Role In Skeletal Muscle Development Research Articles

Reciprocal Inhibition between MyoD and STAT3 in the Regulation of Growth and Differentiation of Myoblasts

The development of myoblasts is regulated by various growth factors as well as by intrinsic muscle-specific transcriptional factors. In this study, we analyzed the roles for STAT3 in the growth and differentiation of myoblasts in terms of cell cycle regulation and interaction with MyoD using C2C12 cells. Here we found that STAT3 inhibited myogenic differentiation induced by low serum or MyoD as efficiently as the Ras/mitogen-activated protein kinase cascade. As for this mechanism, we found that STAT3 not only promoted cell cycle progression through the induction of c-myc but also inhibited MyoD activities through direct interaction. STAT3 inhibited not only DNA binding activities of MyoD but also its transcriptional activities. However, the inhibited transcriptional activities were restored by the supplement of p300/CBP and PCAF, suggesting that STAT3 might deprive MyoD of these transcriptional cofactors. In addition, we found that MyoD inhibited DNA binding activities of STAT3, thereby inhibiting STAT3-dependent cell growth and survival of Ba/F3 cells. These results suggest that the development of muscle cells is regulated by the coordination of cytokine signals and intrinsic transcription factors.

Deletion of vitamin D receptor gene in mice results in abnormal skeletal muscle development with deregulated expression of myoregulatory transcription factors.

Although rachitic/osteomalacic myopathy caused by impaired vitamin D actions has long been described, the molecular pathogenesis remains elusive. To determine physiological roles of vitamin D actions through vitamin D receptor (VDR) in skeletal muscle development, we examined skeletal muscle in VDR gene deleted (VDR -/-) mice, an animal model of vitamin D-dependent rickets type II, for morphological changes and expression of myoregulatory transcription factors and myosin heavy chain isoforms. We found that each muscle fiber was small and variable in size in hindlimb skeletal muscle from VDR -/- mice, although overall myocyte differentiation occurred normally. These abnormalities were independent of secondary metabolic changes such as hypocalcemia and hypophosphatemia, and were accompanied by aberrantly high and persistent expression of myf5, myogenin, E2A, and early myosin heavy chain isoforms, which are normally down-regulated at earlier stages. Moreover, treatment of VDR-positive myoblastic cells with 1,25(OH)2D3 in vitro caused down-regulation of these factors. These results suggest that VDR plays a physiological role in skeletal muscle development, participating in temporally strict down-regulation of myoregulatory transcription factors. The present study can form a molecular basis of VDR actions on muscle and should help further establish the physiological roles of VDR in muscle development as well as pharmacological effects of vitamin D on muscle functions.

Myf-5 Is Transiently Expressed in Nonmuscle Mesoderm and Exhibits Dynamic Regional Changes within the Presegmented Mesoderm and Somites I–IV

Myf-5 is one of four myogenic regulatory factors that play important roles in skeletal muscle development. This study provides detailed analysis of Myf-5 expression during early chick development using an in situ hybridization technique that has been optimized to detect low level Myf-5 transcripts. This facilitated detection of heretofore unrecognized dynamic changes in Myf-5 expression patterns. Myf-5 expression is first detected at stage 3 in the primitive streak and exhibits transient low-level expression in nonmyogenic mesoderm. Myf-5 is later expressed in the presegmented mesoderm (psm) in a reiterating pattern that is coordinated with somitogenesis and also colocalizes with the Notch ligand C-Delta-1. In somites (S) I–IV, Myf-5 expression exhibits dynamic regional changes, and in somites rostral to S IV, Myf-5 is expressed at higher levels in muscle precursors in the dorsomedial somite. Semiquantitative comparison of Myf-5 mRNA levels in the psm and in myotome-containing somites indicates about a 10-fold difference. The expression pattern of Myf-5 differs from that of MyoD, which we find is expressed only in the dorsomedial somite. These data reveal that Myf-5 is expressed at low levels several stages before muscle differentiation occurs and suggest that only a subset of cells that initially express Myf-5 will upregulate its expression and differentiate as muscle.

Blocking gap junctional intercellular communication in myoblasts inhibits myogenin and MRF4 expression.

Cells rely heavily on cues from their extracellular environment and other cells to coordinate normal physiological processes, and the exchange of molecules via gap junctions has been suggested as on important avenue for cell-cell communication. Gap junctions are found in virtually all mammalian tissues with the notable exception of adult skeletal muscle. However, since functional gap junctions have been detected during the early stages of muscle development, gap junctional intercellular communication (GJIC) may play on important role in myogenesis. In this study, GJIC in normal 16 myoblasts was inhibited using the known blockers l-octanol and beta-glycyrrhetinic acid (beta-GA). Under differentiation promoting conditions, 16 cells fused to form multinucleated myotubes, but when treated with either octanol or beta-GA, no fusion was observed. The expression of two muscle regulatory factors (MRFs), myogenin and MRF4, was examined in both the blocked and control cells. As expected, the activation of both the myogenin and MRF4 genes coincided with the onset of differentiation in the control 16 cells. Neither of these genes were turned on in the blocked cells, even when grown under low serum conditions. This inhibition of differentiation by octanol and beta-GA was reversible, since the activation of both MRF genes as well as myoblast fusion were observed when the blocking medium was replaced with normal differentiating medium. These results suggest that intercellular communication via gap junctions plays an important role in skeletal muscle development and perhaps in the cell signaling events that trigger the activation of muscle-specific MRF genes.

Functional redundancy of the muscle-specific transcription factors Myf5 and myogenin.

The myogenic basic helix-loop-helix transcription factors, Myf5, MyoD, myogenin and MRF4, play key roles in skeletal muscle development. All of them induce myogenic differentiation in cultured non-muscle cells, suggesting that they might be functionally redundant. But the genes are expressed at different times during embryogenesis and mice carrying a mutation in any of the genes have different phenotypes. A rib cage defect was observed in Myf5-deficient mice, which die perinatally. We investigated whether the rib cage defect was due to the failure of the early activation of the gene or to the unique interactions of Myf5 with specific downstream targets. For this we inserted a myogenin complementary DNA into the Myf5 locus by homologous recombination which simultaneously disrupted Myf5 function. We report here that mice homozygous for this myogenin gene knock-in (ki) developed a normal rib cage and were viable, therefore demonstrating functional redundancy of Myf5 and myogenin for rib formation.