Two nuclear localization signals present in the basic-helix 1 domains of MyoD promote its active nuclear translocation and can function independently.

MyoD, a member of the family of helix-loop-helix myogenic factors that plays a crucial role in skeletal muscle differentiation, is a nuclear phosphoprotein. Using microinjection of purified MyoD protein into rat fibroblasts, we show that the nuclear import of MyoD is a rapid and active process, being ATP and temperature dependent. Two nuclear localization signals (NLSs), one present in the basic region and the other in the helix 1 domain of MyoD protein, are demonstrated to be functional in promoting the active nuclear transport of MyoD. Synthetic peptides spanning these two NLSs and biochemically coupled to IgGs can promote the nuclear import of microinjected IgG conjugates in muscle and nonmuscle cells. Deletion analysis reveals that each sequence can function independently within the MyoD protein since concomittant deletion of both sequences is required to alter the nuclear import of this myogenic factor. In addition, the complete cytoplasmic retention of a beta-galactosidase-MyoD fusion mutant protein, double deleted at these two NLSs, argues against the existence of another functional NLS motif in MyoD.

cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of striated muscle characterized in 60% of cases by a t(2;13)(q35;q14). This results in the fusion of PAX3, a developmental transcription factor required for limb myogenesis, with FKHR, a member of the forkhead family of transcription factors. The resultant PAX3-FKHR gene possesses transforming properties; however, the effects of this chimeric oncogene on gene expression are largely unknown. To investigate the actions of these transcription factors, both Pax3 and PAX3-FKHR were introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA microarray containing 2,225 elements. We found that PAX3-FKHR but not PAX3 activated a myogenic transcription program including the induction of transcription factors MyoD, Myogenin, Six1, and Slug as well as a battery of genes involved in several aspects of muscle function. Notable among this group were the growth factor gene Igf2 and its binding protein Igfbp5. Relevance of this model was suggested by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in alveolar rhabdomyosarcoma cell lines. This study utilizes cDNA microarrays to elucidate the pattern of gene expression induced by an oncogenic transcription factor and demonstrates the profound myogenic properties of PAX3-FKHR in NIH 3T3 cells.

The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G00 arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis.

Combinatorial control of muscle development by basic helix-loop-helix and MADS-box transcription factors.

Members of the MyoD family of muscle-specific basic helix-loop-helix (bHLH) proteins function within a genetic pathway to control skeletal muscle development. Mutational analyses of these factors suggested that their DNA binding domains mediated interaction with a coregulator required for activation of muscle-specific transcription. Members of the myocyte enhancer binding factor 2 (MEF2) family of MADS-box proteins are expressed at high levels in muscle and neural cells and at lower levels in several other cell types. MEF2 factors are unable to activate muscle gene expression alone, but they potentiate the transcriptional activity of myogenic bHLH proteins. This potentiation appears to be mediated by direct interactions between the DNA binding domains of these different types of transcription factors. Biochemical and genetic evidence suggests that MEF2 factors are the coregulators for myogenic bHLH proteins. The presence of MEF2 and cell-specific bHLH proteins in other cell types raises the possibility that these proteins may also cooperate to regulate other programs of cell-specific gene expression. We present a model to account for such cooperative interactions.

Role of insulin-like growth factors and myogenin in the altered program of proliferation and differentiation in the NFB4 mutant muscle cell line.

In the present study we used the mutant muscle cell line NFB4 to study the balance between proliferation and myogenic differentiation. We show that removal of serum, which induced the parental C2C12 cells to withdraw from the cell cycle and differentiate, had little effect on NFB4 cells. Gene products characteristic of the proliferation state, such as c-Jun, continued to accumulate in the mutant cells in low serum, whereas those involved in differentiation, like myogenin, insulin-like growth factor II (IGF-II), and IGF-binding protein 5 (IGFBP-5) were undetectable. Moreover, NFB4 cells displayed a unique pattern of tyrosine phosphorylated proteins, especially in low serum, suggesting that the signal transduction pathway(s) that controls differentiation is not properly regulated in these cells. Treatment of NFB4 cells with exogenous IGF-I or IGF-II at concentrations shown to promote myogenic differentiation in wild-type cells resulted in activation of myogenin but not MyoD gene expression, secretion of IG-FBP-5, changes in tyrosine phosphorylation, and enhanced myogenic differentiation. Similarly, transfection of myogenin expression constructs also enhanced differentiation and resulted in activation of IGF-II expression, showing that myogenin and IGF-II cross-activate each other's expression. However, in both cases, the expression of Jun mRNA remained elevated, suggesting that IGFs and myogenin cannot overcome all aspects of the block to differentiation in NFB4 cells.

Transdifferentiation of myoblasts by the adipogenic transcription factors PPAR gamma and C/EBP alpha.

Skeletal muscle and adipose tissue development often has a reciprocal relationship in vivo, particularly in myodystrophic states. We have investigated whether determined myoblasts with no inherent adipogenic potential can be induced to transdifferentiate into mature adipocytes by the ectopic expression of two adipogenic transcription factors, PPAR gamma and C/EBP alpha. When cultured under optimal conditions for muscle differentiation, murine G8 myoblasts expressing PPAR gamma and C/EBP alpha show markedly reduced levels of the myogenic basic helix-loop-helix proteins MyoD, myogenin, MRF4, and myf5 and are completely unable to differentiate into myotubes. Under conditions permissive for adipogenesis including a PPAR activator, these cells differentiate into mature adipocytes that express molecular markers characteristic of this lineage. Our results demonstrate that a developmental switch between these two related but highly specialized cell types can be controlled by the expression of key adipogenic transcription factors. These factors have an ability to inhibit myogenesis that is temporally and functionally separate from their ability to stimulate adipogenesis.