Identification of a regulatory function for an orphan receptor in muscle: COUP-TF II affects the expression of the myoD gene family during myogenesis.

COUP-TF II is an 'orphan steroid receptor' that binds a wide variety of AGGTCA repeats and represses thyroid hormone (T3) and retinoid dependent trans-activation; however, very little is known of its functional and/or developmental role during mammalian cell differentiation. T3 and retinoids have been demonstrated to promote terminal muscle differentiation via activation of the muscle specific myoD gene family (myoD, myogenin, myf-5 and MRF-4). The myoD gene family can direct the fate of mesodermal cell lineages, repress proliferation, activate differentiation and the contractile phenotype. Hence, we investigated the expression and functional role of COUP-TF II during muscle differentiation. Proliferating C2C12 myoblasts expressed COUP-TF II mRNA which was repressed when cells were induced to differentiate into post-mitotic multinucleated myotubes by serum withdrawal. Concomitant with the decrease of COUP-TF II mRNA was the appearance of muscle specific mRNAs (e.g. myogenin, alpha-actin). We show that Escherichia coli expressed full length and truncated COUP-TF II bound in a sequence specific manner to the T3 response elements (TREs) in the myoD and myogenin regulatory HLH genes [Olson (1992) Dev. Biol. 154, 261-272]; and the TRE in the skeletal alpha-actin contractile protein gene. COUP-TF II diminished the homodimeric binding of the thyroid hormone receptor and the heterodimeric binding of thyroid hormone and retinoid X receptor complexes to these TREs. Constitutive over-expression of COUP-TF II cDNA in mouse C2C12 myogenic cells suppressed the levels of myoD mRNA and blocked the induction of myogenin mRNA, whereas constitutive expression of anti-sense COUP-TF II cDNA significantly increased the steady state levels of myoD mRNA and hyper-induced myogenin mRNA. These studies demonstrate for the first time (i) that COUP-TF II, functions as a physiologically relevant antagonistic regulator of myogenesis via direct effects on the myoD gene family and (ii) direct evidence for the developmental role of COUP-TF II during mammalian cell differentiation.     

Stac3 Inhibits Myoblast Differentiation into Myotubes

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.     

The multiple ADP/ATP translocase genes are differentially expressed during human muscle development.

The expression of the genes encoding the three isoforms of the human ADP/ATP translocase (T1, T2, and T3) has been analyzed at different stages of myogenic differentiation in an in vitro muscle cell system and compared with that in mature muscle. The results indicate that the three stages of muscle differentiation corresponding to myoblast proliferation, myotube formation, and mature muscle fibers are characterized by a different pattern of expression of the ADP/ATP translocase genes. In particular, the two T2-specific mRNAs are present at high, similar levels in myoblasts and myotubes and markedly decrease in amount in mature adult muscle. By contrast, the T3-specific mRNA is present in high amount in growing myoblasts, decreases markedly in myotubes, and is barely detectable in adult muscle. Finally, the T1-specific mRNA is present at a high level in adult muscle and is not detectable in either myoblasts or myotubes. Therefore, T1 gene expression appears to be a marker of a late stage in myogenesis. A parallel investigation of expression of the myosin heavy chain mRNA revealed absence of hybridization with the specific probe in RNA from proliferating myoblasts, a significant hybridization in myotube RNA, and a strong signal in adult muscle RNA.     

Effect of sodium butyrate on gene expression in a rat myogenic cell line

Sodium butyrate, when added in millimolar concentration to a culture of myoblasts of the L6 cell line, inhibits reversibly cell proliferation and differentiation. In the present work, we have studied the effect of Na butyrate on the translational efficiency of the overall poly (A)+ RNA. The mRNA from treated cells was translated in vitro as efficiently as proliferating myoblasts mRNA, while a decrease of translation efficiency was observed with myotubes mRNA. In addition this RNA directs the synthesis of several new polypeptides. on the switch on of alpha actin and myosin heavy chains (MHC), muscle specific genes by the dot blot and Northern blot techniques using cloned probes. Na butyrate prevented the expression of MHC and allowed the switch on of alpha actin gene but at a lesser extent than in normal myotubes. In addition the drug prevented the translocation of alpha actin mRNA into the cytoplasm.     

Nucleotide sequence and hormonal regulation of mouse glycerophosphate dehydrogenase mRNA during adipocyte and muscle cell differentiation

We have studied the structure and regulation of glycerophosphate dehydrogenase (GPD) mRNA during mouse adipocyte and muscle cell differentiation. This message has a size of 2.8 kilobases that includes a coding segment of 1050 bases and a large untranslated 3' end of about 1700 bases. There is a high degree of amino acid homology (91%) between the mouse adipocyte and rabbit muscle GPD proteins. GPD mRNA is not detected in myoblasts, but, as in adipogenesis, it is expressed upon differentiation into myotubes. The modulation of GPD mRNA by cyclic AMP analogues and tumor necrosis factor has been examined in both adipocytes and myotubes. Dibutyryl cAMP or 8-bromo-cAMP causes a large reduction of GPD mRNA levels in both cell types, with less than 20% remaining after 18 h of treatment. Tumor necrosis factor effects a dramatic and rapid reduction in GPD mRNA in fat cells and a slower but significant decrease in the level of this mRNA in muscle cells. These results indicate that GPD gene expression is linked to cell differentiation in both fat and muscle cells, and suggest certain similarities in hormonal modulation in both cell types.     

Role of cell division in differentiation of myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus.

The relationship between a potential requirement for cell DNA synthesis and the expression of differentiated muscle cell functions was investigated using primary chicken embryo myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus (RSV). Under optimized conditions, transformed myoblasts growing at the permissive temperature could differentiate into multinucleated myotubes, express muscle-specific myosin, desmin and acetylcholine receptors in the absence of DNA synthesis and cell division following a shift to the non-permissive temperature. Furthermore, the experiments demonstrate that individual RSV-infected myoblasts have two options: either to divide and express the transformed phenotype or to withdraw from the cell cycle and differentiate into myotubes. The choice between these options appears to depend on the protein-kinase activity of pp60src, the src gene product.     

Coexpression of α-sarcomeric actin, α-smooth muscle actin and desmin during myogenesis in rat and mouse embryos I. Skeletal muscle

Expression of vimentin, desmin, alpha-sarcomeric and alpha-smooth muscle actins in embryonic tissues of rat and mice was examined using an immunohistochemical approach. The results showed a similarity in the expression of desmin and alpha-actin isoforms (alpha-sr and alpha-sm) in skeletal muscle cells during murine feto-embryonic development. In the two species, coexpression of alpha-sr and alpha-sm actins has been observed in cardiomyoblasts, myotomal myoblasts and myotubes. The intensity of alpha-sm actin expression decreased during the terminal steps of myogenesis and disappeared completely in mature cardiomyocytes and myofibres. Desmin was expressed in all prefusion myoblasts (type 1 and 2 myoblasts), myotubes, and in myofibres. The appearance of desmin in myoblasts of somites preceded by a few hours the expression of the alpha-actins (alpha-sr and alpha-sm). Our study on vimentin expression, limited to rat embryos, revealed that somite premyoblasts expressed only vimentin, type 1 myoblasts expressed vimentin and desmin, and type 2 myoblasts (rhabdomyoblasts) expressed desmin and alpha-actins (alpha-sr and alpha-sm). Our study demonstrates the resemblance between feto-embryonic myogenesis and myogenic neoplastic differentiation: desmin appears before the alpha-actins in embryonic myoblasts, and can be considered as a marker of an initial step in myogenic differentiation. alpha-sm actin, considered as a striated muscle cell feto-embryonic actin, is expressed transiently in skeletal myoblasts and cardiomyoblasts during development and reappears during neoplastic transformation of skeletal muscle.     

Effects of dimethyl sulphoxide and dexamethasone on mRNA expression of myogenesis- and muscle proteolytic system-related genes in mouse myoblastic C2C12 cells

We examined the time course of mRNA expression of myogenic cell differentiation- and muscle proteolytic system-related genes in cultures of C2C12 cells during differentiation from myoblasts to myotubes. Furthermore, we treated C2C12 myotubes with dimethyl sulphoxide (DMSO) and dexamethasone (Dex), and examined changes in these mRNA levels. Myogenin (Myog), Atrogin1, forkhead box O1 (Foxo1) and Capn1 mRNA levels increased in C2C12 cells differentiating from myoblasts to myotubes, whereas Myf5 mRNA levels decreased. Although genes such as MRF4, Foxo3a, UbB, Capn1 and MuRF1 mRNAs in the myotubes were affected by DMSO exposure, mRNA levels of other genes were not markedly affected by exposure to 0.02% or 0.5% DMSO. Myf5, MRF4, Atrogin1, Foxo3 and MuRF1 mRNA levels were elevated by Dex at all time points, Cbl and Capn1 mRNA levels were significantly elevated by Dex at 8 h, and Myog mRNA levels were significantly elevated by Dex at 24 h. However, CtsH mRNA levels decreased significantly with Dex at 24 h. This study provides a useful database of gene profiles that are differentially expressed throughout myogenic cell differentiation and the muscle proteolytic system.     

Phosphofructokinase isozyme expression during myoblast differentiation

Isozyme expression of phosphofructokinase (PFK), the key regulatory enzyme for glycolysis, was studied during differentiation of mouse C2 myoblasts to myotubes. The total PFK activity increased 20-fold during in vitro myogenesis. The rate of synthesis, relative to the rate of total protein synthesis, measured by pulse labeling and immunoprecipitation was lowest for muscle PFK (PFK-A), 0.008% in myoblasts, while those for liver (PFK-B) and brain (PFK-C) PFK were 0.017 and 0.014%, respectively. The relative rate of PFK-A synthesis increased sharply (5-fold) at an initial period of differentiation (8 h) and reached maximum of 10-fold at 48 h, to make PFK-A the major isoform synthesized in myotubes. The relative rates of synthesis for both PFK-B and PFK-C did not change drastically, decreasing slightly at 8 h, but were restored to 1.5-2-fold of myoblasts. cDNA sequences coding for mouse muscle PFK were cloned and used along with those for mouse liver PFK, which we have previously cloned, to measure by Northern blot analysis under highly stringent conditions the steady-state mRNA concentrations for muscle and liver PFK during C2 differentiation. The hybridizable mRNA level for PFK-A increased gradually, reaching 13-fold at 48 h when 80% of cells was fused to myotubes. The PFK-A mRNA level at 96 h was 90-fold of that for myoblasts. In contrast, the mRNA level for PFK-B increased slightly during differentiation, showing a maximum of 4-fold at 96 h. These results indicate isozyme-specific control of muscle PFK gene expression during C2 myoblast differentiation.