Enhanced growth and myogenic differentiation of spheroid-derived C2C12 cells.

Among many factors of controlling stem cell differentiation, the key transcription factor upregulation via physical force is a good strategy on the lineage-specific differentiation of stem cells. The study aimed to compare growth and myogenic potentials between the parental cells (PCs) and the 1-day-old C2C12 spheroid-derived cells (SDCs) in two-dimensional (2D) and three-dimensional (3D) culture conditions through examination of the cell proliferation and the expression of myogenic genes. The data showed that 1-day-old spheroids had more intense expression of MyoD gene with respect to the PCs. The proliferation of the SDCs is significantly higher than the PCs in a time-dependent manner. The SDCs had also significantly higher myogenic potential than the PCs in 2D and 3D culture conditions. The results suggest that MyoD gene upregulation through cell-cell contacts is the good approach for preparation of seed cells in muscle tissue engineering.

The MyoD family inhibitor domain-containing protein enhances the chemoresistance of cancer stem cells in the epithelial state by increasing ß-catenin activity.

Cancer cells with mesenchymal attributes potentially display chemoresistance. Cancer stem cells (CSCs), which are intrinsically resistant to most chemotherapy agents, exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal states. However, the drug response of CSCs in the epithelial and mesenchymal states has not been completely investigated. In this study, we found that epithelial-type (E-cadherinhigh/CD133high) CSCs displayed a higher sphere formation ability and chemoresistance than mesenchymal-type (E-cadherinlowCD133high) CSCs. Gene expression profiling of the CSC and non-CSC subpopulations with distinct epithelial-to-mesenchymal transition (EMT) states showed that MyoD family inhibitor domain-containing (MDFIC) was selectively upregulated in epithelial-type CSCs. Knockdown of MDFIC sensitized epithelial-type CSCs to chemotherapy agents. Ectopic expression of MDFIC increased the chemoresistance of mesenchymal-type CSCs. In a tissue microarray, high MDFIC expression was associated with poor prognosis of non-small cell lung cancer (NSCLC) patients. A mechanistic study showed that the MDFIC p32 isoform, which is located in the cytoplasm, interacted with the destruction complex, Axin/GSK-3/ß-catenin. This interaction stabilized ß-catenin by inhibiting ß-catenin phosphorylation at S33/37 and increased the nuclear translocation and transcriptional activity of ß-catenin. Knockdown of ß-catenin decreased MDFIC-enhanced chemoresistance. These results suggested that the upregulation of MDFIC enhanced the chemoresistance of epithelial-type CSCs by elevating ß-catenin activity. Thus, targeting MDFIC-regulated ß-catenin signaling of epithelial-type CSCs may be a potential strategy to overcome chemoresistance in NSCLC.

Galectin-3- Mediated Transdifferentiation of Pulmonary Artery Endothelial Cells Contributes to Hypoxic Pulmonary Vascular Remodeling.

BACKGROUND/AIMS: Vascular muscularity is a key event in vessel remodeling during pulmonary artery hypertension (PAH). Endothelial-mesenchymal transdifferentiation (EndMT) has been increasingly reported to play a role in disease occurrence. Galectin-3, a carbohydrate-binding protein regulates cell proliferation, differentiation, migration and neovascularization. However, whether galectin-3 controls endothelial cell transdifferentiation during the development of PAH is unknown. METHODS: Rats were exposed to normoxic or hypoxic conditions (fraction of inspired O2 0.10) for 21 d to establish PAH models. Hemodynamic changes were evaluated through surgery of the right jugular vein and ultrasound biomicroscopy inviVue. And vessel pathological alterations were detected by H&E staining. Galectin-3 (Gal-3)-induced pulmonary artery endothelium cell (PAEC) dynamic alterations were measured by MTT assays, Cell immunofluorescence, Flow cytometry, Real-time PCR and Western blot. RESULTS: Our study demonstrated that Gal-3 was expressed in hypoxic pulmonary vascular adventitia and intima. The increased Gal-3 expression was responsible for hypoxic vessel remodeling and PAH development in vivo. Gal-3 was found to inhibit cell proliferation and apoptosis in cultured endothelial cells. Meanwhile endothelial cell morphology was altered and exhibited smooth muscle-like cell features as demonstrated by the expression of α-SMA after Gal-3 treatment. Gal-3 activated Jagged1/Notch1 pathways and induced MyoD and SRF. When MyoD or SRF were silenced with siRNAs, Gal-3-initiated transdifferentiation in endothelial cells was blocked as indicated by a lack of α-SMA. CONCLUSION: These results suggest that Gal-3 induces PAECs to acquire an α-SMA phenotype via a transdifferentiation process which depends on the activation of Jagged1/Notch1 pathways that mediate MyoD and SRF expression.

MUNC, an Enhancer RNA Upstream from the MYOD Gene, Induces a Subgroup of Myogenic Transcripts in trans Independently of MyoD.

MyoD upstream noncoding RNA (MUNC) initiates in the distal regulatory region (DRR) enhancer of MYOD and is formally classified as an enhancer RNA (DRReRNA). MUNC is required for optimal myogenic differentiation, induces specific myogenic transcripts in trans (MYOD, MYOGENIN, and MYH3), and has a functional human homolog. The vast majority of eRNAs are believed to act in cis primarily on their neighboring genes (1, 2), making it likely that MUNC action is dependent on the induction of MYOD RNA. Surprisingly, MUNC overexpression in MYOD-/- C2C12 cells induces many myogenic transcripts in the complete absence of MyoD protein. Genomewide analysis showed that, while many genes are regulated by MUNC in a MyoD-dependent manner, there is a set of genes that are regulated by MUNC, both upward and downward, independently of MyoD. MUNC and MyoD even appear to act antagonistically on certain transcripts. Deletion mutagenesis showed that there are at least two independent functional sites on the MUNC long noncoding RNA (lncRNA), with exon 1 more active than exon 2 and with very little activity from the intron. Thus, although MUNC is an eRNA of MYOD, it is also a trans-acting lncRNA whose sequence, structure, and cooperating factors, which include but are not limited to MyoD, determine the regulation of many myogenic genes.

The transcriptional co-repressor TLE3 regulates myogenic differentiation by repressing the activity of the MyoD transcription factor.

Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity.

Skeletal muscle programming and re-programming.

The discovery of the transcription factor MyoD and its ability to induce muscle differentiation was the first demonstration of genetically programmed cell transdifferentiation. MyoD functions by activating a feed-forward circuit to regulate muscle gene expression. This requires binding to specific E-boxes throughout the genome, followed by recruitment of chromatin modifying complexes and transcription machinery. MyoD binding can be modified by both cooperative factors and inhibitors, including microRNAs that may serve as important developmental switches. Recent studies indicate that epigenetic regulation of MyoD binding sites is another important mechanism for controlling MyoD activity, which may ultimately limit its ability to induce transdifferentiation to cells with permissive epigenetic 'landscapes.'

Myostatin suppression of Akirin1 mediates glucocorticoid-induced satellite cell dysfunction.

Glucocorticoids production is increased in many pathological conditions that are associated with muscle loss, but their role in causing muscle wasting is not fully understood. We have demonstrated a new mechanism of glucocorticoid-induced muscle atrophy: Dexamethasone (Dex) suppresses satellite cell function contributing to the development of muscle atrophy. Specifically, we found that Dex decreases satellite cell proliferation and differentiation in vitro and in vivo. The mechanism involved Dex-induced upregulation of myostatin and suppression of Akirin1, a promyogenic gene. When myostatin was inhibited in Dex-treated mice, Akirin1 expression increased as did satellite cell activity, muscle regeneration and muscle growth. In addition, silencing myostatin in myoblasts or satellite cells prevented Dex from suppressing Akirin1 expression and cellular proliferation and differentiation. Finally, overexpression of Akirin1 in myoblasts increased their expression of MyoD and myogenin and improved cellular proliferation and differentiation, theses improvements were no longer suppressed by Dex. We conclude that glucocorticoids stimulate myostatin which inhibits Akirin1 expression and the reparative functions of satellite cells. These responses attribute to muscle atrophy. Thus, inhibition of myostatin or increasing Akirin1 expression could lead to therapeutic strategies for improving satellite cell activation and enhancing muscle growth in diseases associated with increased glucocorticoid production.

Coordination of cell cycle, DNA repair and muscle gene expression in myoblasts exposed to genotoxic stress.

Upon exposure to genotoxic stress, skeletal muscle progenitors coordinate DNA repair and the activation of the differentiation program through the DNA damage-activated differentiation checkpoint, which holds the transcription of differentiation genes while the DNA is repaired. A conceptual hurdle intrinsic to this process relates to the coordination of DNA repair and muscle-specific gene transcription within specific cell cycle boundaries (cell cycle checkpoints) activated by different types of genotoxins. Here, we show that, in proliferating myoblasts, the inhibition of muscle gene transcription occurs by either a G 1- or G 2-specific differentiation checkpoint. In response to genotoxins that induce G 1 arrest, MyoD binds target genes but is functionally inactivated by a c-Abl-dependent phosphorylation. In contrast, DNA damage-activated G 2 checkpoint relies on the inability of MyoD to bind the chromatin at the G 2 phase of the cell cycle. These results indicate an intimate relationship between DNA damage-activated cell cycle checkpoints and the control of tissue-specific gene expression to allow DNA repair in myoblasts prior to the activation of the differentiation program.

P/CAF rescues the Bhlhe40-mediated repression of MyoD transactivation.

Previously, we found that MRFs (myogenic regulatory factors) regulated the expression of PGC-1alpha (peroxisome-proliferator-activated receptor gamma co-activator 1alpha) by targeting a short region, from nt -49 to +2 adjacent to the transcription initiation site, that contained two E-boxes. However, only the E2-box had significant affinity for MRFs, and the E1-box was predicted to be the target of Bhlhe40 (basic helix-loop-helix family, member e40, also known as Stra13, Bhlhb2, DEC1 and Sharp2), a transcriptional repressor implicated in the regulation of several physiological processes. In the present study, by using EMSA (electrophoresis mobility-shift assay), we confirmed that Bhlhe40 targeted the E1-box and formed a complex with the basic helix-loop-helix transcription factor MyoD (myogenic differentiation factor D) on the PGC-1alpha core promoter. We demonstrate that Bhlhe40 binds to the promoters of PGC-1alpha and myogenic genes in vivo and that Bhlhe40 represses the MyoD-mediated transactivation of these promoters. Furthermore, we found that this repression could be relieved by P/CAF (p300/CBP-associated factor) in a dose-dependent manner, but not by CBP [CREB (cAMP-response-element-binding protein)-binding protein]. Bhlhe40 interacted with P/CAF and this interaction disrupted the interaction between P/CAF and MyoD. These results suggest that Bhlhe40 functions as a repressor of MyoD by binding to adjacent E-boxes and sequestering P/CAF from MyoD.

Interaction of the porcine reproductive and respiratory syndrome virus nucleocapsid protein with the inhibitor of MyoD family-a domain-containing protein.

Porcine reproductive and respiratory syndrome (PRRS) virus is an RNA virus that replicates in the cytoplasm, but the viral nucleocapsid (N) protein localizes specifically in the nucleus and nucleolus of virus-infected cells. Nuclear localization of N is non-essential for PRRSV replication in cultured cells but has been shown to modulate the pathogenesis of virus in pigs, suggesting that N plays an accessory role in the nucleus during infection. We identified by yeast two-hybrid screening the inhibitor of MyoD family-a (I-mfa) domain-containing protein (HIC) as a cellular partner for PRRS virus (PRRSV) N protein. This protein is a homolog of human HIC, a recently identified cellular transcription factor. The specific interaction of PRRSV N with HIC was confirmed in cells by mammalian two-hybrid assay and co-immunoprecipitation and in vitro by GST pull-down assay. HIC is a zinc-binding protein and confocal microscopy demonstrated co-localization of N with the HIC-p40 isomer in the nucleus and nucleolus, and in the cytoplasm with HIC-p32, which is the N-terminal truncation of HIC-p40. The porcine homolog of HIC is universally expressed in pig tissues including alveolar macrophages. The interaction of viral capsid with the cellular transcription factor implicates a possible regulation of host cell gene expression by the N protein during PRRSV infection.

Osterix overexpression enhances osteoblast differentiation of muscle satellite cells in vitro.

Muscle satellite cells have long been considered a distinct myogenic lineage responsible for postnatal growth, repair and maintenance of skeletal muscle. Recent studies have demonstrated that they are multi-potential. Osterix (Osx), a novel zinc-finger-containing transcription factor of the sp family, is required for osteoblast differentiation and bone formation. It was hypothesized that Osx overexpression would enhance osteoblast differentiation of muscle satellite cells in vitro. Recombinant adenovirus-mediated Osx gene (Ad-Osx) was constructed and used to transfect muscle satellite cells. Osx overexpression inhibited myogenesis, as demonstrated by suppression of MyoD and myogenin mRNA levels and reduced myotube formation. Muscle satellite cells transduced with Ad-Osx exhibited apparent osteoblast differentiation as determined by the expression of related osteoblastic genes, increased activity of alkaline phosphatase and the formation of mineralized nodules. These results confirmed the ability of Osx to enhance osteoblast differentiation of muscle satellite cells in vitro, and the competence of muscle satellite cells as promising seed cells for bone tissue engineering.

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.

The hedgehog regulated oncogenes Gli1 and Gli2 block myoblast differentiation by inhibiting MyoD-mediated transcriptional activation.

The mechanism by which activation of the Hedgehog (Hh) pathway modulates differentiation and promotes oncogenesis in specific tissues is poorly understood. We therefore, analysed rhabdomyosarcomas from mice that were haploinsufficient for the Hh-binding protein, Hip1, or for the Hh receptor, Patched 1 (Ptch1). Transfection of the Hh-regulated transcription factor Gli1, which is expressed in a subset of mouse and human rhabdomyosarcomas, suppressed differentiation of myogenic rhabdomyosarcoma lines generated from Hip1+/- and Ptch1+/- mice. The closely related factor, Gli2, had similar effects. Gli1 and Gli2 inhibited myogenesis by repressing the capacity of MyoD to activate transcription. Deletion analysis of Gli1 indicated that multiple domains of Gli1 are required for efficient inhibition of MyoD. Gli1 reduced the ability of MyoD to heterodimerize with E12 and bind DNA, providing one mechanism whereby the Gli proteins modulate the activity of MyoD. This novel activity of Gli proteins provides new insights into how Hh signaling modulates terminal differentiation through inhibition of tissue-specific factors such as MyoD. This mechanism may contribute to the broad role of Hh signaling and the Gli proteins in differentiation decisions and cancer formation.

Histone methyltransferase Suv39h1 represses MyoD-stimulated myogenic differentiation.

Suv39h1 is a histone H3 lysine-9 (H3-K9) specific methyltransferase (HMT) that is associated with gene silencing through chromatin modification. The transition from proliferation into differentiation of muscle cell is accompanied by transcriptional activation of previously silent muscle genes. I report Suv39h1 interaction with myogenic regulator MyoD in proliferating muscle cells and its HMT activity, which is associated with MyoD, diminishes as differentiation proceeds. The Suv39h1-MyoD complex was detected on the chromatin regulatory regions of a silent differentiation signal muscle gene myogenin and that Suv39h1 presence correlated with H3-K9 methylation. Increased Suv39h1 expression repressed MyoD-dependent muscle gene expression and this property required its HMT activity. This repression required Suv39h1 association with MyoD as well as sustained methylation of H3-K9 on myogenin promoter. Suv39h1 was required for muscle gene repression because its abrogation by siRNA activates these gene expressions by MyoD. These findings suggest that Suv39h1 presence in association with MyoD on the promoter of muscle genes silences gene transcription, providing a necessary checkpoint between proliferation and differentiation.

Ran binding protein 9 interacts with Raf kinase but does not contribute to downstream ERK1/2 activation in skeletal myoblasts.

Raf kinase is the upstream activator of MEK1/2 leading to phosphorylation and activation of ERK1/2. Sustained activation of Raf represses skeletal muscle-specific reporter gene transcription and formation of multinucleated myofibers. Inhibition of myogenesis by activated Raf involves downstream ERK1/2 as well as undefined mediators. To identify Raf-interacting proteins that may influence repression of muscle formation, a yeast two-hybrid screen was performed using a MEK1-binding defective Raf (RafBXB-T481A) as bait. Twenty cDNAs coding for Raf-interacting proteins were identified including Ran binding protein 9 (RanBP9), a protein previously reported to interact with receptor tyrosine kinases. Forced expression of RanBP9 in myogenic cells did not alter myogenesis. Co-expression of RanBP9 with constitutively active RafBXB, but not RafBXB-T481A, synergistically inhibited MyoD-directed muscle reporter gene transcription. Knockdown of RanBP9 expression did not restore the differentiation program to Raf-expressing myoblasts. Thus, RanBP9 physically associates with Raf but does not substantially contribute to the inhibitory actions of the kinase.

c-Myb inhibits myogenic differentiation through repression of MyoD.

c-Myb, known to play a central role in hematopoiesis, is also an important factor involved in myogenesis. Here, we found that the c-myb gene is expressed in proliferating C2C12 myoblasts and turned off in differentiating cells. Detailed analysis of c-myb RNAs revealed that the cell density is the essential factor determining c-myb expression. Both c-myb and its alternatively spliced form c-mybE9A RNAs are down-regulated in confluent cells. Constitutive expression of exogenous c-myb in C2C12 cells inhibits their terminal differentiation. It is shown that the c-Myb protein physically interacts with MyoD, the key regulator of myogenesis, and inhibits MyoD-dependent transcription. The interaction domains are the DNA binding domain of c-Myb and the bHLH motif of MyoD. Our data suggest that in proliferating cells c-Myb binds MyoD and inhibits its transcriptional activity until cell-cell contacts are established and c-myb expression is switched off. Thus, the c-Myb protein may be one of factors ensuring that proliferating myoblasts remain undifferentiated.

Proinflammatory cytokine impairment of insulin-like growth factor I-induced protein synthesis in skeletal muscle myoblasts requires ceramide.

GH and IGF-I control over 80% of postnatal growth. We recently established that TNFalpha impairs the ability of IGF-I to increase protein synthesis and promote expression of myogenin in myoblasts. Here we extend these results by showing that ceramide, a second messenger in both TNFalpha and IL-1beta receptor signaling pathways, is a key downstream sphingosine-based lipid that leads to IGF-I resistance. A cell-permeable ceramide analog, C2-ceramide, inhibits IGF-I-induced protein synthesis by 65% and blocks the ability of IGF-I to increase expression of two key myogenic factors, myogenin and MyoD. Identical results were obtained with both TNFalpha and IL-1beta (1 ng/ml). Consistent with these data, neutral sphingomyelinase (N-SMase), an enzyme that catalyzes formation of ceramide from sphingomyelin, blocks IGF-I-induced protein synthesis and expression of both myogenin and MyoD. The possibility that cytokine-induced ceramide production is required for disruption of IGF-I biologic activity was confirmed by treating C2C12 myoblasts with inhibitors of all three ceramide-generating pathways. A N-SMase inhibitor, glutathione, as well as an acidic sphingomyelinase (A-SMase) inhibitor, D609, reverse the cytokine inhibition of IGF-I-induced protein synthesis by 80% and 45%, respectively. Likewise, an inhibitor of de novo ceramide synthesis, FB1, causes a 50% inhibition. Similarly, all three inhibitors significantly impair the ability of both TNFalpha and IL-1beta to suppress IGF-I-driven expression of myogenin. These experiments establish that ceramide, derived both from sphingomyelin and de novo synthesis, is a key intermediate by which proinflammatory cytokines impair the ability of IGF-I to promote protein synthesis and expression of critical muscle-specific transcription factors.

Hypoxia inhibits myogenic differentiation through accelerated MyoD degradation.

Cells undergo a variety of biological responses when placed in hypoxic conditions, including alterations in metabolic state and growth rate. Here we investigated the effect of hypoxia on the ability of myogenic cells to differentiate in culture. Exposure of myoblasts to hypoxia strongly inhibited multinucleated myotube formation and the expression of differentiation markers. We showed that hypoxia reversibly inhibited MyoD, Myf5, and myogenin expression. One key step in skeletal muscle differentiation involves the up-regulation of the cell cycle-dependent kinase inhibitors p21 and p27 as well as the product of the retinoblastoma gene (pRb). Myoblasts cultured under hypoxic conditions in differentiation medium failed to up-regulate both p21 and pRb despite the G1 cell cycle arrest, as evidenced by p27 accumulation and pRb hypophosphorylation. Hypoxia-dependent inhibition of differentiation was associated with MyoD degradation by the ubiquitin-proteasome pathway. MyoD overexpression in C2C12 myoblasts overrode the differentiation block imposed by hypoxic conditions. Thus, hypoxia by inducing MyoD degradation blocked accumulation of early myogenic differentiation markers such as myogenin and p21 and pRb, preventing both permanent cell cycle withdraw and terminal differentiation. Our study revealed a novel anti-differentiation effect exerted by hypoxia in myogenic cells and identified MyoD degradation as a relevant target of hypoxia.

Functional characterization of an amino-terminal region of HDAC4 that possesses MEF2 binding and transcriptional repressive activity.

Like the full-length histone deacetylase (HDAC) 4, its amino terminus (amino acids 1-208) without the carboxyl deacetylase domain is also known to effectively bind and repress myocyte enhancer factor 2 (MEF2). Within this repressive amino terminus, we further show that a stretch of 90 amino acids (119-208) displays MEF2 binding and repressive activity. The same region is also found to associate specifically with HDAC1 which is responsible for the repressive effect. The amino terminus of HDAC4 can associate with the DNA-bound MEF2 in vitro, suggesting that it does not repress MEF2 simply by disrupting the ability of MEF2 to bind DNA. In vivo, MEF2 induces nuclear translocation of both the full-length HDAC4 and HDAC4-(1-208), whereas the nuclear HDAC4 as well as HDAC4-(1-208) in turn specifically sequesters MEF2 to distinct nuclear bodies. In addition, we show that MyoD and HDAC4 functionally antagonize each other to regulate MEF2 activity. Combined with data from others, our data suggest that the full-length HDAC4 can repress MEF2 through multiple independent repressive domains.

Transactivation of capn2 by myogenic regulatory factors during myogenesis.

The calcium-activated cysteine protease m-calpain plays a pivotal role during the earlier stages of myogenesis, particularly during fusion. The enzyme is a heterodimer, encoded by the genes capn2, for the large subunit, and capn4, for the small subunit. To study the regulation of m-calpain, the DNA sequence upstream of capn2 was analyzed for promoter elements, revealing the existence of five consensus-binding sites (E-box) for several myogenic regulatory factors and one binding site for myocyte enhancer factor-2 (MEF-2). Transient transfections with reporter gene constructs containing the E-box revealed that MyoD presents a high level of transactivation of reporter constructs containing this region, in particular the sequences including the MEF-2/E4-box. In addition, over-expression of various myogenic factors demonstrated that MyoD and myogenin with much less efficiency, can up-regulate capn2, both singly and synergistically, while Myf5 has no effect on synthesis of the protease. Experiments with antisense oligonucleotides directed against each myogenic factor revealed that MyoD plays a specific and pivotal role during capn2 regulation, and cannot be replaced wholly by myogenin and Myf5.