EGF responsiveness and receptor regulation in normal and differentiation-defective mouse myoblasts

The interrelationship between cell proliferation and terminal myogenic differentiation has been analyzed by studying a differentiation-defective subclone (DD-1) of the permanent mouse myoblast line MM14. Parental MM14 myoblasts withdraw irreversibly from the cell cycle and initiate terminal differentiation when they are deprived of certain mitogens. In contrast, DD-1 cells become quiescent in a mitogen-depleted environment and less than 0.4% of the cells differentiate. When refed with mitogen-rich medium quiescent DD-1 cells resume proliferation. Expression of this differentiation-defective phenotype is apparently coupled to an alteration in mitogen sensitivity: MM14 myoblasts require horse serum plus either chick embryo extract or fibroblast growth factor (FGF) to sustain cell growth: DD-1 variants are responsive to FGF, but also proliferate in response to serum alone or to reduced serum plus epidermal growth factor (EGF). Interestingly, EGF also appears to retard DD-1 cell differentiation in a manner similar to the FGF repression of differentiation in normal myoblasts. Normal and differentiation-defective myoblasts which have been maintained under growth-promoting conditions exhibit similar EGF binding, internalization, and degradation. However, whereas the EGF binding capacity of MM14 myoblasts declines to less than 5% of its initial level within 24 hr of FGF removal, DD-1 variants exhibit an increase in EGF binding when switched to an FGF-depleted medium. The relationship of altered EGF receptor regulation to changes in mitogen sensitivity and differentiation capacity of the DD-1 variant is discussed, and implications for general in vivo processes governing cell proliferation and differentiation are considered.     

Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor

Analysis of MM14 mouse myoblasts demonstrates that terminal differentiation is repressed by pure preparations of both acidic and basic fibroblast growth factor (FGF). Basic FGF is approximately 30-fold more potent than acidic FGF and it exhibits half maximal activity in clonal assays at 0.03 ng/ml (2 pM). FGF repression occurs only during the G1 phase of the cell cycle by a mechanism that appears to be independent of ongoing cell proliferation. When exponentially growing myoblasts are deprived of FGF, cells become postmitotic within 2-3 h, express muscle-specific proteins within 6-7 h, and commence fusion within 12-14 h. Although expression of these three terminal differentiation phenotypes occurs at different times, all are initiated by a single regulatory "commitment" event in G1. The entire population commits to terminal differentiation within 12.5 h of FGF removal as all cells complete the cell cycle and move into G1. Differentiation does not require a new round of DNA synthesis. Comparison of MM14 behavior with other myoblast types suggests a general model for skeletal muscle development in which specific growth factors serve the dual role of stimulating myoblast proliferation and directly repressing terminal differentiation.     

Triiodothyronine influences quail myoblast proliferation and differentiation*

The influence of triiodothyronine (T3) on avian myoblast proliferation and differentiation was studied in secondary cultures using plating densities of 2500 and 7000 cells/cm². Culture media were depleted of T3 (control myoblasts) and increasing amounts were then added to concentrations of 0.6, 3 and 15 nM T3 (treated myoblasts). Independent of the cell density, T3 induced a dose-related decrease in myoblast proliferation measured by cell number, doubling time and ³H-thymidine incorporation. However, with the lower plating density, this influence was delayed, occurring only after the third day of culture for 0.6 nM T3-treated myoblasts and simultaneous with the onset of myosin heavy chain accumulation. Moreover, when myoblasts were exposed to BrdU for 48 h, the T3 growth inhibitory effect disappeared, thus showing that this effect was clearly linked to differentiation. In addition, we have shown that T3 induced an early fusion of myoblasts: 65% of the maximal value of the fusion index was reached on day 3 in the T3-treated cells in comparison to 25% in the control myoblasts. This hormone also enhanced accumulation of muscle-specific proteins (connectin, acetylcholine receptors, myosin heavy chain), tested by cytoimmunofluorescence, ELISA, binding experiments and Western blot. All these results show that T3 increased myoblast differentiation through a pathway including myoblast withdrawal from the cell cycle. The influence of T3 could partly explain its previously reported positive effect on the number of muscle fibers.     

Fine regulation of RhoA and Rock is required for skeletal muscle differentiation

The RhoA GTPase controls a variety of cell functions such as cell motility, cell growth, and gene expression. Previous studies suggested that RhoA mediates signaling inputs that promote skeletal myogenic differentiation. We show here that levels and activity of RhoA protein are down-regulated in both primary avian myoblasts and mouse satellite cells undergoing differentiation, suggesting that a fine regulation of this GTPase is required. In addition, ectopic expression of activated RhoA in primary quail myocytes, but not in mouse myocytes, inhibits accumulation of muscle-specific proteins and cell fusion. By disrupting RhoA signaling with specific inhibitors, we have shown that this GTPase, although required for cell identity in proliferating myoblasts, is not essential for commitment to terminal differentiation and muscle gene expression. Ectopic expression of an activated form of its downstream effector, Rock, impairs differentiation of both avian and mouse myoblasts. Conversely, Rock inhibition with specific inhibitors and small interfering RNA-mediated gene silencing leads to accelerated progression in the lineage and enhanced cell fusion, underscoring a negative regulatory function of Rock in myogenesis. Finally, we have reported that Rock acts independently from RhoA in preventing myoblast exit from the cell cycle and commitment to differentiation and may receive signaling inputs from Raf-1 kinase.     

Anchorage-dependent control of muscle-specific gene expression in C2C12 mouse myoblasts

Summary Our previous studies have demonstrated that expression of growth-associated genes is regulated by the adhesive state of the cell. To understand the role of cell adhesion in regulating the switch from growth to differentiation, we are studying the differentiation of mouse myoblasts into multinucleated contractile myotubes. In this report, we describe a novel means of culturing C₂C₁₂ myoblasts that permits an analysis of the role of cell adhesion in regulating the sequential induction of muscle-specific genes that control myogenesis. Suspension of an asynchronous, proliferating population of myoblasts in a viscous gel of methylcellulose dissolved in medium containing 20% serum induces growth arrest in G₀ phase of the cell cycle without a concomitant induction of muscle-specific genes. Reattachment to a solid substratum in 20% serum, 0.5nM bFGF, or 10 nM IGF-1 rapidly activates entry of the quiescent cells into G₁ followed by a synchronous progression of the cell population through into S phase. bFGF or IGF-1 added separately facilitate only one passage through the cell cycle, whereas 20% serum or the two growth factors added together support multiple cell divisions. Adhesion of suspended cells in DMEM alone or with 3 nM IGF-1 induces myogenesis as evidenced by the synthesis of myogenin and myosin heavy chain (MHC) proteins followed by fusion into myotubes. bFGF completely inhibits this differentiation process even in the presence of myogenic doses of IGF-1. Addition of 3 nM IGF-1 to quiescent myoblasts maintained in suspension culture in serum-free conditions does not induce myogenin or MHC expression. Thus, adhesion is a requirement for the induction of muscle gene expression in mouse myoblasts. The development of a muscle cell culture environment in which proliferating myoblasts can be growth arrested in G₀ without activating muscle-specific gene expression provides a means of analyzing the synchronous activation of either the myogenic or growth programs and how adhesion affects each process, respectively. Supported by training grant T32-HL07035     

Manipulation of myogenesis in vitro: Reversible inhibition by DMSO

A system has been developed for the detailed analysis of the transition from proliferative myoblast to differentiated muscle cell. Dimethylsulfoxide (DMSO) prevents the terminal differentiation of L8 myoblasts in vitro, and its effect is reversible. DMSO (2%) inhibits the fusion of myoblasts to form multinucleate myotubes, the normal increases in activity of creatine phosphokinase (CPK) and acetylcholinesterase, and the synthesis of α-actin and acetylcholine receptor protein. Upon removal of DMSO from the medium, a lag precedes the onset of differentiation. The potential to inhibit muscle differentiation reversibly is not specific to DMSO, but is shared by a number of compounds, including dimethylformamide, hexamethylbisacetamide and butyric acid, all potent inducers of gene expression in Friend erythroleukemia cells. L8 cells routinely cease DNA synthesis and initiate fusion and muscle protein synthesis once they are confluent. In the presence of DMSO, however, nearly all cells continue DNA synthesis, even several days after reaching confluence. Protein synthetic patterns of DMSO-inhibited cells are almost indistinguishable from those of untreated myoblasts and distinct from differentiated myotubes. It appears that cells exposed to DMSO are locked indefinitely in a proliferative myoblast stage of development and are unable to enter the G₀ phase of the cell cycle necessary for initiation of differentiation. DMSO coordinately inhibits all the differentiative parameters measured. In contrast, cytochalasin B uncouples normally linked differentiative events so that fusion is inhibited while muscle-specific protein synthesis proceeds. DMSO has similar effects on both cytochalasin B-treated and fusing control cultures, suggesting that its primary effect is exerted not at the level of fusion but earlier in the differentiative timetable. Once fusion and the synthesis of muscle-specific proteins are well under way, the addition of DMSO is ineffective and differentiation continues in its presence. The potential to manipulate muscle gene expression in vitro makes this system particularly useful for the detailed analysis of the processes involved in the transition to the differentiated state and for determining the linkage of developmental events.     

Myogenic differentiation in permanent clonal mouse myoblast cell lines: Regulation by macromolecular growth factors in the culture medium

A permanent clonal cell line of mouse myoblasts (MM14) has been used to study the transition from proliferation to terminal differentiation. Results indicate that the transition is strictly dependent on the culture medium environment. Evidence from clonal density cultures suggests that (1) specific macromolecular mitogenic components of the culture medium stimulate mouse myoblast proliferation and prevent differentiation, (2) mouse myoblasts eliminate mitogenic activity from the culture medium before differentiating, and (3) lowered activity of specific mitogens stops mouse myoblast proliferation and triggers the program of terminal differentiation leading to the elaboration of muscle specific gene products and formation of myotubes. Evidence for the regulatory role of specific mitogens is the stimulation of proliferation and delay of differentiation by the addition of nanomolar concentrations of fibroblast growth factor to mitogen-depleted, differentiation-promoting, culture medium, whereas the addition of other purified mitogens has no effect. The results support and extend evidence from other muscle culture systems that stimulation of proliferation delays myoblast differentiation, and they provide an experimental basis for controlling the synchronous differentiation of pure populations of clonally derived mouse myoblasts.     

Quantal Division and a Postmitotic State in Myoblast Differentiation

The reversible arrest of myoblast differentiation by ethidium bromide (EB) has been used to examine the nature of the transition from the proliferative state to terminal differentiation resulting in fusion into muscle fibers. If EB is introduced at the time that myoblasts are shifted to medium that induces fusion, all apparent cytodifferentiation is suspended. When such EB arrested myoblasts are released from EB inhibition they fuse without reentering the cell cycle. If EB arrested myoblasts are released into proliferation promoting medium rather than medium that induces fusion they neither fuse nor proliferate. In this case they remain quiescent in the proliferating medium for an extended period, however, if these myoblasts are subsequently shifted to medium that induces fusion, they fuse without reentering the cell cycle. Apparently the myoblasts have become postmitotic and competent to fuse into muscle fibers during their initial exposure to fusion inducing medium, even though cytodifferentiation has been blocked. Exposure of these postmitotic fusion competent myoblasts to proliferation promoting medium does not stimulate them to reenter the cell cycle but does prevent fusion into muscle fibers. These results are most consistent with a quantal division model of myoblast differentiation rather than a gradual transition from the proliferative state to a state in which fusion occurs.     

Quantal division and a postmitotic state in myoblast differentiation.

The reversible arrest of myoblast differentiation by ethidium bromide (EB) has been used to examine the nature of the transition from the proliferative state to terminal differentiation resulting in fusion into muscle fibers. If EB is introduced at the time that myoblasts are shifted to medium that induces fusion, all apparent cytodifferentiation is suspended. When such EB arrested myoblasts are released from EB inhibition they fuse without reentering the cell cycle. If EB arrested myoblasts are released into proliferation promoting medium rather than medium that induces fusion they neither fuse nor proliferate. In this case they remain quiescent in the proliferating medium for an extended period, however, if these myoblasts are subsequently shifted to medium that induces fusion, they fuse without reentering the cell cycle. Apparently the myoblasts have become postmitotic and competent to fuse into muscle fibers during their initial exposure to fusion inducing medium, even though cytodifferentiation has been blocked. Exposure of these postmitotic fusion competent myoblasts to proliferation promoting medium does not stimulate them to reenter the cell cycle but does prevent fusion into muscle fibers. These results are most consistent with a quantal division model of myoblast differentiation rather than a gradual transition from the proliferative state to a state in which fusion occurs.     

Viral transformation of rat myoblasts: effects on fusion and surface properties.

Lines of rat myoblasts infected by avian sarcoma viruses have been isolated, cloned, and used to study the effects of viral transformation on myogenic differentiation and the surface changes associated with differentiation. The lines transformed by sarcoma viruses failed to fuse into myotubes and did not show the increase in myosin synthesis normally associated with fusion. The parental nontransformed line showed, subsequent to fusion, a surface alteration detectable by external labeling methods. This alteration, an increase in the level of an external protein of MW > 200 × 10³, is similar to that observed in fibroblasts arrested in the G₁ phase of the cell cycle. This protein was absent or greatly reduced on the surfaces of the myoblast lines that had been transformed by sarcoma viruses. Therefore, viral transformation causes loss of several properties normally associated with arrest of myoblasts in G₁.     

Regulation of creatine kinase induction in differentiating mouse myoblasts.

The regulation of creatine kinase (CK) induction during muscle differentiation was analyzed with MM14 mouse myoblasts. These cells withdraw from the cell cycle and commit to terminal differentiation when fed with mitogen-depleted medium. Myoblasts contained trace amounts of an isozyme of brain CK (designated BB-CK), but differentiation was accompanied by the induction of two other isozymes of muscle and brain CKs (designated MM-CK and MB-CK). Increased CK activity was detectable within 6 h of mitogen removal, 3 h after the first cells committed to differentiation and 6 h before fusion began. By 48 h, MM-CK activity increased more than 400-fold, MB-CK activity increased more than 150-fold, and BB-CK activity increased more than 10-fold. Antibodies prepared against purified mouse MM-CK cross-reacted with muscle and brain CKs (designated M-CK and B-CK, respectively) from a variety of species and were used to demonstrate that the increase in enzymatic activity was paralleled by an increase in the protein itself. CK antibodies were also used to aid in identifying cDNA clones to M-CK. cDNA sequences which corresponded to protein-coding regions cross-hybridized with B-CK mRNA; however, a subclone containing the 3'-nontranslated region was unique and was used to quantitate M-CK mRNA levels during myoblast differentiation. M-CK mRNA was not detectable in myoblasts, but within 5 to 6 h of mitogen withdrawal (6 to 7 h before fusion begins) it accumulated to about 30 molecules per cell. By 24 h, myotubes contained approximately 1,100 molecules per nucleus of M-CK mRNA.     

c-Myb Inhibits Myoblast Fusion

Satellite cells represent a heterogeneous population of stem and progenitor cells responsible for muscle growth, repair and regeneration. We investigated whether c-Myb could play a role in satellite cell biology because our previous results using satellite cell-derived mouse myoblast cell line C2C12 showed that c-Myb was expressed in growing cells and downregulated during differentiation. We detected c-Myb expression in activated satellite cells of regenerating muscle. c-Myb was also discovered in activated satellite cells associated with isolated viable myofiber and in descendants of activated satellite cells, proliferating myoblasts. However, no c-Myb expression was detected in multinucleated myotubes originated from fusing myoblasts. The constitutive expression of c-Myb lacking the 3′ untranslated region (3′ UTR) strongly inhibited the ability of myoblasts to fuse. The inhibition was dependent on intact c-Myb transactivation domain as myoblasts expressing mutated c-Myb in transactivation domain were able to fuse. The absence of 3′ UTR of c-Myb was also important because the expression of c-Myb coding region with its 3′ UTR did not inhibit myoblast fusion. The same results were repeated in C2C12 cells as well. Moreover, it was documented that 3′ UTR of c-Myb was responsible for downregulation of c-Myb protein levels in differentiating C2C12 cells. DNA microarray analysis of C2C12 cells revealed that the expression of several muscle-specific genes was downregulated during differentiation of c-Myb-expressing cells, namely: ACTN2, MYH8, TNNC2, MYOG, CKM and LRRN1. A detailed qRT-PCR analysis of MYOG, TNNC2 and LRRN1 is presented. Our findings thus indicate that c-Myb is involved in regulating the differentiation program of myogenic progenitor cells as its expression blocks myoblast fusion.     

Possible role of prostaglandins in the regulation of mouse myoblasts

A differentiation-defective mouse myoblast subclone (DD-1), cells of which do not fuse into myotubes nor synthesize muscle-specific proteins, was employed to help define the role of eicosanoids in mouse myoblast differentiation. We observed by hplc, tlc, and radioimmunoassay that the DD-1 cells release strikingly higher levels of cyclooxygenase pathway products prostaglandin E2 and F2 alpha into the culture medium than the parental non-differentiation-defective cells (DZ). In contrast, the levels of 15-hydroxyeicosatetraenoic acid (15-HETE), a lipoxygenase product, and a putatively identified second lipoxygenase product (LLP) did not differ greatly in the two cell types. The DD-1 cells also have strikingly higher levels of cyclooxygenase activity than the parental cells as determined by intact and broken cell assays. Additional fusion-defective clones were isolated on the basis of their flattened appearance and ability to grow in "mitogen-poor" medium and these cells also released strikingly higher levels of prostaglandins E2 and F2 alpha into the growth medium. The "turn on" of the cyclooxygenase pathway in the DD-1 cells and other fusion-defective cells is consistent with the hypothesis that the products of this pathway contribute to the inability of myoblasts to fuse with one another. This hypothesis is supported by the observation that there is a dose-dependent decrease in fusion of DZ cells when PGE2 is added to commitment medium.     

In differentiating prefusion myoblasts connexin43 gap junction coupling is upregulated before myoblast alignment then reduced in post-mitotic cells

Previously we have shown that during in vivo muscle regeneration differentiating rat primary myoblasts transiently upregulate connexin43 (Cx43) gap junctions and leave cell cycle synchronously. Here, we studied the temporal regulation of Cx expression in relation to functional dye coupling in allogenic primary myoblast cultures using western blotting, immuno-confocal microscopy and dye transfer assays. As in vivo, Cx43 was the only Cx isotype out of Cx26, 32, 37, 40, 43 and 45 found in cultured rat myoblasts by immunostaining. Cultured myoblasts showed similar temporal regulation of Cx43 expression and phenotypic maturation to those regenerating in vivo. Cx43 protein was progressively upregulated in prefusion myoblasts, first by the cytoplasmic assembly in sparse myoblast meshworks and then in cell membrane particles in aligned cells. Dye injection using either Lucifer Yellow alone, Cascade Blue with a non-junction permeant FITC-dextran revealed an extensive gap junction coupling between the sparse interacting myoblasts and a reduced communication between the aligned, but still prefused cells. The aligned myoblasts, uniformly upregulate p21^waf1/cip1 and p27^kip1 cell cycle control proteins. Taken together, in prefusion myoblasts less membrane-bound Cx43 was found to mediate substantially more efficient dye coupling in the growing cell fraction than those in the aligned post-mitotic myoblasts. These and our in vivo results in early muscle differentiation are consistent with the role of Cx43 gap junctions in synchronizing cell cycle control of myoblasts to make them competent for a coordinated syncytial fusion.     

The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation.

Differentiation of skeletal muscle involves withdrawal of myoblasts from the cell cycle, fusion to form myotubes, and the coordinate expression of a variety of muscle-specific gene products. Fibroblast growth factor and type beta transforming growth factor specifically inhibit myogenesis; however, the transmembrane signaling pathways responsible for suppression of differentiation by these growth factors remain elusive. Because ras proteins have been implicated in the transduction of growth factor signals across the plasma membrane, we used DNA-mediated gene transfer to investigate the potential involvement of this family of regulatory proteins in the control of myogenesis. Transfection of the mouse skeletal muscle cell line C2 with the oncogenic forms of H-ras or N-ras completely suppressed both myoblast fusion and induction of the muscle-specific gene products nicotinic acetylcholine receptor and creatine kinase. Inhibition of differentiation by activated ras genes occurred at the level of muscle-specific mRNA accumulation. In contrast, proto-oncogenic forms of N-ras or H-ras had no apparent effects on the ability of C2 cells to differentiate. Myoblasts transfected with activated ras genes exhibited normal growth properties and ceased proliferating in the absence of mitogens, indicating that ras inhibited differentiation through a mechanism independent of cell proliferation. These results demonstrate that activated ras gene products mimic the inhibitory effects of fibroblast growth factor and type beta transforming growth factor on myogenic differentiation and suggest that each of these regulators of myogenesis may operate through a common intracellular pathway.