Negative Regulator Of Myogenesis Research Articles

Evaluation of Interferon Response Induced by Anti-Myostatin shRNA Constructs in Goat (Capra hircus) Fetal Fibroblasts by Quantitative Real TIME-Polymerase Chain Reaction

RNAi is an evolutionary conserved, highly efficient, and cost effective technique of gene silencing. It holds considerable promise and success has been achieved both in vitro and in vivo experiments. However, it is not devoid of undesirable side effects as dsRNA can trigger the immune response and can also cause non-specific off-target gene silencing. In the present study, silencing of myostatin gene, a negative regulator of myogenesis, was evaluated in caprine fetal fibroblasts using three different shRNA constructs. Out of these three constructs, two constructs sh1 and sh2 showed, 72% and 50% reduction (p < 0.05) of myostatin mRNA, respectively. Efficient suppression (42–86%) of MSTN gene (p < 0.05) was achieved even by reducing the concentration of shRNA constructs. The induction of classical interferon stimulated gene (Oligoandenylate Synthetase-1, OAS-1) was studied to analyze the immune response against shRNAs. Notably, a reduction in the potency of shRNAs to induce interferon response was observed at lower concentration for OAS1 gene. The results obtained in the study would be helpful in the abrogation of the bystander effects of RNAi for long term stable expression of anti-MSTN expression constructs in the muscle.

Decorin promotes myogenic differentiation and mdx mice therapeutic effects after transplantation of rat adipose-derived stem cells

Adipose-derived stem cells (ADSC) have been considered as attractive candidates for the treatment of Duchenne muscular dystrophy (DMD), but the rate of ADSC myogenesis is very low. Myostatin (Mstn), a negative regulator of myogenesis, is known to be responsible for limiting skeletal muscle regeneration. Decorin could bind Mstn and deactivate it. Decorin has been shown to improve myogenic differentiation in mdx mice. We hypothesized that inhibition of Mstn by using decorin may ameliorate myogenic differentiation of ADSC. Rat ADSC were transfected with the lentivirus-containing green fluorescence protein (GFP) and human decorin gene. The transfected ADSC were induced by 5-azacytidine (5-AzaC). The rates of myogenic differentiation and adipogenesis were detected. The transfected ADSC were injected into mdx mice and the expression of Mstn and decorin detected by Western blot. Dystrophin was detected after transfected ADSC transplantation by immunofluorescence staining and Western blot. Serum creatine kinase (CK) and histologic changes were also evaluated. The optimal multiplicity of infection of ADSC was 10. Decorin improved muscle mass. In accordance with the increased muscle mass, dystrophin expression increased. Following the level of decorin increase, the Mstn expression decreased. Furthermore, serum CK and histologic changes in centrally nucleated fiber (CNF) decreased. Improved myogenic differentiation of ADSC was observed by using decorin. This process was probably the result of decorin inhibiting Mstn. A new method of DMD therapy combining Mstn inhibition (using decorin) and ADSC transplantation is probably feasible.

Ankrd2/ARPP is a novel Akt2 specific substrate and regulates myogenic differentiation upon cellular exposure to H2O2

Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C(2)C(12) murine muscle cells exploiting protein characterization databases in combination with an anti-phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Ser-99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells, where it mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H(2)O(2) triggers this phosphorylation. Moreover, the forced expression of a phosphorylation-defective mutant form of Ankrd2 in C(2)C(12) myoblasts promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Ser-99 in the negative regulation of myogenesis in response to stress conditions.

SNPs detection in largemouth bass myostatin gene and its association with growth traits

Myostatin or GDF-8,a member of the transforming growth factor-β(TGF-β) surperfamily,has been demonstrated to be a negative regulator of myogenesis and inhibited myoblast proliferation during cell cycle and myogenic differentiation in animals.In present research,two SNPs sites(C-1453T and T+33C) were identified from total MSTN gene in larghmouth bass by using PCR-SSCP technique and sequencing;The mutational site of C-1453 was between the regions of E8 box and Octamer(+).the mutation of T+33C was situated in the first extron of the MSTN gene,however the mutation of the first extron has not changed the encoded amino which belonged to the synonymy mutation.The three kinds of genotypes per mutation site were obtained by PCR-RFLP method and the genotype frequency of the mutation was caculated in the population of 128 individuals.A general linear model was used to statistically analyze associations of MSTN gene polymorphisms with major growth traits respectively,and the results indicated that there was no significant difference(P0.05) by all genotypes of single mutation site.Six diplotypes,with the minor allelic frequencies of above 3%,were constructed based on two SNPs in the experiment population.The means of diplotype D2 were over other diplotypes and the means of diplotype D5 were lower other diplotypes in terms of body width,body height,body length,body weight,and snout length therfore associations analysis indicated that there were significant associations between diplotype D2 and D5 in the five growth traits of largemouth bass.The study implies that diplotypes D2 and D5 of MSTN gene could be used as the moleculer genetic marker for selection breeding.

NF-κB Functions in Stromal Fibroblasts to Regulate Early Postnatal Muscle Development

Classical NF-kappaB activity functions as an inhibitor of the skeletal muscle myogenic program. Recent findings reveal that even in newborn RelA/p65(-/-) mice, myofiber numbers are increased over that of wild type mice, suggesting that NF-kappaB may be a contributing factor in early postnatal skeletal muscle development. Here we show that in addition to p65 deficiency, repression of NF-kappaB with the IkappaB alpha-SR transdominant inhibitor or with muscle-specific deletion of IKKbeta resulted in similar increases in total fiber numbers as well as an up-regulation of myogenic gene products. Upon further characterization of early postnatal muscle, we observed that NF-kappaB activity progressively declines within the first few weeks of development. At birth, the majority of this activity is compartmentalized to muscle fibers, but by neonatal day 8 NF-kappaB activity from the myofibers diminishes, and instead, stromal fibroblasts become the main cellular compartment within the muscle that contains active NF-kappaB. We find that NF-kappaB functions in these fibroblasts to regulate inducible nitric-oxide synthase expression, which we show is important for myoblast fusion during the growth and maturation process of skeletal muscle. Together, these data broaden our understanding of NF-kappaB during development by showing that in addition to its role as a negative regulator of myogenesis, NF-kappaB also regulates nitric-oxide synthase expression within stromal fibroblasts to stimulate myoblast fusion and muscle hypertrophy.

Inhibition of myostatin promotes myogenic differentiation of rat bone marrow-derived mesenchymal stromal cells

Mesenchymal stromal cells (MSC) have been thought to be attractive candidates for the treatment of Duchenne muscular dystrophy (DMD), but the rate of MSC myogenesis is very low. Thus MSC treatment for DMD is restricted. Myostatin (Mstn), a negative regulator of myogenesis, is known to be responsible for limiting skeletal muscle regeneration. We hypothesized that inhibition of Mstn by using anti-Mstn antibody (Ab) would ameliorate the myogenic differentiation of MSC in vitro and in vivo. MSC were isolated from rat bone marrow. Induced rat MSC (rMSC) were treated with various concentrations of anti-Mstn Ab. The expression of myogenic differentiation antigen (MyoD), myogenin and myosin heavy chain-type alpha (MHC-alpha) were estimated by immunofluorescence analysis and reverse transcription-polymerase chain reaction (RT-PCR). Adipogenic differentiation of rMSC inhibited by anti-Mstn Ab was evaluated by Oil Red O staining. The expression of dystrophin was detected 16 weeks after anti-Mstn Ab injection and rMSC transplantation by immunofluorescence staining, RT-PCR and Western blot. Motor function, serum creatine kinase (CK) and histologic changes were also evaluated. Five-azacytidine-mediated myogenic differentiation induced significant endogenous Mstn expression. Anti-Mstn Ab improved the expression of MyoD, myogenin and MHC-alpha and inhibited adipocyte formation. Sixteen weeks after transplantation, the inhibition of Mstn had improved motor function and muscle mass. In accordance with the increased motor function and muscle mass, dystrophin expression had increased. Furthermore, serum CK and centrally nucleated fiber (CNF) levels decreased slightly, suggesting specific pathologic features of the dystrophic muscle were partially restored. Using anti-Mstn Ab, we found that inhibition of Mstn improved myogenic differentiation of rMSC in vitro and in vivo. A combination of Mstn blockade and MSC transplantation may provide a pharmacologic and cell-based strategy for the treatment of DMD.

A retrovirus‐based system to stably silence GDF‐8 expression and enhance myogenic differentiation in human rhabdomyosarcoma cells

Myostatin, also called GDF-8, a secreted growth and differentiating factor that belongs to the transforming growth factor-beta superfamily, is a known negative regulator of myogenesis in vivo. Overexpression of GDF-8 contributes to the lack of differentiation in human rhabdomyosarcoma (RMS) cells. We investigated whether a retrovirus-based RNA interference (RNAi) system against GDF-8 expression in human RMS cells would enhance myogenic differentiation. A retrovirus-based RNAi system was developed that utilized the U6-RNA polymerase III promoter to drive efficient expression and deliver the GDF8-specific short hairpin RNAs (shRNAs) in human RMS cell A204. In this system, the retrovirus vector was integrated into the host cell genome and allowed stable expression of shRNAs. GDF-8 expression was determined by real-time polymerase chain reaction and western blotting analysis. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cell proliferation. Myogenic differentiation markers were monitored by western blotting analysis. Cell cycle and apoptosis was determined by propidium iodide staining and analysed in a flow cytometer. In the siGDF8 A204 cell pools, the levels of both GDF-8 mRNA and protein were dramatically reduced by this RNAi system. In differentiation conditions, inhibition of myostatin synthesis led to enhanced cell cycle withdrawal, consequently stimulated myogenic differentiation and increased the rate of tumor cell apoptosis. The results demonstrate that deactivation of myostatin by using retrovirus-based RNAi thus may be useful for therapy in rhabdomyosarcomas.

Ambient glucose levels qualify the potency of insulin myogenic actions by regulating SIRT1 and FoxO3a in C2C12 myocytes

Nutrition availability is one of the major environmental signals influencing cell fate, such as proliferation, differentiation, and apoptosis, often functioning in concert with other humoral factors, including insulin. Herein, we show that low-serum-induced differentiation of C(2)C(12) myocytes is significantly hampered under low glucose (LG; 5 mM) compared with high glucose (HG; 22.5 mM) conditions, concurrently with nuclear accumulation of SIRT1, an NAD(+)-dependent deacetylase, and FoxO3a, both of which are implicated in the negative regulation of myogenesis. Intriguingly, insulin appears to exert opposite actions, depending on glucose availability, with regard to the regulation of SIRT1 and FoxO3a abundance, which apparently contributes to modulating the potency of insulin's myogenic action. Namely, insulin exerts a potent myogenic effect in the presence of sufficient glucose, whereas insulin is unable to exert its myogenic action under LG conditions, since insulin evokes massive upregulation of both SIRT1 and FoxO3a in the absence of sufficient ambient glucose. In addition, the hampered differentiation state under LG is significantly restored by sirtinol, a SIRT1 inhibitor, whereas insulin abolished this sirtinol-dependent restoration, indicating that insulin can function as a negative as well as a positive myogenic factor depending on glucose availability. Taken together, our data reveal the importance of ambient glucose levels in the regulation of myogenesis and also in the determination of insulin's myogenic potency, which is achieved, at least in part, through regulation of the cellular contents and localization of SIRT1 and FoxO3a in differentiating C(2)C(12) myocytes.

Myostatin antisense RNA-mediated muscle growth in normal and cancer cachexia mice

Myostatin is a negative regulator of myogenesis, and inactivation of myostatin leads to muscle growth. Here we have used modified RNA oligonucleotides targeting the myostatin mRNA and examined the therapeutic potential in normal and cancer cachexia mouse models. We found that the RNA oligonucleotides could suppress the myostatin expression in vivo, leading to the increase in muscle growth both in normal and cachectic mice. We also established that the effect of myostatin inhibition caused by the RNA oligonucleotides may be through the MyoD pathway, as evidenced by a significant upregulation of MyoD expression. Taken together, these results demonstrate the feasibility using antisense strategy for the treatment of muscle wasting conditions.

Myostatin gene targeting in cultured China Han ovine myoblast cells

Myostatin (MSTN), a member of the transforming growth factor-β superfamily, has been shown to be a negative regulator of myogenesis. Natural mutation in beef cattle causes double-muscling phenotypes. We report an investigation designed to knockout the MSTN gene by gene targeting in ovine myoblast cells. Two promoter-trap targeting vectors MSTN-green fluorescent protein (GFP) and MSTN-neo were constructed and used to transfect foetal and neonatal ovine primary myoblast cells. Both GFP-expressing cells and drug-resistant cells were obtained. Targeted cells expressing GFP were confirmed by polymerase chain reaction (PCR) assay and drug-resistant cells were characterised by PCR and Southern blot after growing into cell clones.

Signaling pathways weigh in on decisions to make or break skeletal muscle.

Regulation of muscle size is essential for proper development and homeostasis of adult musculature. This regulation is mediated in large part by signal transduction pathways that promote the synthesis or breakdown of skeletal muscle. PI(3)K/Akt, myostatin and NF-kappaB represent three such pathways that will be the focus of this review. Recent reports solidify the requirement of the PI(3)K/Akt pathway in the regulation of muscle hypertrophy. In response to IGF-1, Akt activates downstream effectors, mTOR and p70S6K to stimulate protein synthesis thereby increasing the cytoplasmic compartment in muscle fibers. Tsc2 was also identified as a novel Akt target, whose phosphorylation and inactivation by Akt may lead to an increase in cell size. The mechanisms by which myostatin functions in muscle wasting was recently explored using in-vitro assays of myogenesis. Myostatin was found to repress myogenesis by inhibiting the synthesis and activity of MyoD. Paradoxically, myostatin expression is itself regulated by MyoD binding to the myostatin promoter. The NF-kappaB transcription factor also functions as a negative regulator of myogenesis by inhibiting MyoD. Chronic activation of NF-kappaB has been associated with muscle wasting, but the mechanisms by which this regulation occurs remain for the most part unknown. Recent cell culture and animal studies have provided insight on the mechanisms by which Akt, myostatin, and NF-kappaB signaling pathways regulate muscle size. Clinical intervention to boost Akt signaling or modulate myostatin and NF-kappaB activities may prove useful in diseases associated with chronic muscle wasting.

Myostatin Inhibits Myoblast Differentiation by Down-regulating MyoD Expression

Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3. In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C(2)C(12) myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3.MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negative Smad3 rescued the activity of a MyoD promoter-reporter in C(2)C(12) myoblasts treated with myostatin. Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.

The myostatin gene is a downstream target gene of basic helix-loop-helix transcription factor MyoD.

Myostatin is a negative regulator of myogenesis, and inactivation of myostatin leads to heavy muscle growth. Here we have cloned and characterized the bovine myostatin gene promoter. Alignment of the upstream sequences shows that the myostatin promoter is highly conserved during evolution. Sequence analysis of 1.6 kb of the bovine myostatin gene upstream region revealed that it contains 10 E-box motifs (E1 to E10), arranged in three clusters, and a single MEF2 site. Deletion and mutation analysis of the myostatin gene promoter showed that out of three important E boxes (E3, E4, and E6) of the proximal cluster, E6 plays a significant role in the regulation of a reporter gene in C(2)C(12) cells. We also demonstrate by band shift and chromatin immunoprecipitation assay that the E6 E-box motif binds to MyoD in vitro and in vivo. Furthermore, cotransfection experiments indicate that among the myogenic regulatory factors, MyoD preferentially up-regulates myostatin promoter activity. Since MyoD expression varies during the myoblast cell cycle, we analyzed the myostatin promoter activity in synchronized myoblasts and quiescent "reserve" cells. Our results suggest that myostatin promoter activity is relatively higher during the G(1) phase of the cell cycle, when MyoD expression levels are maximal. However, in the reserve cells, which lack MyoD expression, a significant reduction in the myostatin promoter activity is observed. Taken together, these results suggest that the myostatin gene is a downstream target gene of MyoD. Since the myostatin gene is implicated in controlling G(1)-to-S progression of myoblasts, MyoD could be triggering myoblast withdrawal from the cell cycle by regulating myostatin gene expression.

Myostatin, a Negative Regulator of Muscle Growth, Functions by Inhibiting Myoblast Proliferation

Myostatin, a member of the transforming growth factor-beta (TGF-beta) superfamily, has been shown to be a negative regulator of myogenesis. Here we show that myostatin functions by controlling the proliferation of muscle precursor cells. When C(2)C(12) myoblasts were incubated with myostatin, proliferation of myoblasts decreased with increasing levels of myostatin. Fluorescence-activated cell sorting analysis revealed that myostatin prevented the progression of myoblasts from the G(1)- to S-phase of the cell cycle. Western analysis indicated that myostatin specifically up-regulated p21(Waf1, Cip1), a cyclin-dependent kinase inhibitor, and decreased the levels and activity of Cdk2 protein in myoblasts. Furthermore, we also observed that in myoblasts treated with myostatin protein, Rb was predominately present in the hypophosphorylated form. These results suggests that, in response to myostatin signaling, there is an increase in p21 expression and a decrease in Cdk2 protein and activity thus resulting in an accumulation of hypophosphorylated Rb protein. This, in turn, leads to the arrest of myoblasts in G(1)-phase of cell cycle. Thus, we propose that the generalized muscular hyperplasia phenotype observed in animals that lack functional myostatin could be as a result of deregulated myoblast proliferation.

Phosphatidylinositol 3-Kinase Inhibitors Block Differentiation of Skeletal Muscle Cells

Skeletal muscle differentiation involves myoblast alignment, elongation, and fusion into multinucleate myotubes, together with the induction of regulatory and structural muscle-specific genes. Here we show that two phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, blocked an essential step in the differentiation of two skeletal muscle cell models. Both inhibitors abolished the capacity of L6E9 myoblasts to form myotubes, without affecting myoblast proliferation, elongation, or alignment. Myogenic events like the induction of myogenin and of glucose carrier GLUT4 were also blocked and myoblasts could not exit the cell cycle, as measured by the lack of mRNA induction of p21 cyclin-dependent kinase inhibitor. Overexpresssion of MyoD in 10T1/2 cells was not sufficient to bypass the myogenic differentiation blockade by LY294002. Upon serum withdrawal, 10T1/2-MyoD cells formed myotubes and showed increased levels of myogenin and p21. In contrast, LY294002-treated cells exhibited none of these myogenic characteristics and maintained high levels of Id, a negative regulator of myogenesis. These data indicate that whereas phosphatidylinositol 3-kinase is not indispensable for cell proliferation or in the initial events of myoblast differentiation, i.e. elongation and alignment, it appears to be essential for terminal differentiation of muscle cells.

Constitutive expression of the orphan receptor, Rev-erbA alpha, inhibits muscle differentiation and abrogates the expression of the myoD gene family.

Rev-erbA alpha is an orphan steroid receptor that is expressed in skeletal muscle. Rev-erbA alpha binds to single/tandem copies of an AGGTCA motif, is transcribed on the noncoding strand of the c-erbA- alpha gene locus, and is postulated to modulate the thyroid hormone (T3) response. T3 induces terminal muscle differentiation and regulates fiber type composition via direct activation of the muscle-specific myoD gene family (e.g. myoD, myogenin). The myoD gene family can direct the fate of mesodermal cell lineages and activate muscle differentiation. Hence we investigated the expression and physiological role of Rev-erbA alpha during myogenesis. We observed abundant levels of Rev-erbA alpha mRNA in dividing C2C12 myoblasts, which were suppressed when the cells differentiated into postmitotic multinucleated myotubes. This decrease in Rev-erbA alpha mRNA correlated with the appearance of muscle-specific mRNAs (e.g. myogenin and alpha-actin). Constitutive overexpression of full length Rev-erbA alpha cDNA in the myogenic cells completely abolished differentiation, suppressed myoD mRNA levels, and abrogated the induction of myogenin mRNA. We then demonstrated that 1) GAL4-REV-erbA alpha chimeras that contain the 'AB' region and lack the 'E' region activated transcription of GAL4 response elements in the presence of 8-Br-cAMP and 2) the ligand-binding domain (LBD) contains an active transcriptional silencer. Overexpression of Rev-erbA alpha (delta AB) in myogenic cells had no impact on the ability of these cells to morphologically or biochemically differentiate. Furthermore, this orphan receptor 1) down-regulated thyroid hormone receptor (TR)/T3 mediated transcriptional activity from the myogenin promoter and thyroid hormone response element (TRE) an 2) disrupted TR homodimer and TR/retinoid X receptor (RXR) heterodimer formation on a number of TREs found in the myoD gene family. In conclusion, Rev-erbA alpha functions as a negative regulator of myogenesis by targeting the expression of the myoD gene family. The mechanism of action may involve inhibition of functional TR/RXR heterodimer formation on critical TREs and dominant trans-repression of gene expression.