Induction Of Myogenin Research Articles

SAT004 A Novel Therapeutic Target Of Mitochondrial Protein To Prevent Steroid-induced Muscle Atrophy Through Regulation Of Proteolysis And Myogenesis

Abstract Disclosure: M. Kim: None. I.S. Sinam: None. S. Kim: None. Z. Siddique: None. S. Kwon: None. J. Jeon: None. I. Lee: None. Objectives: Muscle atrophy, defined as a progressive loss of muscle mass and function induced by various etiology. Steroids is well-known muscle atrophy-inducible drug by enhancing of muscle catabolism and proteolysis. Pyruvate dehydrogenase kinase (PDK), a mitochondrial protein that inhibits the pyruvate dehydrogenase complex, is highly upregulated in metabolic diseases, especially pathologic muscle conditions associated with enhanced muscle proteolysis and aberrant myogenesis. Here, we investigated the role of PDK on proteolysis and myogenesis in steroid-induced muscle atrophy. Methods & Materials: Human skeletal (gluteus-maximus) muscles obtained from patients who underwent hip replacement surgery according to steroid administration were used for evaluated at the molecular level by expression of key genes and proteins involved in myogenesis. Steroid-induced muscle atrophy models were developed administered with glucocorticoid (GC) 25 mg/kg, intraperitoneally, for 10 days. Muscle dysfunction was studied in vivo in wild-type and PDK4 knockout mice treated with GC. We used C2C12 myotubes treated with GC for 24 h for in vitro study. Results: The expression of PDK4, as well as muscle-specific E3 ligases: muscle RING finger 1(MuRF1) and muscle atrophy F box (MAFbx), are significantly increased in skeletal muscle of steroid-user. In mouse model of steroid-induced muscle atrophy, expression of PDK4 and MAFbx are also upregulated, whereas knockdown of PDK4 reduces MAFbx expression and ameliorated steroid-induced muscle atrophy; increased muscle strength and muscle fibers. In C2C12 myotubes, knockdown of PDK4 was also associated with reduction of myogenin induction and MAFbx expression. To find out the mechanism, we used coimmunoprecipitation and liquid chromatography-mass spectrometry analysis and found that myogenin is novel substrate of PDK4. Mechanistically, this result suggests that PDK4 promotes MAFbx recruitment to catalyze muscle atrophy-induced MYOG ubiquitination and degradation. Conclusion: Taken together, the results suggest that PDK4-dependent activation of the ubiquitin degradation pathway appears to underly the promotion of muscle atrophy by directly targeting the muscle-specific MAFbx. Therefore, the inhibition of PDK4 represents as a novel therapeutic target to alleviate steroid-induced muscle atrophy. Presentation: Saturday, June 17, 2023

Pyruvate dehydrogenase kinase 4 promotes ubiquitin-proteasome system-dependent muscle atrophy.

Muscle atrophy, leading to muscular dysfunction and weakness, is an adverse outcome of sustained period of glucocorticoids usage. However, the molecular mechanism underlying this detrimental condition is currently unclear. Pyruvate dehydrogenase kinase 4 (PDK4), a central regulator of cellular energy metabolism, is highly expressed in skeletal muscle and has been implicated in the pathogenesis of several diseases. The current study was designed to investigated and delineate the role of PDK4 in the context of muscle atrophy, which could be identified as a potential therapeutic avenue to protect against dexamethasone-induced muscle wasting. The dexamethasone-induced muscle atrophy in C2C12 myotubes was evaluated at the molecular level by expression of key genes and proteins involved in myogenesis, using immunoblotting and qPCR analyses. Muscle dysfunction was studied in vivo in wild-type and PDK4 knockout mice treated with dexamethasone (25mg/kg body weight, i.p., 10days). Body weight, grip strength, muscle weight and muscle histology were assessed. The expression of myogenesis markers were analysed using qPCR, immunoblotting and immunoprecipitation. The study was extended to in vitro human skeletal muscle atrophy analysis. Knockdown of PDK4 was found to prevent glucocorticoid-induced muscle atrophy and dysfunction in C2C12 myotubes, which was indicated by induction of myogenin (0.3271±0.102 vs 2.163±0.192, ****P<0.0001) and myosin heavy chain (0.3901±0.047 vs. 0.7222±0.082, **P<0.01) protein levels and reduction of muscle atrophy F-box (10.77±2.674 vs. 1.518±0.172, **P<0.01) expression. In dexamethasone-induced muscle atrophy model, mice with genetic ablation of PDK4 revealed increased muscle strength (162.1±22.75 vs. 200.1±37.09g, ***P<0.001) and muscle fibres (54.20±11.85% vs. 84.07±28.41%, ****P<0.0001). To explore the mechanism, we performed coimmunoprecipitation and liquid chromatography-mass spectrometry analysis and found that myogenin is novel substrate of PDK4. PDK4 phosphorylates myogenin at S43/T57 amino acid residues, which facilitates the recruitment of muscle atrophy F-box to myogenin and leads to its subsequent ubiquitination and degradation. Finally, overexpression of non-phosphorylatable myogenin mutant using intramuscular injection prevented dexamethasone-induced muscle atrophy and preserved muscle fibres. We have demonstrated that PDK4 mediates dexamethasone-induced skeletal muscle atrophy. Mechanistically, PDK4 phosphorylates and degrades myogenin via recruitment of E3 ubiquitin ligase, muscle atrophy F-box. Rescue of muscle regeneration by genetic ablation of PDK4 or overexpression of non-phosphorylatable myogenin mutant indicates PDK4 as an amenable therapeutic target in muscle atrophy.

Clusterin silencing restores myoblasts viability and down modulates the inflammatory process in osteoporotic disease

BackgroundTargeting new molecular pathways leading to Osteoporosis (OP) and Osteoarthritis (OA) is a hot topic for drug discovery. Clusterin (CLU) is a glycoprotein involved in inflammation, proliferation, cell death, neoplastic disease, Alzheimer disease and aging. The present study focuses on the expression and the role of CLU in influencing the decrease of muscle mass and fiber senescence in OP-OA condition.MethodsVastus lateralis muscle biopsies were collected from 20 women with OP undergoing surgery for fragility hip fracture and 20 women undergoing arthroplasty for hip osteoarthritis.ResultsWe found an overexpression of CLU in degenerated fibers in OP closely correlated with interleukin 6 (IL6) and histone H4 acetylation level. Conversely, in OA muscle tissues we observed a weak expression of CLU but no nuclear histone H4 acetylation. Ex vivo studies on isolated human myoblasts confirmed CLU overexpression in OP as compared to OA (p < 0.001). CLU treatment of isolated OP and OA myoblasts showed: modulation of proliferation, morphological changes, increase of histone H4 acetylation and induction of myogenin (MYOG) activation in OP myoblast only. In OP condition, functional knockdown of CLU by siRNA restores proliferative myoblasts capability and tissue damage repair, carried out by an evident upregulation of Transglutaminase 2 (TGM2). We also observed downmodulation of CX3CR1 expression with consequent impairing of the inflammatory infiltrate recruitment.ConclusionsResults obtained suggest a potential role of CLU in OP by influencing myoblasts terminal differentiation, epigenetic regulation of muscle cell differentiation and senescence. Moreover, CLU silencing points out its role in the modulation of tissue damage repair and inflammation, proposing it as a new diagnostic marker for muscle degeneration and a potential target for specific therapeutic intervention in OP related sarcopenia.

Skeletal muscle atrophy is induced by Fbxw7β via atrogene upregulation.

Muscle atrophy decreases skeletal muscle mass and is induced by inherited cachectic symptoms, genetic disorders, and sarcopenia. However, the molecular pathways associated with the onset of muscle atrophy are still unclear. In this study, we evaluated Fbxw7β, a gene associated with the development of muscle atrophy in vitro and in vivo. Among the three Fbxw7 isoforms, ectopically overexpressed Fbxw7β induced the expression of myogenin and major atrogene markers (atrogin-1 and MuRF-1) and reduced myoblast differentiation. In addition, endogenous expression of Fbxw7β was also upregulated by dexamethasone, which mimics muscle atrophy in vitro, accompanied by induction of myogenin and atrogene expression in primary myoblasts. Functional analysis of Fbxw7β using short hairpin RNA (shRNA) and a dominant-negative mutant (ΔFbox) suggested that Fbxw7β regulated muscle atrophy in vitro and in vivo. In particular, ΔFbox did not reduce the sizes of muscle fibers and did not induce myogenin and atrogene expression in vivo. Therefore, our findings demonstrated, for the first time, that Fbxw7β induced muscle atrophic phenotypes via atrogenes in adult muscle precursor cells and myofibers; this mechanism could be a potential therapeutic target for skeletal muscle atrophy.

Depleting extracellular vesicles from fetal bovine serum alters proliferation and differentiation of skeletal muscle cells in vitro.

BackgroundFetal bovine serum (FBS) contains a wide range of growth factors, hormones, vitamins, amino acids, fatty acids and trace elements required for cell growth. It was shown that animal sera contain also extracellular vesicles (EVs) with important biological properties; thus we wondered whether EVs present in FBS would influence muscle cell phenotype.EVs were removed from sera by ultracentrifugation (18 h). C2C12, L6 and human primary myoblasts, were grown either in classical media (CM) or in EVs-depleted media. Differentiation was induced by replacing the culture medium either with CM or EV-depleted media. qRT-PCR of relevant genes and miRNA involved in proliferation, differentiation, energy metabolism and EVs formation and secretion were performed.ResultsGrowth of myoblasts in EV-free media during proliferation produces the most unfavorable situation for proper myotube formation, when considering C212 and human myoblasts. Removing EVs from serum committed myoblasts to differentiate precociously (induction of myogenin and decreased expression of myomiR involved in myogenesis). C2C12 and human myoblasts, grown constantly in EV-depleted media during proliferation and differentiation, formed less myotubes than in CM. They had a reduced level of myogenin and a strong increase in myostatin expression, a negative regulator of muscle cell differentiation that affects myotube size. This situation was not reversed when confluent myoblasts were switched to CM for differentiation. Like C2C12 and human cells, L6 formed less myotubes in EVs-depleted media. However, as they do not express myostatin, L6 myotubes were larger and expressed higher level of CKTM2 compared to myotubes grown in CM suggesting that they had reached a higher level of differentiation.ConclusionsResearchers studying the role of muscle EVs in culture conditions should consider that depleting EVs from serum alters the phenotype of muscle cells. Interestingly, the cross-talk between myoblasts and myotubes during myogenesis (Forterre 2014, PLoS One. 2014 Jan 2;9(1):e84153) can be recapitulate by using FBS-EVs as well. This implies that EVs can transfer specific signals to cells from unrelated species and that part of serum EV composition is evolutionarily conserved (e.g.; myomiR are detected in FBS-EVs). EVs in body fluids could have an unsuspected function during embryogenesis and in regulation of cellular processes such as hypertrophy and hyperplasia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0262-0) contains supplementary material, which is available to authorized users.

Evaluation of a thermoresponsive polycaprolactone scaffold for in vitro three-dimensional stem cell differentiation.

Tissue engineering (TE) strategies aim at imitating the natural process of regeneration by using bioresorbable scaffolds that support cellular attachment, migration, proliferation, and differentiation. Based on the idea of combining a fully degradable polymer [poly(ɛ-caprolactone)] with a thermoresponsive polymer (polyethylene glycol methacrylate), a scaffold was developed, which liquefies below 20°C and solidifies at 37°C. In this study, this scaffold was evaluated for its ability to support C2C12 cells and human adipose-derived stem cells (ASCs) to generate an expandable three-dimensional (3D) construct for soft or bone TE. As a first step, biomaterial seeding was optimized and cellular attachment, survival, distribution, and persistence within the 3D material were characterized. C2C12 cells were differentiated toward the osteogenic as well as myogenic lineage, while ASCs were cultured in control, adipogenic, or osteogenic differentiation media. Differentiation was examined using quantitative real-time PCR for the expression of osteogenic, myogenic, and adipogenic markers and by enzyme activity and immunoassays. Both cell types attached and were found evenly distributed within the material. C2C12 cells and ASCs demonstrated the potential to differentiate in all tested lineages under 2D conditions. Under 3D osteogenic conditions for C2C12 cells, only osteocalcin expression (fold induction: 16.3±0.2) and alkaline phosphatase (ALP) activity (p<0.001) were increased compared with the control C2C12 cells. Three-dimensional osteogenic differentiation of ASC was limited and donor dependent. Only one donor showed an increase in the osteogenic markers osteocalcin (p=0.027) and osteopontin (p=0.038). In contrast, differentiation toward the myogenic or adipogenic lineage showed expression of specific markers in 3D, at least at the level of the 2D culture. In 3D culture, strong induction of myogenin (p<0.001) as well as myoD (p<0.001) was found in C2C12 cells. The adipogenic differentiation of one donor showed greater expression of peroxisome proliferative-activated receptor gamma (PPARγ) (p=0.004), fatty acid binding protein 4 (FABP4) (p=0.008), and adiponectin (p=0.045) in 3D compared with 2D culture. Leptin levels in the supernatant of the ASC cultures were elevated in the 3D cultures in both donors at day 14 and 21. In conclusion, the thermoresponsive scaffold was found suitable for 3D in vitro differentiation toward soft tissue.

Wnt/β-catenin signaling changes C2C12 myoblast proliferation and differentiation by inducing Id3 expression

Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a β-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/β-catenin induced gene in myoblast cell fate determination.

HMGB1/RAGE regulates muscle satellite cell homeostasis via p38 MAPK/myogenin-dependent repression of Pax7 transcription

Expression of the paired-box 7 (PAX7) transcription factor is regulated during both myoblast proliferation and differentiation: high levels of PAX7 compromise myogenic differentiation because of excess and prolonged proliferation, whereas low levels of PAX7 result in precocious differentiation. We showed that myogenin repressed Pax7 transcription in differentiating myoblasts by binding to specific recognition sites in the Pax7 promoter, and that high-mobility group box 1 (HMGB1)-receptor for advanced glycation end-products (RAGE) signaling was required for myogenin induction and myogenin-dependent repression of Pax7 transcription. In addition, PAX7 negatively and myogenin positively regulated RAGE expression. RAGE, a multiligand receptor of the immunoglobulin superfamily, was not expressed in adult skeletal muscles, and was transiently expressed in activated, proliferating and differentiating satellite cells (SCs) in injured muscles. Compared with wild-type muscles, Rage(-/-) muscles exhibited increased numbers of basal SCs that were further increased in injured Rage(-/-) muscles following elevated myoblast asymmetric division; complete regeneration of injured Rage(-/-) muscles was found to be delayed by ~1 week. Thus, RAGE signaling physiologically repressed Pax7 transcription in SCs by upregulating myogenin, thereby accelerating muscle regeneration and limiting SC self-renewal.

The histone- and PRMT5-associated protein COPR5 is required for myogenic differentiation

Myogenic differentiation requires the coordination between permanent cell cycle withdrawal, mediated by members of the cyclin-dependent kinase inhibitor (CKI) family, and activation of a cascade of myogenic transcription factors, particularly MYOGENIN (MYOG). Recently, it has been reported that the Protein aRginine Methyl Transferase PRMT5 modulates the early phase of induction of MYOG expression. Here, we show that the histone- and PRMT5-associated protein COPR5 (cooperator of PRMT5) is required for myogenic differentiation. C2C12 cells, in which COPR5 had been silenced, could not irreversibly exit the cell cycle and differentiate into muscle cells. This phenotype might be explained by the finding that, in cells in which COPR5 was downregulated, p21 and MYOG induction was strongly reduced and PRMT5 recruitment to the promoters of these genes was also altered. Moreover, we suggest that COPR5 interaction with the Runt-related transcription factor 1 (RUNX1)-core binding factor-β (CBFβ) complex contributes to targeting the COPR5-PRMT5 complex to these promoters. Finally, we present evidence that COPR5 depletion delayed the in vivo regeneration of cardiotoxin-injured mouse skeletal muscles. Altogether, these data extend the role of COPR5 from an adaptor protein required for nuclear functions of PRMT5 to an essential coordinator of myogenic differentiation.

A role for 1-acylglycerol-3-phosphate-O-acyltransferase-1 in myoblast differentiation

AGPAT isoforms catalyze the acylation of lysophosphatidic acid (LPA) to form phosphatidic acid (PA). AGPAT2 mutations are associated with defective adipogenesis. Muscle and adipose tissue share common precursor cells. We investigated the role of AGPAT isoforms in skeletal muscle development. We demonstrate that small interference RNA-mediated knockdown of AGPAT1 expression prevents the induction of myogenin, a key transcriptional activator of the myogenic program, and inhibits the expression of myosin heavy chain. This effect is rescued by transfection with AGPAT1 but not AGPAT2. Knockdown of AGPAT2 has no effect. The regulation of myogenesis by AGPAT1 is associated with alterations on actin cytoskeleton. The role of AGPAT1 on actin cytoskeleton is further supported by colocalization of AGPAT1 to areas of active actin polymerization. AGPAT1 overexpression was not associated with an increase in PA levels. Our observations strongly implicate AGPAT1 in the development of skeletal muscle, specifically to terminal differentiation. These findings are linked to the regulation of actin cytoskeleton.

TGF-β receptors, in a Smad-independent manner, are required for terminal skeletal muscle differentiation

Skeletal muscle differentiation is strongly inhibited by transforming growth factor type β (TGF-β), although muscle formation as well as regeneration normally occurs in an environment rich in this growth factor. In this study, we evaluated the role of intracellular regulatory Smads proteins as well as TGF-β-receptors (TGF-β-Rs) during skeletal muscle differentiation. We found a decrease of TGF-β signaling during differentiation. This phenomenon is explained by a decline in the levels of the regulatory proteins Smad-2, -3, and -4, a decrease in the phosphorylation of Smad-2 and lost of nuclear translocation of Smad-3 and -4 in response to TGF-β. No change in the levels and inhibitory function of Smad-7 was observed. In contrast, we found that TGF-β-R type I (TGF-β-RI) and type II (TGF-β-RII) increased on the cell surface during skeletal muscle differentiation. To analyze the direct role of the serine/threonine kinase activities of TGF-β-Rs, we used the specific inhibitor SB 431542 and the dominant-negative form of TGF-β-RII lacking the cytoplasmic domain. The TGF-β-Rs were important for successful muscle formation, determined by the induction of myogenin, creatine kinase activity, and myosin. Silencing of Smad-2/3 expression by specific siRNA treatments accelerated myogenin, myosin expression, and myotube formation; although when SB 431542 was present inhibition in myosin induction and myotube formation was observed, suggesting that these last steps of skeletal muscle differentiation require active TGF-β-Rs. These results suggest that both down-regulation of Smad regulatory proteins and cell signaling through the TGF-β receptors independent of Smad proteins are essential for skeletal muscle differentiation.

Overload‐induced skeletal muscle extracellular matrix remodelling and myofibre growth in mice lacking IL‐6

Overloading healthy skeletal muscle produces myofibre hypertrophy and extracellular matrix remodelling, and these processes are thought to be interdependent for producing muscle growth. Inflammatory cytokine interleukin-6 (IL-6) gene expression is induced in overloaded skeletal muscle, and the loss of this IL-6 induction can attenuate the hypertrophic response to overload (OV). Although the OV induction of IL-6 in skeletal muscle may be an important regulator of inflammatory processes and satellite cell proliferation, less is known about its role in the regulation of extracellular matrix remodelling. The purpose of the current study was to examine if OV-induced extracellular matrix remodelling, muscle growth, and associated gene expression were altered in mice that lack IL-6, when compared with wild-type mice. Male C57/BL6 (WT) and C57/BL6 x IL-6(-/-) (IL-6(-/-)) mice (10 weeks of age) were assigned to either a sham control or synergist ablation OV treatments for 3, 21 or 56 days. Plantaris muscle mass increased 59% in WT and 116% in IL-6(-/-) mice after 21 day OV. Myofibre CSA was also increased by 21 day OV in both WT and IL-6(-/-) mice. OV induced a twofold greater increase in the volume of non-contractile tissue in IL-6(-/-) muscle compared to WT. OV also induced a significantly greater accumulation of hydroxyproline and procollagen-1 mRNA in IL-6(-/-) muscle, when compared with WT muscle after 21 day OV. Transforming growth factor-beta and insulin-like growth factor-1 mRNA expression were also induced to a greater extent in IL-6(-/-) muscle when compared with WT muscle after 21 day OV. There was no effect of IL-6 loss on the induction of myogenin, and cyclin D1 mRNA expression after 3 day OV. However, MyoD mRNA expression in 3 day OV IL-6(-/-) muscle was attenuated when compared with WT OV mice. IL-6 appears to be necessary for the normal regulation of extracellular matrix remodelling during OV-induced growth.

Regulation of heterochromatin remodelling and myogenin expression during muscle differentiation by FAK interaction with MBD2

Focal adhesion kinase (FAK), a major cell adhesion-activated tyrosine kinase, has an important function in cell adhesion and migration. Here, we report a new signalling of FAK in regulating chromatin remodelling by its interaction with MBD2 (methyl CpG-binding protein 2), underlying FAK regulation of myogenin expression and muscle differentiation. FAK interacts with MBD2 in vitro, in myotubes, and in isolated muscle fibres. Such an interaction, increased in myotubes exposed to oxidative stress, enhances FAK nuclear localization. The nuclear FAK-MBD2 complexes alter heterochromatin reorganization and decrease MBD2 association with HDAC1 (histone deacetylase complex 1) and methyl CpG site in the myogenin promoter, thus, inducing myogenin expression. In line with this view are observations that blocking FAK nuclear localization by expressing dominant negative MBD2 or suppression of FAK expression by its miRNA in C2C12 cells attenuates myogenin induction and/or impairs muscle-terminal differentiation. Together, these results suggest an earlier unrecognized role of FAK in regulating chromatin remodelling that is important for myogenin expression and muscle-terminal differentiation, reveal a new mechanism of MBD2 regulation by FAK family tyrosine kinases, and provide a link between cell adhesion and chromatin remodelling.

Impaired nuclear functions lead to increased senescence and inefficient differentiation in human myoblasts with a dominant p.R545C mutation in the LMNA gene

We have studied myoblasts from a patient with a severe autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) caused by an arginine 545 to cystein point mutation (p.R545C) in the carboxy-terminal domain of the lamin A/C gene. This mutation has pleiotropic cellular effects on these myoblasts as demonstrated by nuclear structural defects, exhibiting lobulations which increase with cell passages in culture. The organization of both lamin A/C and its inner nuclear membrane partner emerin are altered, eventually showing a honeycomb pattern upon immunofluorescence microscopy. In addition, the distribution of histone H3 trimethylated at lysine 27 and of phosphorylated RNA polymerase II, markers of inactive and active chromatin domains, respectively, are altered suggesting an impact on gene expression. Patient myoblasts also presented a high index of senescence in ex vivo culture. Moreover, our data show for the first time in an AD-EDMD context that the 20S core particle of the proteasome was inactivated. With cell passages, the 20S core protein progressively accumulated into discrete nuclear foci that largely colocalized with promyelocytic leukemia (PML) bodies while p21 accumulated throughout the nuclear compartment. Proteasome inactivation has been linked to normal cellular ageing. Our data indicate that it may also contribute to premature senescence in AD-EDMD patient myoblasts. Finally, when transferred to low-serum medium, patient myoblasts were deficient in ex vivo differentiation, as assessed by the absence of myotube formation and myogenin induction. Altogether, these data suggest that the LMNA mutation p.R545C impairs both proliferation and differentiation capacities of myoblasts as part of the pathogenesis of AD-EDMD.

Cooperative Synergy between NFAT and MyoD Regulates Myogenin Expression and Myogenesis

Calcineurin/NFAT signaling is involved in multiple aspects of skeletal muscle development and disease. The myogenic basic helix-loop-helix transcription factors, MyoD, myogenin, Myf5, and MRF4 specify the myogenic lineage. Here we show that calcineurin/NFAT (nuclear factor of activated T cells) signaling is required for primary myogenesis by transcriptional cooperation with the basic helix-loop-helix transcription factor MyoD. Calcineurin/NFAT signaling is involved in myogenin expression in differentiating myoblasts, where the myogenic regulatory factor MyoD synergistically cooperates with NFATc2/c3 at the myogenin promoter. Using gel shift and chromatin immunoprecipitation assays, we identified two conserved NFAT binding sites in the myogenin promoter that were occupied by NFATc3 upon skeletal muscle differentiation. The transcriptional integration between NFATc3 and MyoD is crucial for primary myogenesis in vivo, as myogenin expression is weak in myod:nfatc3 double null embryos, whereas myogenin expression is unaffected in embryos with null mutations for either factor alone. Thus, the combined findings provide a novel transcriptional paradigm for the first steps of myogenesis, where a calcineurin/NFATc3 pathway regulates myogenin induction in cooperation with MyoD during myogenesis.

Differentiation of C2C12 myoblasts expressing lamin A mutated at a site responsible for Emery–Dreifuss muscular dystrophy is improved by inhibition of the MEK–ERK pathway and stimulation of the PI3-kinase pathway

Mutation R453W in A-type lamins, that are major nuclear envelope proteins, generates Emery–Dreifuss muscular dystrophy. We previously showed that mouse myoblasts expressing R453W-lamin A incompletely exit the cell cycle and differentiate into myocytes with a low level of multinucleation. Here we attempted to improve differentiation by treating these cells with a mixture of PD98059, an extracellular-regulated kinase (ERK) kinase (also known as mitogen-activated kinase, MEK) inhibitor, and insulin-like growth factor-II, an activator of phosphoinositide 3-kinase. We show that mouse myoblasts expressing R453W-lamin A were sensitive to the drug treatment as shown by (i) an increase in multinucleation, (ii) downregulation of proliferation markers (cyclin D1, hyperphosphorylated Rb), (iii) upregulation of myogenin, and (iv) sustained activation of p21 and cyclin D3. However, nuclear matrix anchorage of p21 and cyclin D3 in a complex with hypophosphorylated Rb that is critical to trigger cell cycle arrest and myogenin induction was deficient and incompletely restored by drug treatment. As the turn-over of R453W-lamin A at the nuclear envelope was greatly enhanced, we propose that R453W-lamin A impairs the capacity of the nuclear lamina to serve as scaffold for substrates of the MEK–ERK pathway and for MyoD-induced proteins that play a role in the differentiation process.

Insulin-like growth factor-I has different effects on myogenin induction and cell cycle progression in human alveolar and embryonal rhabdomyosarcoma cells

Alveolar rhabdomyosarcoma (RMS) has a much poorer outcome than embryonal RMS. In this study, we found that IGF-I affected the induction of myogenin and cell cycle progression in alveolar RMS cells, but not in embryonal RMS cells. IGF-I enhanced the induction of myogenin protein in alveolar RMS SJ-Rh30 and KP-RMS-MS cells as it did in myoblast C2C12 cells, but not in embryonal RMS RD or KP-RMS-KH cells. IGF-I induction of myogenin protein was blocked by anti-IGF-IR monoclonal antibody alphaIR-3 and the mTOR-specific inhibitor rapamycin. In Rh30mTOR-rr cells, which stably express a rapamycin-resistant mutant mTOR, rapamycin did not inhibit IGF-I induction of myogenin protein. These data suggest that IGF-I induces myogenin in alveolar RMS cells through the IGF-IR/mTOR pathway. In C2C12 cells, IGF-I induces myogenin protein followed by cell cycle arrest leading to myogenic differentiation. IGF-I promoted G1-S cell cycle progression without any signs of terminal differentiation in alveolar RMS cells. On the other hand, IGF-I promoted neither cell cycle arrest nor G1-S cell cycle progression in embryonal RMS cells. In alveolar RMS SJ-Rh30 cells, 4E-BP1, one of two effectors downstream of mTOR, was continuously hyperphosphorylated by IGF-I, whereas in embryonal RMS RD cells, 4E-BP1 was only transiently hyperphosphorylated. These findings suggest that the different effects of IGF-I on myogenin induction and cell cycle progression in alveolar and embryonal RMS cells are due to a difference of phosphorylation status of 4E-BP1. These different responses to IGF-I help to explain immunohistochemical and clinical behavioral differences between alveolar and embryonal RMS.

Reciprocal inhibition between Pax7 and muscle regulatory factors modulates myogenic cell fate determination

Postnatal growth and regeneration of skeletal muscle requires a population of resident myogenic precursors named satellite cells. The transcription factor Pax7 is critical for satellite cell biogenesis and survival and has been also implicated in satellite cell self-renewal; however, the underlying molecular mechanisms remain unclear. Previously, we showed that Pax7 overexpression in adult primary myoblasts down-regulates MyoD and prevents myogenin induction, inhibiting myogenesis. We show that Pax7 prevents muscle differentiation independently of its transcriptional activity, affecting MyoD function. Conversely, myogenin directly affects Pax7 expression and may be critical for Pax7 down-regulation in differentiating cells. Our results provide evidence for a cross-inhibitory interaction between Pax7 and members of the muscle regulatory factor family. This could represent an additional mechanism for the control of satellite cell fate decisions resulting in proliferation, differentiation, and self-renewal, necessary for skeletal muscle maintenance and repair.

Sequential Recruitment of PCAF and BRG1 Contributes to Myogenin Activation in 12-O-Tetradecanoylphorbol-13-acetate-induced Early Differentiation of Rhabdomyosarcoma-derived Cells

Myogenin and its upstream regulator MyoD are known to be required for myogenic cell differentiation. Although both of them can be expressed in rhabdomyosarcoma-derived RD cells, the cells are unable to undergo full-scale terminal myogenic differentiation. 12-O-Tetradecanoylphorbol-13-acetate (TPA) has been found to be functional in the induction of RD cell differentiation, whereas its mechanism is not fully understood. By using quantitative real-time-based chromatin immunoprecipitation and real-time reverse transcription-PCR-based promoter activity assays, we examined the activation mechanism of the myogenin gene during TPA-induced differentiation of the RD cells. We have shown that a histone acetyltransferase PCAF and ATPase subunit BRG1 of the SWI/SNF chromatin remodeling complex are sequentially recruited to the promoter of the myogenin gene. Both PCAF and BRG1 are also involved in the activation of the myogenin gene. In addition, we have found that the p38 mitogen-activated protein kinase is required for BRG1 recruitment in TPA-mediated myogenin induction. We propose that there are two distinct activation steps for the induction of myogenin in TPA-induced early differentiation of RD cells: 1) an early step that requires PCAF activity to acetylate core histones and MyoD to initiate myogenin gene expression, and 2) a later step that requires p38-dependent activity of the SWI/SNF remodeling complex to provide an open conformation for the induction of myogenin. Our studies reveal an essential role for epigenetic regulation in TPA-induced differentiation of RD cells and provide potential drug targets for future treatment of the rhabdomyosarcoma.

Enhancer of Polycomb1, a Novel Homeodomain Only Protein-binding Partner, Induces Skeletal Muscle Differentiation

Homeodomain only protein, Hop, is an unusual small protein that modulates target gene transcription without direct binding to DNA. Here we show that Hop interacts with Enhancer of Polycomb1 (Epc1), a homolog of a Drosophila polycomb group gene product that regulates transcription, to induce the skeletal muscle differentiation. Yeast two-hybrid assay with the human adult heart cDNA library revealed that Hop can associate with Epc1. The amino-terminal domain of Epc1 as well as full Epc1 physically interacted with Hop in mammalian cells and in yeast. Epc1 is highly expressed in the embryonic heart and adult skeletal muscles. Serum deprivation induced differentiation of H9c2, a myoblast cell line, into skeletal myocytes, and Epc1 was up-regulated. Differentiation of H9c2 was induced by Epc1 overexpression, although it was severely impaired in Epc1-knockdown cells. Co-transfection of Hop potentiated Epc1-induced transactivation of myogenin and myotube formation. Hop knock-out mice elicited a decrease in myosin heavy chain and myogenin expressions in skeletal muscle and showed delay in hamstring muscle healing after injury. Differentiation was impaired in skeletal myoblasts from Hop knock-out mice. These results suggest that Epc1 plays a role in the initiation of skeletal muscle differentiation, and its interaction with Hop is required for the full activity.