23A2 cell line. C2C12 myoblasts (4) .... using a stable, myogenic, C3H1OT1/2-pEMC11s cell line ... pEMC11s. Shari Menke and Robyn Kline for expert technical.
MOLECULAR AND CELLULAR BIOLOGY. Aug. 1989. p. 3576-3579 0270-7306/89/083576-04$02.00/0 Copyright © 1989. American Society for Microbiology
Vol. 9. No. 8
Fibroblast Growth Factor and Transforming Growth Factor 3 Repress Transcription of the Myogenic Regulatory Gene MyoDi TUSHAR B. VAIDYA, SIMON J. RHODES, ELIZABETH J. TAPAROWSKY. AND STEPHEN F. KONIECZNY" Depairtment of 'Biological Sc iences, Piticdlie Unei-vrsity, West L aftivette, Inldianaa 47907 Received 28 March 1989/Accepted 19 May 1989
In this report, we demonstrate that myogenic cultures inhibited from differentiating by treatment with fibroblast growth factor or transforming growth factor 11 show reduced levels of MyoDl mRNA. Although this repression may contribute to the inhibition of myogenesis by growth factors, additional regulatory pathways must be affected, since inhibition still occurs in cultures engineered to constitutively express MyoDI mRNA.
Skeletal muscle differentiation involves the transcriptional activation of the contractile protein gene set and the formation of multinucleated muscle fibers. The contractile protein genes are controlled by cis and tran.s regulatory systems which appear to respond to exogenous growth factors, since addition of fibroblast growth factor (FGF) or transforming growth factor 3 (TGF-3) to differentiating muscle cells in culture inhibits both fusion and the accumulation of these muscle-specific proteins (7, 9, 13, 14, 16, 21). It has been hypothesized that FGF and TGF-, may influence one or perhaps several master regulatory pathways. Although the regulatory genes that control the induction of skeletal myogenesis have not been defined completely, at least three genes, MyoDl (8, 20), inyd (18), and myogenin (22). play an important role in establishing the myogenic lineage and inducing the expression of muscle-specific genes. To determine whether these regulatory genes are modulated by exogenous growth factors, we investigated the transcriptional activity of the MyoDl gene in cells exposed to FGF or TGF-3. 23A2 myoblasts (12) were induced to differentiate in medium containing insulin, transferrin, and selenium (ITS) (23) to establish the normal expression pattern of MyoDl and the contractile protein gene, troponin I (TnI), during skeletal myogenesis. RNA was isolated from proliferating and differentiation-induced 23A2 cultures, electrophoresed through formaldehyde-agarose gels, and hybridized with nick-translated TnI (cM113) (10) and MyoD1 (pEMC11s) (8) probes as described previously (11). TnI mRNA was detected within 24 h after ITS induction and reached maximal levels by 48 h (Fig. 1). In contrast, MyoDl mRNA was present in undifferentiated 23A2 myoblasts and remained expressed in myofibers, although a slight decrease was evident after 48 h in ITS. We next compared MyoDl mRNA levels in control 23A2 cultures and in 23A2 cultures induced to differentiate in the presence of FGF or TGF-3 to investigate whether MyoD1 gene expression is affected by the same growth factors that inhibit myoblast fusion and contractile protein gene expression. 23A2 myoblasts expressed high levels of TnI and MyoD1 mRNAs within 48 h of ITS addition (Fig. 2). In contrast, 23A2 cultures fed ITS containing 10 ng of basic FGF or 10 ng of human TGF-f1 (R+D Systems. Inc.) per ml
showed no TnI mRNA (Fig. 2), no myosin heavy-chain protein, and no fusion (data not shown). Interestingly, MyoD1 mRNA levels also were reduced in growth-factortreated cultures so that by 48 h, MyoD1 mRNA was no longer detected. This growth-factor-induced inhibition of differentiation is reversible, however, since removal of FGF from inhibited cultures restored MyoD1 expression, increased TnI mRNA levels (Fig. 2), and led to a resumption of myoblast fusion. Similar results were obtained when TGF-, was removed, although the recovery time was delayed (data not shown). The growth-factor-induced repression of MyoDi mRNA appears to be specific, since the control pAL15 gene (2, 3) was expressed equally in all experimental groups. In addition. the effects of FGF and TGF-3 on myogenesis and MyoD1 expression are not unique to the 23A2 cell line. C2C12 myoblasts (4) behaved in an identical fashion when treated with these growth factors (Fig. 2). The reduction in MyoD1 mRNA observed in FGF- and TGF-,-treated myoblasts suggests that these growth factors inhibit the transcription of the MyoD1 gene. To examine this directly, nuclei isolated from control 23A2 myofibers and from parallel cultures treated with FGF or TGF-P were used in in vitro nuclear run-on assays as previously described (5). The MyoDl and TnI genes were actively transcribed in control myofibers, while transcription from the MyoD1 and TnI genes was not detected in '3A2 cells treated with FGF or TGF-4 for 48 h (Fig. 3). 23A2 myoblasts treated with FGF for 24 h also exhibited reduced MyoDl and TnI expression patterns (8 and 4% of control MyoD1 and Tnl activities, respectively). As expected, removal of FGF from treated cultures resulted in a rapid increase in MyoD1 and TnI gene transcription (Fig. 3). The control gene, pAL15, was transcribed equally under all experimental conditions. The studies described above suggest that FGF and TGF-P may inhibit skeletal muscle differentiation simply by repressing expression of the myogenic regulatory gene, MyoDl. However, it is possible that these growth factors influence additional regulatory signals, such as posttranslational modifications of the MyoD1 protein. which also may be essential for terminal differentiation. Therefore, we investigated whether FGF and TGF-r could inhibit myogenesis in cells constitutively expressing the MyoDl cDNA clone, pEMC11s (8). C3H1OT1/2 fibroblasts (19) were transfected transiently
Corresponding author. 3576
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FIG. 3. Transcriptional activity of the MyoDl and TnI genes in 23A2 muscle cells induced to differentiate in the presence or absence of TGF-P or FGF. Cells were treated with growth factors for the indicated times as described in the legend to Fig. 2. After treatment, nuclei were isolated, subjected to nuclear run-on assays as described previously (5), and hybridized to MyoDl, TnI, pUC19, and pAL15 plasmids immobilized on nitrocellulose filters. (A) Autoradiograph of the hybridized filters. Transcriptionally active MyoDl and Tnl genes were detected in control myofibers, whereas TGF-1and FGF-treated cells repress expression of these genes. Removal of FGF led to a rapid resumption of MyoD1 and Tnl transcription within 24 h. Plasmids pUC19 and pAL15 were included as negative and positive controls, respectively. (B) Relative expression values of the MyoDl and Tnl genes obtained by quantitative densitometry of the nuclear run-on assays depicted in panel A. MyoDl and Tnl values were normalized to the pAL15 signal (set at 100) within each experimental group.
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B. with pEMC11s as described by Yutzey et al. (23). After 48 h in growth medium, cultures were fed ITS medium or ITS supplemented with FGF or TGF-1. After an additional 48 h, RNA was isolated and analyzed by Northern (RNA) hybridizations, and parallel cultures were fixed and stained with MF-20, an antibody directed against the myosin heavy-chain protein (1). After ITS treatment, MyoDl and Tnl mRNAs were not detected in control transfected C3H1OT1/2 cells, while pEMClls-transfected C3H1OT1/2 cells expressed very high levels of MyoDl and a low but detectable level of Tnl
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FIG. 2. Regulation of MyoDl and Tnl mRNAs in 23A2 and C2C12 cultures induced to differentiate in the presence or absence of basic FGF or human TGF-,1l. Control cells were treated with ITS plus 5% horse serum for 48 h. TGF-P and FGF cultures were fed ITS plus 5% horse serum containing 10 ng of TGF-1B or 10 ng of FGF per ml for 24 or 48 h, as indicated. Both TGF-,B and FGF dramatically reduced the level of MyoDl and Tnl mRNAs in these cultures. However, within 24 h after removal of FGF, MyoDl and Tnl mRNAs accumulated. As an internal control, the same blots were rehybridized with a nick-translated probe for the control pAL15 mRNA, which has been shown to be constitutively expressed during adipose (2, 3) and muscle (S.F.K., unpublished results) differentiation.
(Fig. 4). As expected, MyoDl mRNA persisted in the FGFand TGF-1-treated cells, although a slight decrease in MyoDl was evident in these cultures. At this time we are unable to determine whether this reduction is due to a negative effect that these growth factors may have on the transcription of the transfected cDNA construct or on the transcription of the endogenous MyoDl gene, which Tapscott et al. (20) have suggested may be active in C3H1OT1/2 cells after transfection with pEMC11s. However, on the basis of previous experiments in our laboratory in which myoblasts expressing low levels of MyoDl continue to differentiate (11), it is clear that the level of MyoDl mRNA that remains in these transfected cultures is sufficient to induce myogenesis. Surprisingly, Tnl expression was not detected in the TGF-4- and FGF-treated cultures (Fig. 4). Furthermore, when these cultures were immunochemically stained with MF-20, 5% of the pEMClls-transfected cells fed ITS expressed skeletal myosin, whereas only 0.5% of the FGF-treated and 0.2% of the TGF-,B-treated cells stained positive (Fig. 5). Removal of FGF restored the original number of MF-20 positive cells (4%) in the treated cultures
3578
MOL. CELILI. BIOL.
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FIG. 4. Northern analysis of MyoDl and Tnl expression in C3H10T1/2 cells transiently transfected with pUC19 or pEMC11s plasmids. C3H10T1/2 cells were transfected with 10 ,ug of pUC19 or 5 ,ug of pEMC11s plus 5 jig of pUC19 per 100-mm-diameter dish. Transfected cells were maintained in growth medium for 48 h and then induced to differentiate in the presence or absence of TGF-, and FGF as described in the legend to Fig. 2. After 48 h. total RNA was isolated and analyzed by Northern hybridization as described in the legends to Fig. 1 and 2. with the exception that 30 ,ug of RNA obtained from the transiently transfected cultures was analyzed. MyoDl and Tnl mRNAs were not detected in control transfected C3H1OT1/2 cells. The pEMClls-transfected cells, however, expressed very high levels of MyoDl in all experimental groups. Tnl was expressed in the control MyoDl-transfected cultures but repressed in the TGF-p- and FGF-treated cells. 23A2 myofiber (MF) RNA (10 ,ug) was included as a positive control. was
within 24 h (Fig. 5). Identical results have been obtained by stable, myogenic, C3H1OT1/2-pEMC11s cell line treated with FGF and TGF-4 (data not shown). We conclude that FGF and TGF-r are likely to inhibit muscle differentiation by a molecular mechanism independent of the transcriptional regulation of the MyoDl gene. A number of growth-promoting agents inhibit skeletal muscle differentiation, including FGF (7, 13, 21). TGF-4 (9, 14, 16), and the oncogenic form of ras p21 (11. 15, 17). In all cases, fusion and the expression of the contractile protein genes are repressed. In addition, transcription of the myogenic regulatory gene MyoDl becomes down regulated in these cells (11: this study). Interestingly. constitutive expression of the MyoDi cDNA overrides the H-ra-.s-induced inhibition of 23A2 myogenic differentiation (11) but appears unable to block the antagonistic effects of FGF and TGF-f. This suggests that these myogenic inhibitors, although they similarly affect several molecular events critical to myogenesis, do not act via identical intracellular pathways. Our results suggest that at least two levels of regulation are involved in controlling skeletal muscle differentiation. The first involves the transcriptional regulation of the MyoD1 gene, and the second involves the regulation of additional molecular events, such as posttranslational modifications of the MyoD1 protein, that may be required for the transactivation of muscle-specific genes (6). Our future studies will attempt to define further the biological activities of the MyoDi (8, 20), invd (18). and myogenin (22) proteins during muscle development and establish at what point myogenic inhibitors, such as growth factors and oncogene products, alter the program of skeletal myogenesis. using a
4.
FIG. 5. Myosin heavy-chain expression in C3H1OT1/2 cells transiently transfected with the MyoD1 expression vector. pEMC11s. Parallel cultures from the experiment depicted in Fig. 4 were immunochemically stained with MF-20 (1) as described previously (11. 23) to detect the presence of myosin heavy-chain proteins. All cultures were induced to differentiate in the presence or absence of TGF-1 or FGF. (A) C3H10T1/2 cells transfected with the control plasmid pUC19: (B) C3H1OT1/2 cells transfected with pUC19 and treated with FGF; (C) C3H1OT1/2 cells transfected with pEMC11s and induced to differentiate in ITS medium: (D) C3H1OT1/2 cells transfected with pEMC11s and induced to differentiate in the presence of TGF--,B (E) same as in panel D. except that cells were induced to differentiate in the presence of FGF; (F) C3H1OT1/2 cells transfected with pEMC11s. induced to differentiate in the presence of FGF for 48 h and then fed ITS medium without FGF for an additional 24 h. Arrows in panels D and E indicate single. rare. MF-20-positive cells in these cultures.
We thank Andrew Lassar for generously supplying plasmid pEMC11s. Shari Menke and Robyn Kline for expert technical assistance, and Connie Philbrook for the preparation of the manuscript.
This work was supported by Public Health Service grant HD 24489 from the National Institutes of Health to S.F.K.
LITERATURE CITED 1. Bader, D., T. Masaki, and D. A. Fischman. 1982. Immunochemical analysis of myosin heavy chain during avian myogenesis in iw o and in litlo. J. Cell Biol. 95:763-770. 2. Bernlohr, D. A., C. W. Angus, M. D. Lane, M. A. Bolanowski, and T. J. Keliv, Jr. 1984. Expression of specific mRNAs during adipose differentiation: identification of an mRNA encoding a homologue of myelin P2 protein. Proc. Natl. Acad. Sci. USA 81:5468-5472. 3. Bernlohr, D. A., M. A. Bolanowski, T. J. Kelly, Jr., and M. D. Lane. 1985. Evidence for an increase in transcription of specific mRNAs during differentiation of 3T3-L1 preadipocytes. J. Biol. Chem. 260:5563-5567. 4. Blau, H. M., C.-P. Chiu, and C. Webster. 1983. Cytoplasmic activation of human nuclear genes in stable heterokaryons. Cell 32:1171-1180. 5. Bucher, E. A., P. C. Maisonpierre, S. F. Konieczny, and C. P. Emerson, Jr. 1988. Expression of the troponin complex genes: transcriptional coactivation during myoblast differentiation and
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VOL. 9, 1989
6.
7.
8. 9.
10.
11.
12.
13. 14.
independent control in heart and skeletal muscles. Mol. Cell. Biol. 8:4134-4142. Buskin, J. B., A. B. Lassar, R. L. Davis, H. Weintraub, and S. D. Hauschka. 1988. Is the myogenic determination factor MyoD identical to the myocyte-specific DNA binding factor MEF 1? J. Cell Biol. 107:98a. Clegg, C. H., C. A. Linkhart, B. B. Olwin, and S. D. Hauschka. 1987. Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in Gl phase and is repressed by fibroblast growth factor. J. Cell Biol. 105: 949-956. Davis, R. L., H. Weintraub, and A. B. Lassar. 1987. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51:987-1000. Florini, J. R., A. B. Roberts, D. Z. Ewton, S. L. Falen, K. C. Flanders, and M. B. Sporn. 1986. Transforming growth factor-P. J. Biol. Chem. 261:16509-16513. Hastings, K. E. M., P. L. Hallauer, A. C. Peterson, G. Karpati, and R. Koppe. 1989. Differential expression of skeletal muscle troponin I genes, p. 759-766. In L. H. Kedes and F. E. Stockdale (ed.), Cellular and molecular biology of muscle development. Alan R. Liss, Inc., New York. Konieczny, S. F., B. L. Drobes, S. L. Menke, and E. J. Taparowsky. 1989. Inhibition of myogenic differentiation by the H-ras oncogene is associated with the down regulation of the MyoDl gene. Oncogene 4:473-481. Konieczny, S. F., and C. P. Emerson, Jr. 1984. 5-Azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells: evidence for regulatory genes controlling determination. Cell 38:791-800. Lathrop, B., E. Olson, and L. Glaser. 1985. Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line. J. Cell Biol. 100:1540-1547. Massague, J., S. Cheifetz, T. Endo, and B. Nadal-Ginard. 1986.
15. 16.
17.
18.
19.
20.
21. 22. 23.
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Type ,B transforming growth factor is an inhibitor of myogenic differentiation. Proc. Natl. Acad. Sci. USA 83:8206-8210. Olson, E. N., G. Spizz, and M. A. Tainsky. 1987. The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation. Mol. Cell. Biol. 7:2104-2111. Olson, E. N., E. Sternberg, J. S. Hu, G. Spizz, and C. Wilcox. 1986. Regulation of myogenic differentiation by type P transforming growth factor. J. Cell Biol. 103:1799-1805. Payne, P. A., E. N. Olson, P. Hsiau, R. Roberts, M. B. Perryman, and M. D. Schneider. 1987. An activated c-Ha-ras allele blocks the induction of muscle-specific genes whose expression is contingent on mitogen withdrawal. Proc. Natl. Acad. Sci. USA 84:8956-8960. Pinney, D. F., S. H. Pearson-White, S. F. Konieczny, K. E. Latham, and C. P. Emerson, Jr. 1988. Myogenic lineage determination and differentiation: evidence for a regulatory gene pathway. Cell 53:781-793. Reznikoff, C. A., D. W. Brankow, and C. Heidelberger. 1973. Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division. Cancer Res. 33:3231-3238. Tapscott, S. J., R. L. Davis, M. J. Thayer, P.-F. Cheng, H. Weintraub, and A. B. Lassar. 1988. MyoDl: a nuclear phosphoprotein requiring a myc homology region to convert fibroblasts to myoblasts. Science 242:405-411. Wice, B., J. Milbrandt, and L. Glaser. 1987. Control of muscle differentiation in BC3H1 cells by fibroblast growth factor and vanadate. J. Biol. Chem. 262:1810-1817. Wright, W. E., D. A. Sassoon, and V. K. Lin. 1989. Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell 56:607-617. Yutzey, K. E., R. L. Kline, and S. F. Konieczny. 1989. An internal regulatory element controls troponin I gene expression. Mol. Cell. Biol. 9:1397-1405.
