (n = 111) à participer à une enquête sur leurs perceptions de l'importance de la RS dans leur recherche, leur formation et leur future carrière. Résultats: Plus de 80 % (n = 87) des répondants ont reconnu la pertinence de la RS et se sont engagés à l'intégrer dans leurs futures activités de recherche, mais seul un nombre limité d'étudiants se sont sentis confiants et/ou soutenus dans leurs capacités à intégrer la RS dans leur recherche. Conclusions: Une formation propre à la RS dans le cadre des études supérieures est nécessaire pour que les étudiants puissent intégrer efficacement des éléments de la RS dans leur recherche, et ainsi promouvoir les mandats de RS de leurs établissements de formation. Nous estimons que la sensibilisation aux principes de la RS formalise les normes professionnelles des chercheurs biomédicaux et qu'elle est donc fondamentale pour l'élaboration d'un programme de professionnalisme dans les programmes d'études supérieures en médecine. Nous proposons d'élargir le pentagone du partenariat de l'Organisation mondiale de la santé (OMS) pour y inclure les partenaires de l'écosystème de la recherche (partenaires financiers, organismes de certification) qui collaborent avec les chercheurs biomédicaux pour rendre la recherche socialement responsable.

CCAAT/enhancer binding protein beta is expressed in satellite cells and controls myogenesis.

Upon injury, muscle satellite cells become activated and produce skeletal muscle precursors that engage in myogenesis. We demonstrate that the transcription factor CCAAT/enhancer binding protein beta (C/EBPß) is expressed in the satellite cells of healthy muscle. C/EBPß expression is regulated during myogenesis such that C/EBPß is rapidly and massively downregulated upon induction to differentiate. Furthermore, persistent expression of C/EBPß in myoblasts potently inhibits differentiation at least in part through the inhibition of MyoD protein function and stability. As a consequence, myogenic factor expression, myosin heavy chain expression, and fusogenic activity were reduced in C/EBPß-overexpressing cells. Using knockout models, we demonstrate that loss of Cebpb expression in satellite cells results in precocious differentiation of myoblasts in growth conditions and greater cell fusion upon differentiation. In vivo, loss of Cebpb expression in satellite cells resulted in larger muscle fiber cross-sectional area and improved repair after muscle injury. Our results support the notion that C/EBPß inhibits myogenic differentiation and that its levels must be reduced to allow for activation of MyoD target genes and the progression of differentiation.

Retinoic acid-induced Smad3 expression is required for the induction of osteoblastogenesis of mesenchymal stem cells.

Mesenchymal stem cells are pluripotent precursor cells that can differentiate into osteoblasts, adipocytes, chondrocytes and myocytes. Despite their important therapeutic potential little is known about the transcriptional cascades that govern lineage decisions in these cells. Treatment of C3H10T1/2 mouse mesenchymal stem cells with retinoic acid (RA) inhibits adipogenesis and enhances osteoblastogenesis. In particular, RA treatment stimulates the expression of the osteoblast master regulator, runt-related transcription factor 2 (Runx2), whose expression is necessary for the formation of bone. We have shown previously in mesenchymal stem cells that RA acts to stimulate osteoblastogenesis by interfering with the actions of the bzip transcription factor CCAAT/Enhancer Binding Protein beta (C/EBPß), where it binds to a negative regulatory element within the Runx2 promoter and inhibits its expression. Herein we show that Smad3, whose expression is stimulated by RA, relays the effects of RA on differentiation by initiating the displacement of C/EBPß from the Runx2 promoter. In addition to stimulating Smad3 expression, RA also stimulated the nuclear localization of this factor, such that in the absence of RA, ectopic Smad3 was unable to drive osteoblastogenesis. While not sufficient to promote osteoblastogenesis, knockdown of Smad3 using a specific shRNA prevented the RA-mediated stimulation of differentiation and displacement of C/EBPß from the Runx2 P1 promoter. Taken together, these data indicate that Smad3 is an important mediator of RA activity during mesenchymal stem cell differentiation and is necessary for the stimulation of osteoblastogenesis.

From quiescence to repair: C/EBPß as a regulator of muscle stem cell function in health and disease.

CCAAT/Enhancer Binding protein beta (C/EBPß) is a transcriptional regulator involved in numerous physiological processes. Herein, we describe a role for C/EBPß as a regulator of skeletal muscle stem cell function. In particular, C/EBPß is expressed in muscle stem cells in healthy muscle where it inhibits myogenic differentiation. Downregulation of C/EBPß expression at the protein and transcriptional level allows for differentiation. Persistence of C/EBPß promotes stem cell self-renewal and C/EBPß expression is required for mitotic quiescence in this cell population. As a critical regulator of skeletal muscle homeostasis, C/EBPß expression is stimulated in pathological conditions such as cancer cachexia, which perturbs muscle regeneration and promotes myofiber atrophy in the context of systemic inflammation. C/EBPß is also an important regulator of cytokine expression and immune response genes, a mechanism by which it can influence muscle stem cell function. In this viewpoint, we describe a role for C/EBPß in muscle stem cells and propose a functional intersection between C/EBPß and NF-kB action in the regulation of cancer cachexia.

C/EBPß promotes the expression of atrophy-inducing factors by tumours and is a central regulator of cancer cachexia.

BACKGROUND: CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor whose high expression in human cancers is associated with tumour aggressiveness and poor outcomes. Most advanced cancer patients will develop cachexia, characterized by loss of skeletal muscle mass. In response to secreted factors from cachexia-inducing tumours, C/EBPß is stimulated in muscle, leading to both myofibre atrophy and the inhibition of muscle regeneration. Involved in the regulation of immune responses, C/EBPß induces the expression of many secreted factors, including cytokines. Because tumour-secreted factors drive cachexia and aggressive tumours have higher expression of C/EBPß, we examined a potential role for C/EBPß in the expression of tumour-derived cachexia-inducing factors. METHODS: We used gain-of-function and loss-of-function approaches in vitro and in vivo to evaluate the role of tumour C/EBPß expression on the secretion of cachexia-inducing factors. RESULTS: We report that C/EBPß overexpression up-regulates the expression of 260 secreted protein genes, resulting in a secretome that inhibits myogenic differentiation (-31%, P < 0.05) and myotube maturation [-38% (fusion index) and -25% (myotube diameter), P < 0.05]. We find that knockdown of C/EBPß in cachexia-inducing Lewis lung carcinoma cells restores myogenic differentiation (+25%, P < 0.0001) and myotube diameter (+90%, P < 0.0001) in conditioned medium experiments and, in vivo, prevents muscle wasting (-51% for small myofibres vs. controls, P < 0.01; +140% for large myofibres, P < 0.01). Conversely, overexpression of C/EBPß in non-cachectic tumours converts their secretome into a cachexia-inducing one, resulting in reduced myotube diameter (-41%, P < 0.0001, EL4 model) and inhibition of differentiation in culture (-26%, P < 0.01, EL4 model) and muscle wasting in vivo (+98% small fibres, P < 0.001; -76% large fibres, P < 0.001). Comparison of the differently expressed transcripts coding for secreted proteins in C/EBPß-overexpressing myoblasts with the secretome from 27 different types of human cancers revealed ~18% similarity between C/EBPß-regulated secreted proteins and those secreted by highly cachectic tumours (brain, pancreatic, and stomach cancers). At the protein level, we identified 16 novel secreted factors that are present in human cancer secretomes and are up-regulated by C/EBPß. Of these, we tested the effect of three factors (SERPINF1, TNFRSF11B, and CD93) on myotubes and found that all had atrophic potential (-33 to -36% for myotube diameter, P < 0.01). CONCLUSIONS: We find that C/EBPß is necessary and sufficient to induce the secretion of cachexia-inducing factors by cancer cells and loss of C/EBPß in tumours attenuates muscle atrophy in an animal model of cancer cachexia. Our findings establish C/EBPß as a central regulator of cancer cachexia and an important therapeutic target.

Transcription factor Smad3 is required for the inhibition of adipogenesis by retinoic acid.

The process of adipocyte differentiation is driven by a highly coordinated cascade of transcriptional events that results in the development of the mature adipocyte and in lipid accumulation. One of the early events of differentiation is the up-regulation of CCAAT/enhancer-binding protein beta (C/EBPbeta) expression. C/EBPbeta then acts to up-regulate the expression of adipogenic factors such as C/EBPalpha, which control the late stage of adipogenesis. Retinoic acid (RA) is a potent inhibitor of adipogenesis, and its action appears to block C/EBPbeta transcriptional potential early during differentiation. Using preadipocytes and mesenchymal stem cell models, we show that RA specifically blocks the occupancy of C/EBPbeta of the Cebpa promoter, thereby abrogating the differentiation process. RA does not act directly on C/EBPbeta but rather stimulates the expression of the transforming growth factor beta-effector protein Smad3, which can interact with C/EBPbeta via its Mad homology 1 domain and can interfere with C/EBPbeta DNA binding. The RA-induced increase in Smad3 expression results in increased cytoplasmic and nuclear Smad3, an important event as ectopic expression of Smad3 in preadipocytes in the absence of RA treatment only modestly inhibits adipogenesis and C/EBPbeta DNA binding, suggesting that Smad3 alone is not sufficient to completely recapitulate the effects of retinoic acid treatment during differentiation. However, in the absence of Smad3, RA is not able to inhibit adipocyte differentiation or to elicit a decrease in C/EBPbeta DNA occupancy suggesting that Smad3 is necessary to convey the inhibitory effects of retinoic acid during adipogenesis.

Web-based software to assist in the localization of neuroanatomical lesions.

OBJECTIVE: To evaluate the educational effectiveness of a novel, web-based neuroanatomical localization application. METHODS: A prototype version of a neuroanatomical localization application was developed, limited to lesions involving Cranial Nerve (CN) VII. Second year medical students at the University of Ottawa were recruited to participate in the study. Participants were exposed to a didactic teaching session on CN VII anatomy. They were subsequently randomized to two groups - one group was granted access to the localization application (the "intervention group"), while the other group was given a booklet of standard textbook resources (the "control group"). Participants then completed a case-based multiple choice test on localization of neurologic lesions associated with CN VII, followed by a questionnaire regarding the experience. RESULTS: Thirty-nine students volunteered to participate. Twenty were randomized to the intervention group and 19 to the control group. There was a mean test score difference of 1.3 (CI.95=0.2, 2.3) that was significantly higher in the intervention group when compared to the control group. Significance was determined by a Wilcoxon rank test (p=0.028). Questionnaire results were similar for both groups, showing an overall favourable evaluation of the localization application. CONCLUSIONS: The results support our hypotheses that students using the application would perform better on the multiple choice question (MCQ) test and there would be an overall preference for its use. The demonstrated educational benefit of the application, in addition to the demand for such a resource expressed by the participants, warrant further investigation into the development of a neurological localization application.

Contaminating reactivity of a monoclonal CCAAT/Enhancer Binding Protein ß antibody in differentiating myoblasts.

OBJECTIVE: CCAAT/Enhancer Binding proteins (C/EBPs) are transcription factors involved in the regulation of a variety of cellular processes. We used the Abcam Recombinant Anti-C/EBP beta antibody (E299) to detect C/EBPß expression during myogenesis. Though the antibody is monoclonal, and the immunogen used is highly specific to C/EBPß, we identified an intense band at 23 kDa on western blot that did not correspond to any of the known isoforms of C/EBPß, or family members predicted to cross-react. Absent in myoblast cells overexpressing C/EBPß, the band was present when C/EBPß was knocked down, confirming specificity for a protein other than C/EBPß. The objective of this work was to identify the contaminating reactivity. RESULTS: We performed immunoprecipitation followed by mass spectrometry to identified myosin light chain 4 (MYL4) as the unknown band, suggesting that the Abcam monoclonal antibody directed against C/EBPß is not pure, but contains a contaminating antibody against MYL4. Caution should be used when working in cells lines that express MYL4 to not confound the detection of MYL4 with that of C/EBPß isoforms.

CCAAT/Enhancer binding protein beta abrogates retinoic acid-induced osteoblast differentiation via repression of Runx2 transcription.

Runx2/CBFA1/AML3 is a master regulator of the osteoblast lineage and has been shown to directly control the transcription of numerous osteoblast-specific genes including alkaline phosphatase, osteopontin, and type I collagen. In its absence, ossification does not occur during development resulting in animals with cartilaginous skeletons and no osteoblasts. In humans, loss of one copy of Runx2 causes cleidocranial dysplasia characterized by malformations of the facial and cranial bones and the clavicle. Despite its important role in osteoblast biology, relatively little is known about the transcriptional regulation of the Runx2 gene. In the present study, we show that CCAAT/enhancer binding protein beta (C/EBPbeta) is a negative regulator of Runx2 expression and acts by directly binding a C/EBP element located at -591/-576 within the osteoblast-specific Runx2 P1 promoter. Ectopic expression of C/EBPbeta in C3H10T1/2 cells causes a reduction in Runx2 expression concomitant with a decrease in osteogenic potential during all-trans retinoic acid (ATRA)-induced differentiation. In nondifferentiating cells, C/EBPbeta can be found occupying the C/EBP negative response element within the Runx2 P1 promoter. ATRA, the effects of which are mediated by retinoic acid receptor alpha and gamma in C3H10T1/2 cells, stimulates the dissociation of C/EBPbeta from this element and promotes Runx2 expression. Thus, ATRA initiates osteoblastic differentiation of C3H10T1/2 cells, at least in part, by triggering the dissociation of C/EBPbeta from the Runx2 promoter.

CCAAT/Enhancer Binding Protein ß inhibits myogenic differentiation via ID3.

Myogenesis is regulated by the coordinated expression of muscle regulatory factors, a family of transcription factors that includes MYOD, MYF5, myogenin and MRF4. Muscle regulatory factors are basic helix-loop-helix transcription factors that heterodimerize with E proteins to bind the regulatory regions of target genes. Their activity can be inhibited by members of the Inhibitor of DNA binding and differentiation (ID) family, which bind E-proteins with high affinity, thereby preventing muscle regulatory factor-dependent transcriptional responses. CCAAT/Enhancer Binding protein beta (C/EBPß) is a transcription factor expressed in myogenic precursor cells that acts to inhibit myogenic differentiation, though the mechanism remains poorly understood. We identify Id3 as a novel C/EBPß target gene that inhibits myogenic differentiation. Overexpression of C/EBPß stimulates Id3 mRNA and protein expression, and is required for C/EBPß-mediated inhibition of myogenic differentiation. Misexpression of C/EBPß in myogenic precursors, such as in models of cancer cachexia, prevents the differentiation of myogenic precursors and we show that loss of Id3 rescues differentiation under these conditions, suggesting that the stimulation of Id3 expression by C/EBPß is an important mechanism by which C/EBPß inhibits myogenic differentiation.

SOX7 Is Required for Muscle Satellite Cell Development and Maintenance.

Satellite cells are skeletal-muscle-specific stem cells that are activated by injury to proliferate, differentiate, and fuse to enable repair. SOX7, a member of the SRY-related HMG-box family of transcription factors is expressed in quiescent satellite cells. To elucidate SOX7 function in skeletal muscle, we knocked down Sox7 expression in embryonic stem cells and primary myoblasts and generated a conditional knockout mouse in which Sox7 is excised in PAX3+ cells. Loss of Sox7 in embryonic stem cells reduced Pax3 and Pax7 expression. In vivo, conditional knockdown of Sox7 reduced the satellite cell population from birth, reduced myofiber caliber, and impaired regeneration after acute injury. Although Sox7-deficient primary myoblasts differentiated normally, impaired myoblast fusion and increased sensitivity to apoptosis in culture and in vivo were observed. Taken together, these results indicate that SOX7 is dispensable for myogenesis but is necessary to promote satellite cell development and survival.

Induction of CCAAT/Enhancer-Binding Protein ß Expression With the Phosphodiesterase Inhibitor Isobutylmethylxanthine Improves Myoblast Engraftment Into Dystrophic Muscle.

UNLABELLED: Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is the most common muscular dystrophy. Characterized by rounds of muscle degeneration and regeneration, DMD features progressive muscle wasting and is fatal. One approach for treatment is transplantation of muscle progenitor cells to repair and restore dystrophin expression to damaged muscle. However, the success of this approach has been limited by difficulties in isolating large numbers of myogenic progenitors with strong regenerative potential, poor engraftment, poor survival of donor cells, and limited migration in the diseased muscle. We demonstrate that induction of the transcription factor CCAAT/enhancer-binding protein ß (C/EBPß) using the cyclic adenosine monophosphate phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) results in enhanced myoblast expansion in culture and increased satellite cell marker expression. When equal numbers of IBMX-treated cells were transplanted into dystrophic muscle, they contributed to muscle repair more efficiently than did vehicle-treated cells and engrafted into the satellite cell niche in higher numbers, demonstrating improved cell migration from the site of injury and enhanced survival after transplantation. Thus, pharmacologic stimulation of C/EBPß expression reprograms myoblasts to a more stem cell-like state, promotes expansion in culture, and improves engraftment such that better transplantation outcomes are achieved. SIGNIFICANCE: Duchenne muscular dystrophy is a genetic disorder for which no cure exists. One therapeutic approach is transplantation of myogenic progenitors to restore dystrophin to damaged muscle, but this approach is limited by poor engraftment of cultured myoblasts. Transient upregulation of CCAAT/enhancer-binding protein ß in primary myoblasts using the phosphodiesterase isobutylmethylxanthine (IBMX) increases satellite cell marker expression in cultured myoblasts, improves their migration, and increases their survival after transplantation. When transplanted into C57BL/10ScSn-mdx/J mice , IBMX-treated myoblasts restored dystrophin expression and were able to occupy the satellite cell niche more efficiently than controls. A myoblast culture approach that reprograms myoblasts to a more primitive state, resulting in improved transplantation outcomes and reinvigorating research into myoblast transplantation as a viable therapeutic approach, is described.

CCAAT/enhancer binding protein ß is required for satellite cell self-renewal.

BACKGROUND: Postnatal growth and repair of skeletal muscle relies upon a population of quiescent muscle precursor cells, called satellite cells that can be activated to proliferate and differentiate into new myofibers, as well as self-renew to replenish the satellite cell population. The balance between differentiation and self-renewal is critical to maintain muscle tissue homeostasis, and alterations in this equilibrium can lead to chronic muscle degeneration. The transcription factor CCAAT/enhancer binding protein beta (C/EBPß) is expressed in Pax7+ satellite cells of healthy muscle and is downregulated during myoblast differentiation. Persistent expression of C/EBPß upregulates Pax7, inhibits MyoD, and blocks myogenic differentiation. METHODS: Using genetic tools to conditionally abrogate C/EBPß expression in Pax7+ cells, we examined the role of C/EBPß in self-renewal of satellite cells during muscle regeneration. RESULTS: We find that loss of C/EBPß leads to precocious differentiation at the expense of self-renewal in primary myoblast and myofiber cultures. After a single muscle injury, C/EBPß-deficient satellite cells fail to self-renew resulting in a reduction of satellite cells available for future rounds of regeneration. After a second round of injury, muscle regeneration is impaired in C/EBPß conditional knockout mice compared to wild-type control mice. We find that C/EBPß can regulate Notch2 expression and that restoration of Notch activity in myoblasts lacking C/EBPß prevents precocious differentiation. CONCLUSIONS: These findings demonstrate that C/EBPß is a novel regulator of satellite cell self-renewal during muscle regeneration acting at least in part through Notch2.