of mesenchymal-epithelial interactions (Heine et al., 1987). TGF-β may play an important role in myogenic differen- tiation (Olson et al., 1986). TGF-β is thought ...
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Development 120, 1085-1095 (1994) Printed in Great Britain © The Company of Biologists Limited 1994
Inhibition of myogenic differentiation in myoblasts expressing a truncated type II TGF-β receptor Ellen H. Filvaroff, Reinhard Ebner and Rik Derynck* Departments of Growth and Development, and Anatomy, Programs in Cell Biology and Developmental Biology, University of California at San Francisco, San Francisco, CA 94143-0640, USA *Author for correspondence
SUMMARY Transforming growth factor-β (TGF-β) is thought to play a role in mesenchymal cell development and, specifically, in muscle differentiation, yet its precise role in the latter process remains unclear. TGF-β has been shown to both inhibit and induce myoblast maturation in vitro, depending on the culture conditions. Whether the type I or type II TGF-β receptor mediates the various TGF-β effects on myogenesis is not known. In the present study, C2C12 myoblasts were transfected with an expression vector for a truncated type II TGF-β receptor, which has been shown to act as a dominant negative inhibitor of type II receptor signaling. In contrast to the parental cells, the transfected clones did not efficiently form myotubes or induce expression of MyoD, myogenin and several other differentiation markers following incubation in low serum media. However, some muscle differentiation markers continued to be expressed in the transfected cells suggesting that at least two pathways are involved in muscle cell differentiation. These cells could still growth arrest in low serum media, showing that decreased proliferation can be disso-
ciated from differentiation. Unlike several oncogenes known to block myogenic differentiation, expression of the truncated TGF-β receptor did not result in myoblast transformation. Injection of the parental or the transfected C2C12 cells into the limb muscle of nude mice revealed quantitative and qualitative differences in their behavior, and suggested that myoblasts expressing the truncated TGF-β receptor cannot fuse in vivo. Finally, retrovirusmediated expression of MyoD in the transfected cells restored their ability to form myotubes in vitro, indicating that inhibition of myoblast differentiation by the truncated TGF-β receptor may depend on decreased MyoD expression. We propose that TGF-β signaling through the type II receptor is required for several distinct aspects of myogenic differentiation and that TGF-β acts as a competence factor in this multistep process.
INTRODUCTION
TGF-β may play an important role in myogenic differentiation (Olson et al., 1986). TGF-β is thought to be involved in cardiomyogenesis (Potts et al., 1989, 1991) and its expression is induced during experimental myocardial infarction (Thompson et al., 1988; Lefer et al., 1990). However, the role of TGF-β in skeletal myoblast differentiation is unclear, since exogenous TGF-β has both positive and negative effects on muscle cell development in vitro. On the one hand, treatment of skeletal myoblast cell lines or primary muscle cells with TGF-β in low serum inhibits terminal differentiation (Massagué et al., 1986). In addition, TGF-β blocks the expression and function of two muscle-specific transcription factors, MyoD (Vaidya et al., 1989) and myogenin (Brennan et al., 1991), thereby preventing expression of downstream muscle transcripts. On the other hand, TGF-β in normal serum can induce differentiation of myoblasts (Zentella and Massagué, 1992) and treatment of embryonic stem cells with TGF-β results in preferential differentiation of cells into muscle (Slager et al., 1993). TGF-β acts by binding to a set of specific receptors
The decision of a cell to proliferate, differentiate and migrate during development is intimately connected to its environment. Transplantation and ablation studies have shown the importance of cell-cell interactions and the extracellular milieu in the determination of cell fate (Greenwald and Rubin, 1992; Gurdon, 1992). Several peptide growth factors have been shown to play a role in inductive processes that give rise to differentiated cell types (Jessell and Melton, 1992). Transforming growth factor-β (TGF-β), one such factor, affects the growth and differentiation of many cell types in vitro, especially those of mesenchymal origin (Ignotz and Massagué, 1985; Rosen et al., 1988; Torti et al., 1989). Its expression pattern during mouse development also suggests an important function in specific morphogenetic and differentiation events in vivo (Heine et al., 1987; Millan et al., 1991; Pelton et al., 1991). In particular, TGF-β is highly expressed during periods of morphogenesis or remodeling of mesenchyme and at sites of mesenchymal-epithelial interactions (Heine et al., 1987).
Key words: TGF-β, differentiation, muscle, myoblasts, signaling, growth factor
1086 E. H. Filvaroff, R. Ebner and R. Derynck (Massagué, 1992). Of these, the type I and type II TGF-β receptors mediate most of its biological effects (Laiho et al., 1990a, 1991; Geiser et al., 1992). The cytoplasmic domains of the cloned type I and type II TGF-β receptors have sequences characteristic of serine-threonine kinases (Lin et al., 1992; Ebner et al., 1993a, Franzén et al., 1993), and the type II receptor has been shown to be a functional kinase (Wrana et al., 1992). A physical association between the type I and type II receptors has been proposed (Wrana et al., 1992), and the type I receptor may require the type II receptor for its activity (Ebner et al., 1993a,b, Franzén et al., 1993, Bassing et al., 1994). Functional inactivation of the type II receptor in an epithelial cell line has indicated that the type II receptor is required for the antiproliferative effect of TGF-β and that its effect on extracellular matrix protein synthesis may be mediated through the type I receptor (Chen et al., 1993). Which of these two receptors mediates the effects of TGF-β on myogenesis is not yet known. Whereas most cells in culture have both receptors, the type II receptor is expressed in vivo at high levels in undifferentiated mesenchyme and in differentiated muscle tissue (Lawler et al., 1994). The co-localization of TGF-β expression suggests a developmental role for TGF-β during muscle cell differentiation (Heine et al., 1987; Pelton et al., 1991; Lawler et al., 1994). To address the seemingly paradoxical observations on the effects of TGF-β on myoblasts and to gain insight into its role during myogenic differentiation, we transfected C2C12 myoblasts (Yaffe and Saxel, 1977; Blau et al., 1983) with a truncated form of the type II TGF-β receptor. This truncated receptor inhibits signaling by the type II, but not the type I, TGF-β receptor in a dominant negative fashion (Brand et al., 1993; Chen et al., 1993). We show that myoblasts expressing the truncated type II TGF-β receptor do not undergo morphological or biochemical differentiation. This inhibition of differentiation is associated with decreased expression of the myogenic determining genes, MyoD and myogenin, and can be rescued following infection with a MyoD-expressing retrovirus. These studies suggest that some, but not all, changes associated with myotube formation require signaling through the TGF-β receptor, and that TGF-β functions in an autocrine or paracrine fashion as a competence factor for myogenic differentiation.
MATERIALS AND METHODS Cell culture and transfections C2C12 cells which have been clonally purified for reproducible myogenic differentiation (Blau et al., 1983) from C2 cells (Yaffe and Saxel, 1977), were obtained from Dr H. Blau and grown in 20% fetal calf serum (FCS) in DMEM. To induce differentiation into myotubes, the cells were switched into DMEM containing 2% horse serum for 1 to 3 days. C2C12 cells were transfected with pcDNA1Neo (InVitrogen) expressing the truncated type II TGF-β receptor and encoding neomycin resistance (Chen et al., 1993) using the calcium phosphate precipitation method (Sambrook, 1989). The transfected cells were then cultured in medium containing G418 (400 µg/ml). After 14 to 21 days, G418 resistant clones were isolated, expanded and screened by northern hybridization for expression of mRNA for the truncated receptor. The three clones selected for this study were chosen on the basis of cell surface expression of the truncated receptor, as determined by chemical cross-linking using
125I-TGF-β. Five randomly selected neomycin-resistant, controltransfected clones were also analyzed and found to undergo normal myogenic differentiation.
Northern hybridization analysis 3 days after plating equal numbers of cells (determined using a Coulter counter), cells were switched to differentiation media (2% horse serum) or were kept in growth media (20% fetal calf serum) for three more days. RNA was isolated (Chomczynski and Sacchi, 1987) and analyzed by northern analysis (Sambrook, 1989) using cDNA probes radiolabelled by random priming using a commercial kit (Boehringer Mannheim). Following hybridization at 42˚C for 24 hours, the nitrocellulose blots were washed in 0.5× SSC, 0.5% SDS for 20 minutes at 42˚C and 0.1× SSC, 0.5% SDS for 30 minutes at 55˚C. cDNAs for myosin light chain 2, MyoD (Davis et al., 1987) and myosin light chain 1/3 (Periasamy et al., 1984, modified by Dr N. Rosenthal), the acetylcholine receptor α subunit (Isenberg et al., 1986) and myogenin (Wright et al., 1989) were used as hybridization probes to assess the differentiation state. Receptor cross-linking analysis Recombinant human TGF-β1 was 125I-labelled using a slightly modified chloramine T method (Frolik et al., 1984). Cross-linking of parental and transfected C2C12 cells was carried out as described (Gazit et al., 1993). Western analysis Equal quantities of protein (determined by the Biorad colorimetric assay) were electrophoresed in 12% (for troponin T or desmin) or 7.5% (for pRB or myosin heavy chain) denaturing polyacrylamide gels and transferred to nitrocellulose membranes at 40 V for 2 hours. The blots were washed for 10 minutes in TBST (25 mM Tris pH 8.0, 125 mM NaCl, 0.025% Tween 20), incubated in blocking buffer (25 mM Tris pH 8.0, 125 mM NaCl, 0.1% Tween 20, 1% BSA, 0.1% NaN3) for 2 hours at room temperature or 24 hours at 4˚C, and then incubated with the antibody for 2 hours at room temperature. They were then washed three times with blocking buffer (15 minutes per wash) and incubated with a secondary, alkaline phosphatase-conjugated antibody (Promega) for 1 hour. After three 15 minute washes with blocking buffer and three 5 minute washes with TBST, the blots were incubated in phosphatase substrate (BCIP/NBT, Kirkegaard and Perry Laboratories) for 5-30 minutes and, once developed, washed with distilled water. Antibodies for troponin T, myosin heavy chain (i.e. MF20 antibody, Sigma) and desmin were provided by Dr Charles Ordahl (UCSF). The antibodies to pRB (DeCaprio et al., 1988) were purchased from Pharmingen. Immunofluorescence Cells on coverslips were washed three times with phosphate-buffered saline (PBS) and fixed in methanol (for myosin heavy chain antibodies) or in 4% formaldehyde in PBS (for troponin T or desmin antibodies) for 10 minutes at room temperature. After washing with PBS, cells were incubated in 0.1% Triton in PBS for 10 minutes and rinsed again with PBS. The samples were then treated with 3% bovine serum albumin (BSA) in PBS for 15-30 minutes and incubated with primary antibody for 45 minutes at 37˚C. After three washes in PBS, the cells were incubated with a rhodamine-conjugated secondary antibody for 45 minutes at 37˚C. The cells were then washed three times with PBS, hydrated in 70% then 100% ethanol (3 minute washes each), air dried, mounted with Fluoromount-G (Fisher Scientific) and a coverslip, and photographed with a Zeiss Axioplan microscope.. Growth curves On day 0, the cells were counted and plated such that by the day of treatment, all samples would have approximately the same cell number. On day 4, one set of plates was changed to differentiation media (2% horse serum), while the other set remained in growth
Myogenesis and type II TGF-β receptor 1087 media (20% serum). All cells were changed and counted every other day using a Coulter counter. Retrovirus infections To construct the retrovirus encoding hygromycin resistance and βgalactosidase, the β-galactosidase gene was cut from plasmid pML62 (constructed by Drs M. Landowski and G. Martin) with SalI and BamHI and ligated into the SalI and BamHI sites of a derivative of pLXSH (Miller et al., 1993), obtained from Drs M. Lochric and H. Varmus. The packaging cell line, PA317 (Miller et al., 1993), was transfected with this plasmid and the retrovirus-containing conditioned media, harvested 48 hours later, was used to infect another packaging cell line, PE501. These cells were grown for 2 weeks in DMEM/10% serum with hygromycin, and individual colonies were isolated and grown. Viruses produced from these clones were titered using NIH-3T3 fibroblasts, and the highest titer virus stock was used to infect C2C12 cells. 48 hours after infection, C2C12 cells were switched to growth media with hygromycin (500 µg/ml) and selected for two weeks prior to X-gal staining. The high titer MyoD retrovirus was kindly supplied by Dr Dusty Miller (Weintraub et al., 1989). Infections were performed by incubating growing cells overnight with viral stock plus 4 µg/ml of polybrene. In vivo injections and analysis For intramuscular injections into mice, the cells were trypsinized, washed twice and resuspended in PBS at 4˚C. Approximately 4×105 myoblasts (in 20 µl) were delivered via 4 injections (5 µl each) into the hind limbs of anesthetized 3- to 4-week-old nude mice. 2 weeks later, these limbs were removed from euthanized mice and frozen in isopentane, serially sectioned, and fixed and stained for β-galactosidase activity as described (Dhawan et al., 1991). Muscle fibers were examined across multiple serial sections throughout the length of the limb. Sections were mounted in Gel/Mount (Biomeda Corporation) and photographed with a Zeiss Axioplan microscope.
RESULTS Myoblast fusion is inhibited in cells expressing a truncated type II TGF-β receptor The mouse C2C12 myoblast cell line (Yaffe and Saxel, 1977; Blau et al., 1983) is frequently used as a model to study the process of myogenic differentiation in vitro. This clonal cell population has highly reproducible differentiation properties (Blau et al., 1983). These cells proliferate as mononuclear myoblasts in high (20%) serum media (growth media), but become growth arrested and undergo morphological and biochemical differentiation after a switch to low (2%) serum media (differentiation media) (Blau et al., 1983). To examine the role of TGF-β and its signaling through the type II receptor during myogenesis, C2C12 cells were transfected with a truncated type II TGF-β receptor expression vector containing a neomycin-resistance marker. This truncated receptor, which lacks its cytoplasmic domain, has been shown to inhibit signaling specifically through the type II TGF-β receptor in a dominant negative fashion (Chen et al., 1993). After 2 weeks, G418-resistant colonies were isolated, propagated as stable cell lines, and tested for expression of the truncated receptor mRNA by northern blotting (data not shown). To verify expression of the truncated TGF-β receptor protein, the transfected cells were incubated with 125I-labelled TGF-β, and the cross-linked receptors were analyzed by polyacrylamide gel electrophoresis. As shown in Fig. 1A, the truncated receptor
Fig. 1. Binding of 125I-TGF-β to the truncated type II TGF-β receptor at the cell surface in transfected myoblasts. Parental C2C12 cells (lane 1) or clones stably transfected with a truncated type II TGF-β receptor (lanes 2,3) were incubated with 125I-TGF-β, and cross-linked proteins were (A) analyzed by SDS-PAGE or (B) immunoprecipitated with antibodies to an epitope tag in the truncated type II TGF-β receptor expression vector, and then separated on SDS-polyacrylamide gels. The third clone gave similar results (not shown). The type I, type II and type III TGF-β receptors are indicated by Roman numerals. DN and arrow indicate the band corresponding to the truncated receptor.
was expressed at the cell surface of transfected clones and bound TGF-β. The 125I-TGF-β cross-linked receptor could be immunoprecipitated with antibodies against an epitope tag engineered at its carboxy terminus (Fig. 1B). Of the five clones that expressed the truncated receptor mRNA, three showed expression of the corresponding protein at the cell surface. These three clones were used for further analysis and compared with parental and neomycin-resistant control C2C12 cells. Under proliferative conditions, cells transfected with the truncated receptor were morphologically similar to the parental C2C12 cells (Fig. 2A−). To induce differentiation, cells were switched to low serum (2%) media. Within 3 days, the parental C2C12 cells ceased proliferating and fused to form multinucleated fibers. In contrast, in the three C2C12 clones expressing the truncated type II TGF-β receptor, very few (
