Myotonic dystrophy protein kinase is critical for nuclear envelope integrity

Myotonic dystrophy 1 (DM1) is a multisystemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). DMPK is a nuclear envelope (NE) protein that promotes myogenic gene expression in skeletal myoblasts. Muscular dystrophy research has revealed the NE to be a key determinant of nuclear structure, gene regulation, and muscle function. To investigate the role of DMPK in NE stability, we analyzed DMPK expression in epithelial and myoblast cells. We found that DMPK localizes to the NE and coimmunoprecipitates with Lamin-A/C. Overexpression of DMPK in HeLa cells or C2C12 myoblasts disrupts Lamin-A/C and Lamin-B1 localization and causes nuclear fragmentation. Depletion of DMPK also disrupts NE lamina, showing that DMPK is required for NE stability. Our data demonstrate for the first time that DMPK is a critical component of the NE. These novel findings suggest that reduced DMPK may contribute to NE instability, a common mechanism of skeletal muscle wasting in muscular dystrophies.     

Glucocorticoid inhibition of C2C12 proliferation rate and differentiation capacity in relation to mRNA levels of the MRF gene family

The muscle regulatory factors (MRF) gene family regulate muscle fibre development. Several hormones and drugs also affect muscle development. Glucocorticoids are the only drugs reported to have a beneficial effect on muscle degenerative disorders. We investigated the glucocorticoid-related effects on C2C12 myoblast proliferation rate, morphological differentiation, and subsequent mRNA expression patterns of the MRF genes. C2C12 cells were incubated with the glucocorticoids dexamethasone or alpha-methyl-prednisolone. Both glucocorticoids showed comparable effects. Glucocorticoid treatment of C2C12 cells during the proliferative phase reduced the proliferation rate of the cells dose dependently, especially during the third and fourth day of culture, increased MyoD1, myf-5, and MRF4 mRNA levels, and reduced myogenin mRNA level, compared to untreated control cells. Thus, the mRNA level of proliferation-specific MyoD1 and myf-5 expression does not seem to associate with C2C12 myoblast proliferation rate. Glucocorticoid treatment of C2C12 cells during differentiation reduced the differentiation capacity dose dependently, which is accompanied by a dose dependent reduction of myogenin mRNA level, and increased MyoD1, myf-5, and MRF4 mRNA levels compared to untreated control cells. Therefore, we conclude that glucocorticoid treatment reduces differentiation of C2C12 myoblasts probably through reduction of differentiation-specific myogenin mRNA level, while inducing higher mRNA levels of proliferation-associated MRF genes.     

miR-152 regulates the proliferation and differentiation of C2C12 myoblasts by targeting <Emphasis Type="Italic">E2F3</Emphasis>

The development of skeletal muscle is a complex process involving the proliferation, differentiation, apoptosis, and changing of muscle fiber types in myoblasts. Many reports have described the involvement of microRNAs in the myogenesis of myoblasts. In this study, we found that the expression of miR-152 was gradually down-regulated during myoblast proliferation, but gradually up-regulated during the differentiation of myoblasts. Transfection with miR-152 mimics restrained cell proliferation and decreased the expression levels of cyclin E, CDK4, and cyclin D1, but promoted myotube formation and significantly increased the mRNA expression levels of MyHC, MyoD, MRF4, and MyoG in C2C12 myoblasts. However, treatment with miR-152 inhibitors promoted cell proliferation and restrained differentiation. Moreover, over-expression of miR-152 significantly decreased E2F3 production in C2C12 myoblasts. A luciferase assay confirmed that miR-152 could bind to the 3′ UTR of E2F3. In conclusion, this study showed that miR-152 inhibited proliferation and promoted myoblast differentiation by targeting E2F3.     

MBNL1 is the primary determinant of focus formation and aberrant insulin receptor splicing in DM1

In myotonic dystrophy 1 (DM1), aggregation of the mutant DMPK RNA into RNA-protein complexes containing MBNL1 and MBNL2 has been linked to aberrant splicing of the insulin receptor (IR) RNA. In a parallel line of investigation, elevated levels of CUG-binding protein (CUG-BP) have been shown to result in altered IR splicing in DM1. The relative importance of MBNL1, MBNL2, and CUG-BP in DM1 pathogenesis is, however, unclear. Here we have demonstrated that either small interfering RNA-mediated down-regulation of MBNL1 and MBNL2 or the overexpression of CUG-BP in normal myoblasts results in abnormal IR splicing. Our results suggest that CUG-BP regulates the equilibrium of splice site selection by antagonizing the facilitatory activity of MBNL1 and MBNL2 on IR exon 11 splicing in a dose-dependent manner. We have shown that CUG-BP levels are elevated in DM1 cells by mechanisms that are independent of MBNL1 and MBNL2 loss. Importantly, rescue experiments in DM1 myoblasts demonstrated that loss of MBNL1 function is the key event, whereas the overexpression of CUG-BP plays a secondary role in the aberrant alternative splicing of IR RNA in DM1. Small interfering RNA-mediated down-regulation of MBNL1, MBNL2, and CUG-BP in DM1 myoblasts demonstrated that MBNL1 plays a critical role in the maintenance of DM1 focus integrity. Thus, these experiments demonstrate that sequestration of MBNL1 by the expanded CUG repeats is the primary determinant of both DM1 focus formation and the abnormal splicing of the IR RNA in DM1 myoblasts. The data therefore support MBNL1-mediated therapy for DM1.     

Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes. Evidence for TWEAK-independent functions of Fn14 during myogenesis

Fibroblast growth factor-inducible 14 (Fn14), distantly related to tumor necrosis factor receptor superfamily and a receptor for TWEAK cytokine, has been implicated in several biological responses. In this study, we have investigated the role of Fn14 in skeletal muscle formation in vitro. Flow cytometric and Western blot analysis revealed that Fn14 is highly expressed on myoblastic cell line C2C12 and mouse primary myoblasts. The expression of Fn14 was decreased upon differentiation of myoblasts into myotubes. Suppression of Fn14 expression using RNA interference inhibited the myotube formation in both C2C12 and primary myoblast cultures. Fn14 was required for the transactivation of skeletal alpha-actin promoter and the expression of specific muscle proteins such as myosin heavy chain fast type and creatine kinase. RNA interference-mediated knockdown of Fn14 receptor in C2C12 myoblasts decreased the levels of myogenic regulatory factors MyoD and myogenin upon induction of differentiation. Conversely, overexpression of MyoD increased differentiation in Fn14-knockdown C2C12 cultures. Suppression of Fn14 expression in C2C12 myoblasts also inhibited the differentiation-associated increase in the activity of serum response factor and RhoA GTPase. In addition, our data suggest that the role of Fn14 during myogenic differentiation could be independent of TWEAK cytokine. Collectively, our study suggests that the Fn14 receptor is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes.