Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products.

The development of muscle cells involves the action of myogenic determination factors. In this report, we show that human skeletal muscle tissue contains, besides the previously described Myf-5, two additional factors Myf-3 and Myf-4 which represent the human homologues of the rodent proteins MyoD1 and myogenin. The genes encoding Myf-3, Myf-4 and Myf-5 are located on human chromosomes 11, 1, and 12 respectively. Constitutive expression of a single factor is sufficient to convert mouse C3H 10T1/2 fibroblasts to phenotypically normal muscle cells. The myogenic conversion of 10T1/2 fibroblasts results in the activation of the endogenous MyoD1 and Myf-4 (myogenin) genes. This observation suggests that the expression of Myf proteins leads to positive autoregulation of the members of the Myf gene family. Individual myogenic colonies derived from MCA C115 cells (10T1/2 fibroblast transformed by methylcholanthrene) express various levels of endogenous MyoD1 mRNA ranging from nearly zero to high levels. The Myf-5 gene was generally not activated in 10T1/2 derived myogenic cell lines but was expressed in some MCA myoblasts. In primary human muscle cells Myf-3 and Myf-4 mRNA but very little Myf-5 mRNA is expressed. In mouse C2 and P2 muscle cell lines MyoD1 is abundantly synthesized together with myogenin. In contrast, the rat muscle lines L8 and L6 and the mouse BC3H1 cells express primarily myogenin and low levels of Myf-5 but no MyoD1. Myf-4 (myogenin) mRNA is present in all muscle cell lines at the onset of differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The levels of vascular smooth as well as skeletal muscle actin mRNAs differ substantially among both myoblast and fibroblast lines with different skeletal myogenic potentials.

Little is known about the factors which regulate vascular smooth muscle (vsm) actin gene expression during skeletal myogenesis in culture. We have therefore looked for differences in the levels of accumulation of vsm actin mRNA among six mouse cell lines differing in apparent myogenic potential or in the complement of myogenesis determination genes which they express: NIH 3T3 and 10T1/2 non-myogenic fibroblasts and four myogenic lines--3T3-MyoD1 and 10EMc11s, MyoD/myogenin expressing sublines of the fibroblast lines, derived by transfer into the parent lines of a MyoD cDNA expression construct; C2C12, which expresses all four known myogenesis determination genes; and BC3H1, which expresses myf-5, myogenin, little herculin, and no MyoD. In differentiated cells of all four myogenic lines, vsm actin mRNA was expressed at levels dramatically higher than in growth-arrested NIH 3T3 cells, consistent with expression of vsm actin mRNA as an intrinsic part of the skeletal myogenic program somehow directed by myogenesis determination gene products. Interestingly, however, the level of vsm actin mRNA in growth arrested C3H10T1/2 fibroblasts was also dramatically higher than that in NIH 3T3. In view of these findings, and of the relative ease with which 10T1/2 as opposed to NIH 3T3 cells can be converted to myogenic lines, we hypothesize that factors which can act to regulate vsm actin gene expression in the absence of myogenesis determination gene expression may also influence the skeletal myogenic potential of the cells in which they are found. Among the myogenic lines, the ratio of vsm to skm actin mRNA was highest in BC3H1 cells, raising the possibility that were these cells forced to express MyoD and/or more herculin, as do the other myogenic lines, the ratio would decrease. Thus both fibroblast and myogenic lines will be useful for investigating the mechanisms controlling skeletal myogenesis and vsm and skm actin gene expression during myogenesis.     

The levels of vascular smooth as well as skeletal muscle actin mRNAS differ substantially among both myoblast and fibroblast lines with different skeletal myogenic potentials.

Little is known about the factors which regulate vascular smooth muscle (vsm) actin gene expression during skeletal myogenesis in culture. We have therefore looked for differences in the levels of accumulation of vsm actin mRNA among six mouse cell lines differing in apparent myogenic potential or in the complement of myogenesis determination genes which they express: NIH 3T3 and 10T1/2 non-myogenic fibroblasts and four myogenic lines--3T3-MyoD1 and 10EMc11s, MyoD/myogenin expressing sublines of the fibroblast lines, derived by transfer into the parent lines of a MyoD cDNA expression construct; C2C12, which expresses all four known myogenesis determination genes; and BC3H1, which expresses myf-5, myogenin, little herculin, and no MyoD. In differentiated cells of all four myogenic lines, vsm actin mRNA was expressed at levels dramatically higher than in growth-arrested NIH 3T3 cells, consistent with expression of vsm actin mRNA as an intrinsic part of the skeletal myogenic program somehow directed by myogenesis determination gene products. Interestingly, however, the level of vsm actin mRNA in growth arrested C3H10T1/2 fibroblasts was also dramatically higher than that in NIH 3T3. In view of these findings, and of the relative ease with which 10T1/2 as opposed to NIH 3T3 cells can be converted to myogenic lines, we hypothesize that factors which can act to regulate vsm actin gene expression in the absence of myogenesis determination gene expression may also influence the skeletal myogenic potential of the cells in which they are found. Among the myogenic lines, the ratio of vsm to skm actin mRNA was highest in BC3H1 cells, raising the possibility that were these cells forced to express MyoD and/or more herculin, as do the other myogenic lines, the ratio would decrease. Thus both fibroblast and myogenic lines will be useful for investigating the mechanisms controlling skeletal myogenesis and vsm and skm actin gene expression during myogenesis.     

Characterization of myogenic cell lines derived by 5-azacytidine treatment

Three myogenic clonal cell lines were isolated from C3H 10T1/2 C18 cells (10T1/2) treated with 5-azacytidine (5-aza-CR). These lines reproducibly underwent fusion at confluence into functional myotubes capable of contracting in response to acetylcholine. The degree of fusion could be increased two- to threefold if the cells were grown on gelatin-coated dishes. All of the cell lines lost some of their myogenic potential after repeated passaging and the percentage of colonies capable of forming muscle was not increased by permissive media containing 2% horse serum. The 10T1/2 cells expressed only the BB form of creatine phosphokinase but all of the myogenic clones expressed additionally the MM and MB forms of the isozyme after fusion. The overall genomic level of 5-methylcytosine was decreased in some but not all of the cell clones tested. Comparisons between the 10T1/2 cells which never form muscle without 5-aza-CR treatment and clonal derivatives of committed cell types might be of value in understanding the molecular basis of the commitment process.     

The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation

A 60 amino acid domain of the myogenic determination gene MyoD is necessary and sufficient for sequence-specific DNA binding in vitro and myogenic conversion of transfected C3H10T1/2 cells. We show that a highly basic region, immediately upstream of the helix-loop-helix (HLH) oligomerization motif, is required for MyoD DNA binding in vitro. Replacing helix1, helix2, or the loop of MyoD with the analogous sequence of the Drosophila T4 achaete-scute protein (required for peripheral neurogenesis) has no substantial effect on DNA binding in vitro or muscle-specific gene activation in transfected C3H10T1/2 cells. However, replacing the basic region of MyoD with the analogous sequence of other HLH proteins (the immunoglobulin enhancer binding E12 protein or T4 achaete scute protein) allows DNA binding in vitro, yet abolishes muscle-specific gene activation. These findings suggest that a recognition code that determines muscle-specific gene activation lies within the MyoD basic region and that the capacity for specific DNA binding is insufficient to activate the muscle program.     

Myogenic lineage determination and differentiation: evidence for a regulatory gene pathway

Stable myogenic cell lines have been derived at a high frequency by transfection of a cloned multipotential mouse embryo cell line, C3H 10T1/2, with cloned human DNA linked to a selectable neomycin resistance gene. The myogenic phenotype remains linked to neomycin resistance during secondary transfections. Although proliferative in growth conditions, these cell lines maintain the ability to differentiate and express muscle-specific proteins. We conclude that there is a simple genetic basis for myogenic determination and that a single gene, myd, converts 10T1/2 cells to a myoblast lineage. Southern blot analysis demonstrates nonidentity of myd and the MyoD1 gene. Northern blot analysis shows that myd-transfected myogenic lineages express MyoD1 mRNA while parental 10T1/2 cells do not. These results suggest that a dependent regulatory gene pathway mediates myogenic determination and differentiation.     

M-Ras is activated by bone morphogenetic protein-2 and participates in osteoblastic determination, differentiation, and transdifferentiation

The small GTPase M-Ras is highly expressed in the central nervous system and plays essential roles in neuronal differentiation. However, its other cellular and physiological functions remain to be elucidated. Here, we clarify the novel functions of M-Ras in osteogenesis. M-Ras was prominently expressed in developing mouse bones particularly in osteoblasts and hypertrophic chondrocytes. Its expression was elevated in C3H/10T1/2 (10T1/2) mesenchymal cells and in MC3T3-E1 preosteoblasts during differentiation into osteoblasts. Treatment of C2C12 skeletal muscle myoblasts with bone morphogenetic protein-2 (BMP-2) to bring about transdifferentiation into osteoblasts also induced M-Ras mRNA and protein expression. Moreover, the BMP-2 treatment activated the M-Ras protein. Stable expression of the constitutively active M-Ras(G22V) in 10T1/2 cells facilitated osteoblast differentiation. M-Ras(G22V) also induced transdifferentiation of C2C12 cells into osteoblasts. In contrast, knockdown of endogenous M-Ras by RNAi interfered with osteoblast differentiation in 10T1/2 and MC3T3-E1 cells. Osteoblast differentiation in M-Ras(G22V)-expressing C2C12 cells was inhibited by treatment with inhibitors of p38 MAP kinase (MAPK) and c-Jun N-terminal kinase (JNK) but not by inhibitors of MAPK and ERK kinase (MEK) or phosphatidylinositol 3-kinase. These results imply that M-Ras, induced and activated by BMP-2 signaling, participates in the osteoblastic determination, differentiation, and transdifferentiation under p38 MAPK and JNK regulation.     

Transfection of cultured fish cells RBCF-1 with exogenous oncogene and their resistance to malignant transformation

1. Plasmids bearing the G418-resistant gene neo were transfected into cultured fish cells RBCF-1 by electroporation at an efficiency comparable to that in NIH3T3 cells. 2. Transfection of plasmids bearing both neo and activated human c-Ha-ras into NIH3T3 and RBCF-1 cells resulted in the malignant transformation of the former but not of the latter cells.     

Effect of myogenic and adipogenic differentiation on expression of colony-stimulating factor genes.

The influence of cellular differentiation on colony-stimulating factor gene expression was examined in myogenically and adipogenically determined cell lines derived from 5-azacytidine-treated C3H10T1/2 C18 (10T1/2) mouse embryo fibroblasts. These studies demonstrate that colony-stimulating factor gene expression can be modulated by myogenic and adipogenic determination and terminal differentiation.     

The homeodomain protein Barx2 promotes myogenic differentiation and is regulated by myogenic regulatory factors

The homeobox protein Barx2 is expressed in both smooth and skeletal muscle and is up-regulated during differentiation of skeletal myotubes. Here we use antisense-oligonucleotide inhibition of Barx2 expression in limb bud cell culture to show that Barx2 is required for myotube formation. Moreover, overexpression of Barx2 accelerates the fusion of MyoD-positive limb bud cells and C2C12 myoblasts. However, overexpression of Barx2 does not induce ectopic MyoD expression in either limb bud cultures or in multipotent C3H10T1/2 mesenchymal cells, and does not induce fusion of C3H10T1/2 cells. These results suggest that Barx2 acts downstream of MyoD. To test this hypothesis, we isolated the Barx2 gene promoter and identified DNA regulatory elements that might control Barx2 expression during myogenesis. The proximal promoter of the Barx2 gene contained binding sites for several factors involved in myoblast differentiation including MyoD, myogenin, serum response factor, and myocyte enhancer factor 2. Co-transfection experiments showed that binding sites for both MyoD and serum response factor are necessary for activation of the promoter by MyoD and myogenin. Taken together, these studies indicate that Barx2 is a key regulator of myogenic differentiation that acts downstream of muscle regulatory factors.     

Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle.

We have used transient transfections in MM14 skeletal muscle cells, newborn rat primary ventricular myocardiocytes, and nonmuscle cells to characterize regulatory elements of the mouse muscle creatine kinase (MCK) gene. Deletion analysis of MCK 5'-flanking sequence reveals a striated muscle-specific, positive regulatory region between -1256 and -1020. A 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment confers high-level expression in skeletal myocytes but is inactive in myocardiocytes, indicating that skeletal and cardiac muscle MCK regulatory sites are distinguishable. To further delineate muscle regulatory sequences, we tested six sites within the MCK enhancer for their functional importance. Mutations at five sites decrease expression in skeletal muscle, cardiac muscle, and nonmuscle cells. Mutations at two of these sites, Left E box and MEF2, cause similar decreases in all three cell types. Mutations at three sites have larger effects in muscle than nonmuscle cells; an A/T-rich site mutation has a pronounced effect in both striated muscle types, mutations at the MEF1 (Right E-box) site are relatively specific to expression in skeletal muscle, and mutations at the CArG site are relatively specific to expression in cardiac muscle. Changes at the AP2 site tend to increase expression in muscle cells but decrease it in nonmuscle cells. In contrast to reports involving cotransfection of 10T1/2 cells with plasmids expressing the myogenic determination factor MyoD, we show that the skeletal myocyte activity of multimerized MEF1 sites is 30-fold lower than that of the 206-bp enhancer. Thus, MyoD binding sites alone are not sufficient for high-level expression in skeletal myocytes containing endogenous levels of MyoD and other myogenic determination factors.     

JAK2/STAT2/STAT3 are required for myogenic differentiation

Skeletal muscle satellite cell-derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced regeneration. However, the cellular signaling pathways that control proliferation and differentiation of myoblasts remain poorly defined. Recently, we found that JAK1/STAT1/STAT3 not only participate in myoblast proliferation but also actively prevent them from premature differentiation. Unexpectedly, we found that a related pathway consisting of JAK2, STAT2, and STAT3 is required for early myogenic differentiation. Interference of this pathway by either a small molecule inhibitor or small interfering RNA inhibits myogenic differentiation. Consistently, all three molecules are activated upon differentiation. The pro-differentiation effect of JAK2/STAT2/STAT3 is partially mediated by MyoD and MEF2. Interestingly, the expression of the IGF2 gene and the HGF gene is also regulated by JAK2/STAT2/STAT3, suggesting that this pathway could also promote differentiation by regulating signaling molecules known to be involved in myogenic differentiation. In summary, our current study reveals a novel role for the JAK2/STAT2/STAT3 pathway in myogenic differentiation.