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HORMONES, GROWTH FACTORS, Annu. Rev. Physiol. 1991.53:201-216. Downloaded from www.annualreviews.org by University of Massachusetts - Amherst on 04/15/13. For personal use only.
AND MYOGENIC DIFFERENTIATION James R. Florini, Daina Z. Ewton, and Karen A. Magri Biology Department, Syracuse University, Syracuse, New York 13244
KEY WORDS:
myogenesis, insulin-like growth factors, transforming growth factor-/3, fibrob last growth factor, myogenin, MyoDl
INTRODUCTION The import�mce of honnones and growth factors in regulating the growth and differentiation of muscle cells is now widely recognized. This field has been summarized recently by two of the authors of this chapter (26), and other reviews of related topics (65, 69) provide additional infonnation. Since our last review of the field, it has been revolutionized by the discovery of the myogenesis detennination genes, which bring a new focus to the study of myogenesis.
Definitions and Concepts MYOGENESIS DETERMINATION GENES MyoDI (11) and myd (60) were first characterized as genes that control initial determination to the myogenic lineage. Subsequently, Myf-5 (7) and MRF4 (63)/herculin (51) have been added to the family of genes that are found exclusively in skeletal muscle and can convert nonmuscle cells to the myogenic lineage (6). However, MyoDI is not expressed in BC3Hl or L6 myoblasts (6), and MRF4 is not expressed in immortalized myogenic cell lines (63), so it appears that not all of these genes are essential for expression of the myogenic program in all muscle cells. In contrast, m yogenin, which has been identified as a putative controller of tenninal differentiation in both rat (85) and mouse ( 14) muscle cell lines, is expressed in every cell undergoing myogenic differentiation (6, 14, 85). In
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addition, myogenin also induces nonmuscle cells to express muscle-specific genes (14, 85). HORMONES AND GROWTH FACTORS The distinction between protein hor mones and growth factors is arbitrary and has little biological significance. In general, hormones are synthesized and secreted by discrete endocrine glands, while growth factors are secreted by many different kinds of cells. All of these agents are relatively small peptides that act through a receptor on the cell membrane to generate an intracellular signal that is not yet understood. Until recently research on growth factors was severely restricted by the unavailabil ity of purified materials in sufficient quantities. All of the growth factors discussed here are now commercially available, making possible experiments that could not be done in the past. The ready availability of these agents allows study of myogenic differentiation in well-defined systems that give answers much less ambiguous than was possible in the days of fetal bovine serum and chick embryo extract.
Most of the work on control of skeletal myogenesis has been done on three immortalized cell lines. The rat L6 line (86) has probably been the most widely used; it provides a useful combination of properties, with a convenient rate of differentiation that can be modified by medium components. Its major disadvantage is its lack of responsiveness to fibroblast growth factor (FGF). The mouse C2 line (87) differentiates more rapidly than L6 cells, and it is responsive to FGF. The mouse BC3Hl line is unusual in several ways. It was initially isolated (70) from an intracranial tumor, and it does not fuse to form postmitotic myotubes, thus allowing study of the reversibility of myogenesis. In spite of their convenience, these cell lines have limitations. The fact that none of these cells exhibits senescence shows that they are different from normal diploid cells in at least one major property. For this reason, we feel strongly that all major conclusions should be verified in primary cultures of skeletal muscle cells.
MUSCLE CELL LINES
Scope of This Review This chapter concentrates on the differentiation of skeletal muscle and the growth factors that stimulate [insulin-like growth factors (IGFs)] and inhibit [FGF, transforming growth factor (TGF-f3)] it. We mention those instances in which effects of hormones or growth factors on expression of the myogenic determination genes have been reported. The limited space allottment forces us to omit references to many publications that we consider important, and we apologize for the omissions.
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EFFECTS OF SPECIFIC HORMONES AND GROWTH FACTORS
Insulin-like Growth Factors (IGFs) CHEMISTRY AND SECRETION
The IGFs (also known as somatomedins) are
28). As is true of all of the growth factors considered here, the IGFs were initially described as several different agents that were ultimately recognized as identical. The primary agents are IGF-I (70 amino acids, 7649 daltons), IGF-II (67 amino acids, 747 1 daltons), and insulin (52 amino acids, 5733 daltons), listed in order of their potency in stimulating myogenic differentia tion (20). Like the other growth factors described here, IGF-I is synthesized and apparently secreted by a number of cell types, although there is some reason to believe that the primary source of circulating IGFs is the liver (28).
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a family of small peptides similar in structure to proinsulin (reviewed in
IGFs are also synthesized in substantial quantities by cultured muscle cells
(78, 79). ANABOLIC ACTIONS The IGFs exert pleiotypic anabolic actions on skele tal muscle cells (reviewed in 22), as they do on many other kinds of cells (3). These include stimulation of the f ollowing processes: amino acid uptake and incorpor ati on into protein, uridine and thymidine incorporation into nucleic acids, glucose uptake, cell proliferation, etc. IGFs also suppress degradation of proteins. These activities occur in several cell lines as well as in primary myoblast cultures and satellite cells. Unlike FGF, IGFs stimulate myoblast proliferation in completely defined medium in the absence of other serum components.
STIMULATION OF DIFFERENTIATION
The insulin-like hormones are unique
among growth factors and hormones in that they stimulate, rather than inhibit, myogenic differentiation. It was first reported
25 years ago (12) that insulin
(at p.M levels) increased differentiation in chick embryo muscle cells and this
& FIorini (20) first L6 cell differentiation, both at the morphological (fusi on) and biochemical (CK activity) levels, and suggested that the previous observations on insulin represented cross-reaction with the IGF-I receptor. This stimulation is not limited to cell lines; IGFs stimulate differentiation in chick embryo muscle cells (67) and in rat satellite cells (1). Thus the stimulation of differentiation by the insulin-like hormones is a general phenomenon, except in the case of cell lines such as C2 cells that secrete large amounts of these hormones (78, 79) and may thus stimulate their finding was repeated in other laboratories
(26).
Ewton
showed that physiological levels of IGFs stimulated
own differentiation by autocrine/paracrine mechanisms.
A curious aspect of the stimulation by IGFs, at least in L6 cells, is the
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biphasic concentration dependency (25). There is the expected increased stimulation from approximately 0.5 to 20 ng/mI, but this is followed by a rather sharp peak: and lower levels of differentiation down to near control values at very high concentrations (360 ng/mI). Elevation of myogenin mRNA by IGF-I exhibited the same biphasic response (1. FIorini et al, manuscript in preparation). Recent experiments (1. A. Foster, unpublished) show a similar biphasic response to IGF-I in the stimulation of expression of the elastin gene in aortic smooth muscle cells. The mechanism of this biphasic response is not apparent. MECHANISM OF IOF STIMULATION OF DIFFERENTIATION
Some obvious explanations were considered soon after the discovery of the stimulation by IGF-1. One was that increased differentiation was a result of the greater culture density resulting from the mitogenic effects of IGF-1. This possibility was eliminated by two different approaches-use of inhibitors (80) and different plating densities (20). The possibility that the cells differentiated upon treatment with IGF-I because of generally better metabolic condition (the "happy cell" explanation) was eliminated by showing that differentiation was stimulated by IGF-I and IGF-II, even in serum concentrations that allowed high rates of cell prolifera tion (25). An extended study of effects of polyamines led to the conclusion that elevated polyamine levels are necessary but not sufficient for differentia tion (18). Possibilities that have been eliminated
In our view the most likely mechanism of IGF-I action is induction of myogenin, the most universally, expressed of the myogenesis determination genes. Recent work in this laboratory (J. FIorini et aI, manuscript in preparation) has shown that treatment of L6Al cells with IGF-I gives a large (60-fold) increase in myogenin mRNA content at 30 to 40 hr, well before the elevation of creatine kinase. This is the only known case in which expression of the myogenin gene is increased, rather than inhibited, by a well-characterized agent. This elevation exhibits a concentration de pendency like that of the stimulation of differentiation, including the sharply decreased stimulation at higher IGF-I concentrations. An antisense oligomer complementary to the first 15 nucleotides of the translated portion of myoge nin mRNA blocks the stimulation of differentiation, so we conclude that increased myogenin gene expression is the primary mechanism by which IGF-I stimulates differentiation of muscle cells. The rather long time required for this elevation suggests that a rapid single step is not involved, i.e. that a substrate for the IGF-I receptor tyrosine kinase activity does not act directly on an enhancer for the myogenin gene.
Induction of myogenin
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IGF-I receptor Three different receptors-the IOF-I receptor, the IOF-II receptor, and the insulin receptor (for reviews see 62 and 66)-might mediate the stimulation of differentiation. The presence of all three receptors in cultured muscle cells has been demonstrated (2, 5, 13, 19). Our early experiments suggested that two different receptors might be involved in mediating various actions of IOFs on muscle, but we ultimately concluded ( 19) that all actions of IOFs were mediated by the type I receptor that exhibits the specificity IOF-I > IOF-I1 > insulin. Ballard (2) similarly concluded that the stimulation of protein and DNA synthesis in L6 cells was also mediated by the IGF-I receptor. Conclusive support for the primary role of the IGF-I receptor was provided by Kiess et al (37), who developed an antibody that specifically blocked the binding of IOF-II to the IOF-II receptor without affecting the binding of IGF-I to its receptor. This antibody did not alter the effects of IOF-II on amino acid or glucose uptake, or on leucine incorporation into proteins in L6 cells, thus indicating that the IGF-II receptor did not mediate these actions of the IGFs. An important theoretical paper (47) shows that differences in agonist potency for various actions mediated by the same receptor-some of the actions of IOF-I differ more than tenfold in the concentration required for half-maximal effect ( 19)-can be explained by differences in affinities of second messengers for their intracellular receptors. IGF-II receptor The role of the IOF-II receptor remains unclear. It is present in large quantities in L6 cells (5), and it is strikingly elevated during differen tiation in C2 cells (79), but no function has been shown for it in muscle cells. Its surprising identity to the mannose-6-phosphate receptor (which plays a role in targeting of lysosomal enzymes) is well established (52) and suggests that it may be involved in metabolism of IGF-II. This possibility was sup ported by the observation (37) that an antibody to the IOF-II receptor inhibited IGF-II degradation by 90% in L6 myoblasts. Other antibodies to the IOF-II1 mannose-6-phosphate receptor blocked the insulinlIOF-I-induced decrease in protein catabolism of CHO cells, which suggests that this might result from a disruption of movement of lysosomal enzymes to lysosomes (40). IGF binding proteins The IOFs are unlike other peptide hormones in that they circulate bound to a larger protein (3, 4). Initially it was believed that the binding protein functioned to decrease metabolism and regulate the concentra tion of free IOFs, and most early studies showed that binding proteins inhibited IGF actions (89). More recently, stimulatory effects of binding proteins have been reported ( 15), and a number of different binding proteins have been described (4). Human fetal myoblasts (32), as well as L6 and BC3HI myoblasts and porcine smooth muscle cells (49, 50), all secrete IOF binding proteins, and this secretion is regulated by insulin and IOF-I. Tollef-
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sen et al (78, 79) found a 30-fold increase in binding proteins during differen tiation of C2 cells. Thus binding proteins may play an important role in regulating the growth and differentiation of skeletal and smooth muscle,
although the specifics of that role are not yet understood. Effects on oncogene expression Increased oncogene expression is a fre quently reported early effect of growth factors in many cells. In L6 cells, there is a fourfold increase in c10s mRNA by IGF-I; several other oncogenes are not affected (58). The report (61) that elevated c fos expression in transfected L6 myoblasts inhibited differentiation makes this an unlikely mediator of the IGF action considered here. There is induction of c-myc by high levels ( 1 p,g/ml) o f IGF-I ( 16), but a t these levels differentiation of myoblasts is not stimulated (see discussion of the biphasic response above). A listing of oncogenes that exhibit changes during myogenic differentiation (26) and a more extensive treatment of the subject (68) are available in other reviews. The available evidence does not point to any oncogene likely to play a role in the stimulation of myogenic differentiation by IGF, although they may well be involved in proliferative and other anabolic responses.
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-
We have taken a substantial step toward understanding the stimulation of differentiation with the discovery that IGFs stimulate expression of the myo
genin gene. Major questions remain about events that occur between occupan cy of the IGF receptor and increased myogenin mRNA levels that lead to terminal differentiation of muscle cells.
Transforming Growth Factor-{3 (TGF-{3) CHEMISTRY AND SECRETION TGF-f3 was initially characterized as an in ducer of phenotypic transformation of fibroblastic cells, but it has sub sequently been shown to have such a wide variety of actions that its name is clearly unduly restrictive (reviewed in 64). It is a member of a superfamily that includes several forms of TGF-f3, activins, inhibins, and a number of other proteins (64, 84). It has also been found to be identical to several agents, including the myogenesis Differentiation Inhibitor discovered in this labora tory ( 17), that were initially described as having some other activity. At least five isoforms of TGF-f3 have been sequenced, and all members of the family consist of disulfide-linked dimers of 12,500 dalton peptide chains. TGF-f3 is present in relatively large quantities in the Q' granules of platelets, activated lymphocytes, and macrophages, and it has been found (at least in small quantities) in every cell line examined (64).
INHIBITION OF MYOBLAST DIFFERENTIATION
The initial report of inhibi
tion of terminal myogenic differentiation by a crude TGF-f3 preparation was
made by Evinger-Hodges et al ( 17), but the low level of secretion of this growth factor by the BRL cell line prevented its identification as TGF-f3.
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Three independent reports (27, 48, 56) identifying TGF-f3 as an inhibitor of myogenic differentiation appeared virtually simultaneously, and all showed essentially the same results in L6Al, L6E9, C2, and BC3Hl cell lines. The three reports agreed that TGF-f3 blocks all measured aspects of myogenic differentiation (fusion, elevation of creatine kinase activity, appearance of acetylcholine receptors, transition from f3- and y- to a-actin, and expression of other muscle-specific mRNAs and the corresponding proteins) in a con centration-dependent fashion. TGF-f3 has substantial effects on the morpholo gy of L6 myoblasts, inducing formation of stress fibers and foci with numer ous cells arrayed in a stelliform pattern (23). It also blocks the changes in ion transport associated with differentiation (8). The physiological significance of these observations with cell lines was demonstrated by the observation (1) that TGF-f3 also blocks differentiation of satellite cells prepared from adult rat skeletal muscle, as well as that of primary myoblast cultures from rat and quail embryos (17). TGF-f3 is a potent inhibitor, with half-maximal inhibition at 6 pM (0.15 ng/ml) and complete inhibition at 20 pM (0.5 ng/ml) in L6Al cells (27). Like FGF, it acts on a relatively early step (or steps) in the presumed cascade of events that lead to terminal differentiation; it is ineffective if added after the time required for commitment [defined by Nadal-Ginard (54) as attainment of a postmitotic state], and it apparently has no effect on subsequent steps in the pathway to expression of muscle-specific genes (27, 48, 56). We (D. Ewton, J. FIorini, unpublished) find this critical step at about 24 to 30 hr after IGF-I addition for L6Al cells and 18 hr for C2 cells. The inhibition of differentia tion by TGF-f3 is reversible upon removal of the inhibitor with a time course reasonably close to that observed following addition of "differentiation medium" to myoblasts (17, 24, 56). This suggests that TGF-f3 induces a transient signal that is readily reversible, and it does not cause irreversible damage to myoblasts. MECHANISM OF INHIBITION BY TGF- f3
One clue to the action of TGF-f3 is that its effects may be mediated by two oncogenes. In response to TGF-f3, BC3Hl cells showed a dramatic increase in junB and a more modest rise in c-jun mRNA (44), as was also found to occur (59) in nonmuscle cell lines. Induction of ras expression (via an adjacent glucocorticoid response element in stably transfected C2 cells treated with dexamethasone) blocked differenti ation (31), just as does TGF-f3. Activation of ras blocked developmental induction of muscle-specific proteins (a-actin and desmin) and suppressed nonmuscle proteins (f3- and y-actin, and vimentin) (55). This inhibition of differentiation by H-ras is associated with down-regulation of expression of the MyoDl gene (38). Thus all of these observations would be consistent with TGF-f3 acting by inducing expression of jun and/or ras. This may be an oversimplification. Cotransfection with H-ras and MyoDl
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gives at least partial restoration of differentiated functions, in contrast to TGF-/3 (and FGF), which inhibit differentiation in spite of high levels of MyoDI mRNA in aza-C3HlOTl /2-derived myoblasts (38). In C2C12 myo blasts, transfection with r'as decreased myogenin mRNA, but this decrease could be reversed by cotransfection with a constituitively expressed MyoDI (41). There were similar effects of expression of fos in aza-l0Tlf2 myoblasts. These results appear to indicate that either ras is not on the TGF-/3IFGF signaling pathway, or that these inhibtors act by several pathways and ras is involved in only one of them. This view is supported by the report (33) that pertussis toxin inhibited induction of expression of several oncogenes by TGF-/3, but had no effect on the stimulation of extracellular matrix protein gene expression in AKR-2B fibroblasts. Possible roles of the most widely studied intracellular modulators of hor mone actions have been eliminated by a number of investigations. For example, there was no change in intracellular cAMP levels in BC3Hl cells following treatment with either TGF-/3 or FGF (34). Tetrahydrophorbol acetate does not inhibit differentiation in BC3Hl cells (34) or C2 cells, nor do cyclooxygenase inhibitors (K. Magri, 1. FIorini, unpublished observations). There have been some indications of effects of TGF-J3 on established second messengers in other systems (64), but their relevance to muscle differentiation is by no means certain. The actions of TGF-f3 are so varied that is not likely to be useful to extrapolate from other systems, since there is no obvious reason to expect common mechanisms for such disparate actions.
Fibroblast Growth Factor CHEMISTRY AND ACTIONS There are two forms ofFGF, basic FGF (pI 9.6) and acidicFGF (pI 5.6) (reviewed in 29). Both are single peptide chains, with basic FGF composed of 146 amino acids and acidic FGF 140 amino acids. Basic FGF is the more widely distributed and (at least in the case of inhibition of myogenic differentiation) the more potent form. The FGFs are members of a family of related growth factors and have been described by at least 23 synonyms. They are mitogenic for cells of mesoderm- and neuroectoderm derived tissues and have pleiotypic actions similar to those described above for IGFs. Like the other growth factors considered here, basicFGF is secreted by many kinds of cells. FGF is a potent inhibitor of differentiation, with half-maximal inhibition by 0.05 ng/ml basic FGF or 1 ng/ml acidic FGF (9).
RELATIONSHIP BETWEEN MITOGENIC AND DIFFERENTIATION-INHIBITING
The initial suggestion (39) that mitogens block terminal differentiation was based on the observation that differentiation occurred more rapidly in conditioned medium, from which the mitogens had presum ably been metabolized. However, this point was not unambiguously demonACTIONS OF FGF
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strated until it was shown (72) that medium conditioning decreases FGF concentrations from inhibitory levels to barely detectable amounts. Initial reports on inhibition of differentiation by FGF implied and often specifically stated that the effect was associated with its mitogenic activity (implying that other mitogens would also be inhibitors), but it was shown rather early that FGF could block differentiation under conditions in which it was not mitogen ic, and this has been confirmed by several subsequent reports (9, 42, 43, 77). Thus, although the term mitogen removal is still used loosely in describing conditions used to promote myogenic differentiation, it is clear that there is no simple relationship between cell proliferation and differentiation to form postmitotic myotubes, except that the two are mutually exclusive. Indeed, except for two reports on effects of EGF on BC3Hl cells (l0, 82), FGF is the only purified mitogen that has been shown to inhibit myogenic differentiation, and one family of mitogens (the insulin-like growth factors) stimulates the process, as indicated above. FGF blocks an early step (or steps) in the events involved in terminal myogenic differentiation. Initial indications of this (46) have been followed by a report that removal of the growth factor for as little as two and one half hr (before readdition of FGF) allowed half of the MMl4 myoblasts to become committed to terminal differentiation (9). These experiments also show that FGF (like TGF-f3) has no effect on the subsequent expression of muscle-specific genes. It should be kept in mind that all of these studies on FGF actions have been done on cell lines; it has not yet been demonstrated that FGF acts on primary muscle cells by identical mechanisms, andFGF (unlike TGF,B) did not inhibit differentia tion of rat muscle satellite cells when they were stimulated by IGF-I (1).
STAGE OF DIFFERENTIATION INHIBITED BY FGF
MECHANISM(S) OF INHIBITION BY FGF There is no mechanism yet general ly accepted for the actions of FGF on myoblasts, and it has been suggested (75) that FGF actions are mediated by a novel signaling pathway not yet known. Lathrop et al (43) preincubated BC3Hl myoblasts with FGF (in the absence of serum) and showed that this preincubation significantly decreased the lag period before initiation of 3H-thymidine incorporation in response to serum. They concluded that FGF acted by "causing quiescent BC3HI cells to exit from the Go portion of the cell cycle, and to accumulate at a new restriction point 4 to 6 hours into the G1 portion of the cell cycle." It is difficult to define this new restriction point in terms of biochemical mechanisms. One attractive possibility is that increased expression of a cellular oncogene such as c-fos or c-myc in response toFGF (29) is involved in the inhibition of differentiation. However, elevation of c-myc did not repress expression of
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muscle-specific genes in myotubes (16), and persistent c-myc expression was not required for the inhibition of differentiation by FGF (or by TGF-,B) (76). The observation (77) that cycloheximide blocks the down-regulation of CK in differentiated BC3HI cells in response to FGF suggests that the inhibition of differentiation by this growth factor requires protein synthesis. It has been shown by several laboratories that FGF inhibits expression of the myogenesis determination genes MyoDI (81) and myogenin (E. N. Olson, personal communication; J. FIorini et aI, unpublished observations). Thus each of the growth factors considered here affects myogenin expression in a direction consonant with its effect on terminal myogenic differentiation.
OTHER GROWTH FACTORS Our literature searches have yielded few reports of actions of other growth fadors on myogenesis. Platelet-derived growth factor (PDGF) has been shown to have a number of effects on smooth muscle cells, but we (J. R. FIorini, unpublished observations) and C. D. Stiles (personal communication) could' detect no effect of this agent on skeletal muscle cells. It has been reported to be produced by rat skeletal myoblasts (73), but the physiological significance of this is not apparent. As mentioned above, there are two reports that EGF inhibits expression of some differentiated functions in BC3HI cells (10, 82), but it has no effect on primary myoblasts (30) or muscle cell lines (45,25). In many attempts, we have never observed any significant effects of EGF on L6 or C2 cells (25). CHANGES IN RECEPTORS WITH DIFFERENTIATION The finding that binding of EGF by mouse myoblasts decreases as these cells differentiate prompted Hauschka (45) to suggest that loss of growth factor receptors might account for the postmitotic state of myotubes. MM14 cells do not divide in response to EGF, so functional consequences of this decline in receptor number could not be demonstrated. Subsequently it was found that FGF receptors also disappear from the surfaces of differentiated MM l 4 cells (57). TGF-,B receptors also disappear from several muscle cell lines as they differentiate (21). The parallel decrease in TGF-,B-stimulated amino acid uptake in L6A1 myotubes showed that the receptors that disappeared were biologically functional. In contrast, there was only a 50% decrease in binding of TGF-f3 as BC3HI cells differentiated (21), and there was no decrease in FGF receptors (57), perhaps because these cells do not fuse. Uncoupling of biochemical and morphological differentiation by EGTA showed that the down-regulation of TGF-,B receptors is associated with fusion, but not induc tion of muscle-specific proteins (35),
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The generalization that mitogen receptors decrease with differentiation does not extend to the IGFs, which are very active mitogens for muscle cells. Under conditions in which TGF-fJ receptors virtually disappeared, we (21) detected little or no decrease in binding of 125I-IGF-I and no change in responses to IGF-1. Beguinot et al (5) reported a decrease (about 70%) in IGF-I receptors with differentiation in a different subline of L6 cells, but this may be a result of their expressing binding on the basis of protein (which continues to accumulate in myotubes) rather than DNA content of the cul tures. There is no decrease in binding of IGF-I and IGF-II to human muscle cells during differentiation (74), and there is a transient rise in IGF-I binding (78) and a dramatic increase in IGF-II binding (79) as C2 cells differentiate. Thus the decrease in FGF binding mentioned above is just one of many membrane changes that occur during differentiation, and it does not account for the postmitotic state of myotubes.
IN VIVO SIGNIFICANCE All three growth factors considered in detail here are secreted by skeletal muscle, so it is possible that they might act by autocrine/paracrine mecha nisms; current experiments (J. FIorini et aI, unpublished) indicate that IGF-II may play an important role in spontaneous differentiation of C2 cells incu bated in 2% horse serum. Both at the rnRNA and protein levels, genes for the IGFs have been shown to be expressed under conditions in which increased muscle growth occurs. These include regeneration (36) and hypertrophy induced by growth hormone administration (53). Allen & Boxhom (1) show ed that various combinations of IGF-I, TGF-fJ, and FGF could simulate the sequential phases of muscle regeneration. The TGF-fJ gene is expressed in mouse embryos at both the mRNA and protein (64) levels and (with FGF) is thought to play an important role in mesoderm formation in Xenopus oocytes (83). FGF levels in the chick embryo limb bud are high enough to inhibit differentiation of myoblasts (72), and FGF delays the onset of differentiation in muscle precursor cells from limb buds (71). Strohman's group (88) found that FGF is stored in the extracellular matrix of mature muscle and suggested that it plays a role in muscle hypertrophy. Although it is not yet possible to demonstrate conclusively that these agents affect normal growth and develop ment of muscle, it seems very likely that they do. SUMMARY AND CONCLUSIONS
Three families of growth factors/hormones have major effects on the differen tiation of skeletal muscle cells. Two (FGF and TGF-fJ) are potent inhibitors, and the third (IGF) exhibits a biphasic stimulatory action (but is not
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inhibitory even at high concentrations). All of these affect the expression of myogenin, one of the recently discovered family of myogenesis controlling genes, and FGF and TGF-f3 have been shown to inhibit the expression of MyoDI (and probably myf-5 and herculin) as well. These agents inhibit or stimulate (respectively) all measured aspects of myogenic differentiation fusion, expression of a set of muscle-specific genes, and attainment of a postmitotic state-in all cells that are capable of these responses, whether cell lines or primary muscle cell cultures. It now seems clear that the myogenesis
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controlling genes regulate the entire family of muscle-specific proteins. Therefore the demonstration that expression of these genes is controlled (both positively and negatively) by specific growth factors that are now available at high purity and in useful quantities offers the possibility of understanding myogenic differentiation at a level of molecular detail that is very exciting. ACKNOWLEDGMENTS
The preparation of this review was supported by grant HL11551 from the National Institutes of Health, United States Public Health Service. We thank the many colleagues who made manuscripts available to us prior to their publication. Literature Cited 1. Allen, R. E., Boxhorn, L. K. 1989.
Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta. in sulin-like growth factor-I, and fibroblast growth factor. J. Cell. Physiol. 138:
311-15 2. Ballard, F. J. , Read, L. C., Francis, G. L., Bagley, C. J., Wallace, J. C. 1986.
Binding properties and biological pot encies of insulin-like growth factors in L6 myoblasts. Biochem. J. 233:223-
30 3. Baxter, R. C . 1988. The insulin-like growth factors and their binding pro teins. Comp. Biochem. Physiol. 91B:
229-35 4. Baxter, R. C., Martin, J. L. 1989. Bind
ing proteins for the insulin-like growth factors: structure, regulation and func tion. Progr. Growth Factor Res. 1:49-
68 5. Beguinot, F., Kahn, C. R., Moses, A. C., Smith, R. J. 1985. Distinct biologi
cally active receptors for insulin, in sulin-like growth factor I, and insulin like growth factor II in cultured skeletal muscle cells. J. Bioi. Chem. 260:15892-
98 6. Braun, T., Bober, E., Buschhausen Denker, O. Kotz, S., Grzeschik, K.-H.,
Arnold, H. H. 1989. Differential expres sion of myogenic determination genes in muscle cells: possible autoactivation by the myf gene products. EMBO J.
8:3617-25 7. Braun, T., Buschhausen-Denker, G.,
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1989. A novel human muscle factor re
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53 9. Clegg, C. H., Linkhart, T. A . , Olwin , B. B., Hauschka, S. D. 1987. Growth
factor control of skeletal muscle differ entiation: commitment to terminal di fferentiation occurs in G1 phase and is repressed by fibroblast growth factor. 1. Cell BioI. 105:949-56 10. Connolly, J. A., Sarabia, V. E., Kelvin, D. J., Wang, E. 1988. The dis appearance of a cyclin-like protein and the appearance of statin is correlated with the onset of differentiation during
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CONTROLS OF MYOGENESIS myogenesis in vitro. Exp. Cell Res. 174:461-71 11. Davis, R. L., Weintraub, H., Lassar, A. B. 1987. Expression of a single trans feeted eDNA converts fibroblasts to myoblasts. Cell 51:987-1000 12. de la Haba, G., Cooper, G. W., Elting, V. 1966. Hormonal requirements for myogenesis in vitro: insulin and soma totropin. Proc. Natl. Acad. Sci. USA 56:1719-23 13. De Vroede, M. A., Romanus, J. A., Standaert, M. L., Pollett, R. J., Nissley, S. P., Rechler, M. M. 1984. Interaction of insulin-like growth factors with a nonfusing mouse muscle cell line: bind ing, action, and receptor down regulation. Endocrinology 114:1917-29 14. Edmondson, D. G., Olson, E. N. 1989. A gene with homology to the myc sim ilarity region of myoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation pro gram. Genes Dev. 3:62�0 15. Elgin, R. G., Busby, W. H. Jr., Clem mons, D. R. 1987. An insulin-like growth factor (lGF) binding protein en hances the biological response to IGF-I. Proc. Nat!. Acad. Sci. USA 84:3254--58 16. Endo, T., Nadal-Ginard, B. 1986. Transcriptional and posttranscriptional control of c-myc during myogenesis: its rnRNA remains inducible in differenti ated cells and does not suppress the di fferentiated phenotype. Mol. Cell. Bioi. 6:1412-21 17. Evinger-Hodges, M. J., Ewton, D. Z., Seifert, S. C., Fiorini, 1. R. 1982. In hibition of myoblast differentiation in vitro by a protein isolated from liver cell medium. J. Cell Bioi. 93:395-401 18. Ewton, D. Z., Erwin, B. G., Pegg, A. E., Fiorini, J. R. 1984. The role of polyamines in somatomedin-stimulated differentiation of L6 myoblasts. J. Cell. Physiol. 120:263-70 19. Ewton, D. Z., Falen, S. L., FIorini, J. R. 1987. The type II IGF receptor has low affinity for IGF-I analogs: pleiotypic actions of IGFs on myoblasts are apparently mediated by the type I recep tor. Endocrinology 120:115-24 20. Ewton, D. Z., FIorini, J. R. 1981. Effect of somatomedins and insulin on myoblast differentiation in vitro. Dev. Bioi. 86:31-39 21. Ewton, D. Z., Spizz, G., Olson, E. N., FIorini, J. R. 1988. Decrease in transforming growth factor-/3 binding and action during differentiation in mus cle cells. J. Bioi. Chern. 263:4029-32 22. Fiorini, J. R. 1987. Hormonal control of muscle growth. Muscle Nerve 7:577-98
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35. Hu, J. S., Olson, E. N. 1990. Function al receptors for transforming growth fac tor-13 are retained by biochemically dif ferentiated C2 myocytes in growth factor deficient medium containing EGTA, but down-regulated during terminal differ entiation. J. Bioi. Chem. 265:7914--19 36. Jennische, E. , Hansson, H. -A. 1987. Regenerating skeletal muscle cells ex press insulin-like growth factor I. Acta Physiol. Scand. 130:327-32 37. Kiess, W., Haskell, J. F., Greenstein, L. A., Miller, B. E., Aarons, A. L., et al. 1987. An antibody that blocks in sulin-like growth factor (lGF) binding to the type II IGF receptor is neither an agonist nor an inhibitor of IGF stimulated biologic responses in L6 myoblasts. 1. Bioi. Chem. 262:1274551 38. Konieczny, S. F., Drobes, B. L., Menke, S. L. , Taparowsky, E. J. 1989. Inhibition of myogenic differentiation by the H-ras oncogene is associated with the down regulation of the myoD1 gene. Oncogene 4:473-81 39. Konigsberg, I. R. 1971. Diffusion mediated control of myoblast fusion. Dev. Bioi. 26:133-52 40. Kovacina, K. S . , Steele-Perkins, G. 1989. A role for the insulin-like growth factor IIImannose-6-phosphate receptor in the insulin-induced inhibition of pro tein catabolism. Mol. Endocrinol. 3: 901-6 41. Lassar, A. B., Buskin, J. N., Lockshon, D . , Davis, R. L. , Apone, S., et al. 1989. MyoD is a sequence specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer. Cell 58:823-31 42. Lathrop, B., Olson, E., Glaser, L. 1985. Control by fibroblast growth fac tor of differentiation in the BC3Hl mus cle cell line. J. Cell BioI. 100:154047 43. Lathrop, B., Thomas, K., Glaser, L. 1985. Control of myogenic differentia tion by fibroblast growth factor is medi ated by position in the G1 phase of the cell cycle. 1. Cell BioI. 101:2194-98 44. Li, L., Hu, J. S . , Olson, E. N. 1990. Different members of the jun proto oncogene family exhibit distinct patterns of expression in response to type 13 transforming growth factor. 1. Bioi. Chem. 265:1556-62 45. Lim, R. W., Hauschka, S. D. 1984. A rapid decrease in epidermal growth factor-binding capacity accompanies the terminal differentiation of mouse myo blasts in vitro. J. Cell Bioi. 98:739--47
46. Linkhart, T. A., Clegg, C. H., Lim, R. W., Merrill, G. F. , Chamberlain, J. S., Hauschka, S. D. 1982. Control of mouse myoblast commitment to terminal differentiation by mitogens. In Molecu lar and Cellular Control of Muscle De velopment, ed. M. L. Pearson, H. F. Ebstein. 8: 877-82. Cold Spring Harbor Conf. 47. Loeb, J. N . , Strickland, S. 1987. Hor mone binding and coupled response rela tionships in systems dependent on the generation of secondary mediators. Mol. Endocrinol. 1 :75-82 48. Massague, J., Cheifetz, S . , Endo, T., Nadal-Ginard, B. 1986. Type beta transforming growth factor is an in hibitor of myogenic differentiation. Proc. Natl. Acad. Sci. USA 83:8206-10 49. McCusker, R. H., Camacho-Hubner, C., Clemmons, D. R. 1989. Identifica tion of the types of insulin-like growth factor binding proteins that are secreted by muscle cells in vitro. 1. BioI. Chem. 264:7795-7800 50. McCusker, R. H. Clemmons, D. R. 1988. Insulin-like growth factor binding protein secretion by muscle cells: effect of cellular differentiation and prolifera tion. 1. Cell. Physiol. 137:505-12 51. Miner, 1. H., Wold, B. 1990. Herculin, a fourth member of the myoD family of myogenic regulatory genes. Proc. Natl. Acad. Sci. USA 87:1089-93 52. Morgan, D. O., Edman, J. C. , Stand ring, D. N., Fried, V. A., Smith, M. C., et al. 1987. Insulin-like growth factor-II receptor as a multifunctional binding protein. Nature 329:301-7 53. Murphy, L. J., Bell, G. I., Duckworth, M. L., Friesen, H. G. 1987. Identifica tion, characterization, and regulation of a rat complementary deoxyribonucleic acid which encodes insulin-like growth factor-I. Endocrinology 121:684--91 54. Nadal-Ginard, B. 1978. Commitment, fusion, and biochemical differentiation of a myogenic cell line. Cell 15:855-63 55. Olson, E. N., Capetanaki, Y. O. 1989. Developmental regulation of in termediate filament and actin mRNAs during myogenesis is disrupted by oncogenic ras genes. Oncogene 4:90713 56. Olson, E. N. , Sternberg, E., Hu, J. S . , Spizz, G., Wilcox, C. 1986. Regulation of myogenic differentiation by type beta transforming growth factor. 1. Cell Bioi. 103:1799-1806 57. Olwin, B. B . , Hauschka, S. D. 1988. Cell surface fibroblast growth factor and epidermal growth factor receptors are permanently lost during skeletal muscle
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Further
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HORMONES, GROWTH FACTORS, Annu. Rev. Physiol. 1991.53:201-216. Downloaded from www.annualreviews.org by University of Massachusetts - Amherst on 04/15/13. For personal use only.
AND MYOGENIC DIFFERENTIATION James R. Florini, Daina Z. Ewton, and Karen A. Magri Biology Department, Syracuse University, Syracuse, New York 13244
KEY WORDS:
myogenesis, insulin-like growth factors, transforming growth factor-/3, fibrob last growth factor, myogenin, MyoDl
INTRODUCTION The import�mce of honnones and growth factors in regulating the growth and differentiation of muscle cells is now widely recognized. This field has been summarized recently by two of the authors of this chapter (26), and other reviews of related topics (65, 69) provide additional infonnation. Since our last review of the field, it has been revolutionized by the discovery of the myogenesis detennination genes, which bring a new focus to the study of myogenesis.
Definitions and Concepts MYOGENESIS DETERMINATION GENES MyoDI (11) and myd (60) were first characterized as genes that control initial determination to the myogenic lineage. Subsequently, Myf-5 (7) and MRF4 (63)/herculin (51) have been added to the family of genes that are found exclusively in skeletal muscle and can convert nonmuscle cells to the myogenic lineage (6). However, MyoDI is not expressed in BC3Hl or L6 myoblasts (6), and MRF4 is not expressed in immortalized myogenic cell lines (63), so it appears that not all of these genes are essential for expression of the myogenic program in all muscle cells. In contrast, m yogenin, which has been identified as a putative controller of tenninal differentiation in both rat (85) and mouse ( 14) muscle cell lines, is expressed in every cell undergoing myogenic differentiation (6, 14, 85). In
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addition, myogenin also induces nonmuscle cells to express muscle-specific genes (14, 85). HORMONES AND GROWTH FACTORS The distinction between protein hor mones and growth factors is arbitrary and has little biological significance. In general, hormones are synthesized and secreted by discrete endocrine glands, while growth factors are secreted by many different kinds of cells. All of these agents are relatively small peptides that act through a receptor on the cell membrane to generate an intracellular signal that is not yet understood. Until recently research on growth factors was severely restricted by the unavailabil ity of purified materials in sufficient quantities. All of the growth factors discussed here are now commercially available, making possible experiments that could not be done in the past. The ready availability of these agents allows study of myogenic differentiation in well-defined systems that give answers much less ambiguous than was possible in the days of fetal bovine serum and chick embryo extract.
Most of the work on control of skeletal myogenesis has been done on three immortalized cell lines. The rat L6 line (86) has probably been the most widely used; it provides a useful combination of properties, with a convenient rate of differentiation that can be modified by medium components. Its major disadvantage is its lack of responsiveness to fibroblast growth factor (FGF). The mouse C2 line (87) differentiates more rapidly than L6 cells, and it is responsive to FGF. The mouse BC3Hl line is unusual in several ways. It was initially isolated (70) from an intracranial tumor, and it does not fuse to form postmitotic myotubes, thus allowing study of the reversibility of myogenesis. In spite of their convenience, these cell lines have limitations. The fact that none of these cells exhibits senescence shows that they are different from normal diploid cells in at least one major property. For this reason, we feel strongly that all major conclusions should be verified in primary cultures of skeletal muscle cells.
MUSCLE CELL LINES
Scope of This Review This chapter concentrates on the differentiation of skeletal muscle and the growth factors that stimulate [insulin-like growth factors (IGFs)] and inhibit [FGF, transforming growth factor (TGF-f3)] it. We mention those instances in which effects of hormones or growth factors on expression of the myogenic determination genes have been reported. The limited space allottment forces us to omit references to many publications that we consider important, and we apologize for the omissions.
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EFFECTS OF SPECIFIC HORMONES AND GROWTH FACTORS
Insulin-like Growth Factors (IGFs) CHEMISTRY AND SECRETION
The IGFs (also known as somatomedins) are
28). As is true of all of the growth factors considered here, the IGFs were initially described as several different agents that were ultimately recognized as identical. The primary agents are IGF-I (70 amino acids, 7649 daltons), IGF-II (67 amino acids, 747 1 daltons), and insulin (52 amino acids, 5733 daltons), listed in order of their potency in stimulating myogenic differentia tion (20). Like the other growth factors described here, IGF-I is synthesized and apparently secreted by a number of cell types, although there is some reason to believe that the primary source of circulating IGFs is the liver (28).
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a family of small peptides similar in structure to proinsulin (reviewed in
IGFs are also synthesized in substantial quantities by cultured muscle cells
(78, 79). ANABOLIC ACTIONS The IGFs exert pleiotypic anabolic actions on skele tal muscle cells (reviewed in 22), as they do on many other kinds of cells (3). These include stimulation of the f ollowing processes: amino acid uptake and incorpor ati on into protein, uridine and thymidine incorporation into nucleic acids, glucose uptake, cell proliferation, etc. IGFs also suppress degradation of proteins. These activities occur in several cell lines as well as in primary myoblast cultures and satellite cells. Unlike FGF, IGFs stimulate myoblast proliferation in completely defined medium in the absence of other serum components.
STIMULATION OF DIFFERENTIATION
The insulin-like hormones are unique
among growth factors and hormones in that they stimulate, rather than inhibit, myogenic differentiation. It was first reported
25 years ago (12) that insulin
(at p.M levels) increased differentiation in chick embryo muscle cells and this
& FIorini (20) first L6 cell differentiation, both at the morphological (fusi on) and biochemical (CK activity) levels, and suggested that the previous observations on insulin represented cross-reaction with the IGF-I receptor. This stimulation is not limited to cell lines; IGFs stimulate differentiation in chick embryo muscle cells (67) and in rat satellite cells (1). Thus the stimulation of differentiation by the insulin-like hormones is a general phenomenon, except in the case of cell lines such as C2 cells that secrete large amounts of these hormones (78, 79) and may thus stimulate their finding was repeated in other laboratories
(26).
Ewton
showed that physiological levels of IGFs stimulated
own differentiation by autocrine/paracrine mechanisms.
A curious aspect of the stimulation by IGFs, at least in L6 cells, is the
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biphasic concentration dependency (25). There is the expected increased stimulation from approximately 0.5 to 20 ng/mI, but this is followed by a rather sharp peak: and lower levels of differentiation down to near control values at very high concentrations (360 ng/mI). Elevation of myogenin mRNA by IGF-I exhibited the same biphasic response (1. FIorini et al, manuscript in preparation). Recent experiments (1. A. Foster, unpublished) show a similar biphasic response to IGF-I in the stimulation of expression of the elastin gene in aortic smooth muscle cells. The mechanism of this biphasic response is not apparent. MECHANISM OF IOF STIMULATION OF DIFFERENTIATION
Some obvious explanations were considered soon after the discovery of the stimulation by IGF-1. One was that increased differentiation was a result of the greater culture density resulting from the mitogenic effects of IGF-1. This possibility was eliminated by two different approaches-use of inhibitors (80) and different plating densities (20). The possibility that the cells differentiated upon treatment with IGF-I because of generally better metabolic condition (the "happy cell" explanation) was eliminated by showing that differentiation was stimulated by IGF-I and IGF-II, even in serum concentrations that allowed high rates of cell prolifera tion (25). An extended study of effects of polyamines led to the conclusion that elevated polyamine levels are necessary but not sufficient for differentia tion (18). Possibilities that have been eliminated
In our view the most likely mechanism of IGF-I action is induction of myogenin, the most universally, expressed of the myogenesis determination genes. Recent work in this laboratory (J. FIorini et aI, manuscript in preparation) has shown that treatment of L6Al cells with IGF-I gives a large (60-fold) increase in myogenin mRNA content at 30 to 40 hr, well before the elevation of creatine kinase. This is the only known case in which expression of the myogenin gene is increased, rather than inhibited, by a well-characterized agent. This elevation exhibits a concentration de pendency like that of the stimulation of differentiation, including the sharply decreased stimulation at higher IGF-I concentrations. An antisense oligomer complementary to the first 15 nucleotides of the translated portion of myoge nin mRNA blocks the stimulation of differentiation, so we conclude that increased myogenin gene expression is the primary mechanism by which IGF-I stimulates differentiation of muscle cells. The rather long time required for this elevation suggests that a rapid single step is not involved, i.e. that a substrate for the IGF-I receptor tyrosine kinase activity does not act directly on an enhancer for the myogenin gene.
Induction of myogenin
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IGF-I receptor Three different receptors-the IOF-I receptor, the IOF-II receptor, and the insulin receptor (for reviews see 62 and 66)-might mediate the stimulation of differentiation. The presence of all three receptors in cultured muscle cells has been demonstrated (2, 5, 13, 19). Our early experiments suggested that two different receptors might be involved in mediating various actions of IOFs on muscle, but we ultimately concluded ( 19) that all actions of IOFs were mediated by the type I receptor that exhibits the specificity IOF-I > IOF-I1 > insulin. Ballard (2) similarly concluded that the stimulation of protein and DNA synthesis in L6 cells was also mediated by the IGF-I receptor. Conclusive support for the primary role of the IGF-I receptor was provided by Kiess et al (37), who developed an antibody that specifically blocked the binding of IOF-II to the IOF-II receptor without affecting the binding of IGF-I to its receptor. This antibody did not alter the effects of IOF-II on amino acid or glucose uptake, or on leucine incorporation into proteins in L6 cells, thus indicating that the IGF-II receptor did not mediate these actions of the IGFs. An important theoretical paper (47) shows that differences in agonist potency for various actions mediated by the same receptor-some of the actions of IOF-I differ more than tenfold in the concentration required for half-maximal effect ( 19)-can be explained by differences in affinities of second messengers for their intracellular receptors. IGF-II receptor The role of the IOF-II receptor remains unclear. It is present in large quantities in L6 cells (5), and it is strikingly elevated during differen tiation in C2 cells (79), but no function has been shown for it in muscle cells. Its surprising identity to the mannose-6-phosphate receptor (which plays a role in targeting of lysosomal enzymes) is well established (52) and suggests that it may be involved in metabolism of IGF-II. This possibility was sup ported by the observation (37) that an antibody to the IOF-II receptor inhibited IGF-II degradation by 90% in L6 myoblasts. Other antibodies to the IOF-II1 mannose-6-phosphate receptor blocked the insulinlIOF-I-induced decrease in protein catabolism of CHO cells, which suggests that this might result from a disruption of movement of lysosomal enzymes to lysosomes (40). IGF binding proteins The IOFs are unlike other peptide hormones in that they circulate bound to a larger protein (3, 4). Initially it was believed that the binding protein functioned to decrease metabolism and regulate the concentra tion of free IOFs, and most early studies showed that binding proteins inhibited IGF actions (89). More recently, stimulatory effects of binding proteins have been reported ( 15), and a number of different binding proteins have been described (4). Human fetal myoblasts (32), as well as L6 and BC3HI myoblasts and porcine smooth muscle cells (49, 50), all secrete IOF binding proteins, and this secretion is regulated by insulin and IOF-I. Tollef-
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sen et al (78, 79) found a 30-fold increase in binding proteins during differen tiation of C2 cells. Thus binding proteins may play an important role in regulating the growth and differentiation of skeletal and smooth muscle,
although the specifics of that role are not yet understood. Effects on oncogene expression Increased oncogene expression is a fre quently reported early effect of growth factors in many cells. In L6 cells, there is a fourfold increase in c10s mRNA by IGF-I; several other oncogenes are not affected (58). The report (61) that elevated c fos expression in transfected L6 myoblasts inhibited differentiation makes this an unlikely mediator of the IGF action considered here. There is induction of c-myc by high levels ( 1 p,g/ml) o f IGF-I ( 16), but a t these levels differentiation of myoblasts is not stimulated (see discussion of the biphasic response above). A listing of oncogenes that exhibit changes during myogenic differentiation (26) and a more extensive treatment of the subject (68) are available in other reviews. The available evidence does not point to any oncogene likely to play a role in the stimulation of myogenic differentiation by IGF, although they may well be involved in proliferative and other anabolic responses.
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We have taken a substantial step toward understanding the stimulation of differentiation with the discovery that IGFs stimulate expression of the myo
genin gene. Major questions remain about events that occur between occupan cy of the IGF receptor and increased myogenin mRNA levels that lead to terminal differentiation of muscle cells.
Transforming Growth Factor-{3 (TGF-{3) CHEMISTRY AND SECRETION TGF-f3 was initially characterized as an in ducer of phenotypic transformation of fibroblastic cells, but it has sub sequently been shown to have such a wide variety of actions that its name is clearly unduly restrictive (reviewed in 64). It is a member of a superfamily that includes several forms of TGF-f3, activins, inhibins, and a number of other proteins (64, 84). It has also been found to be identical to several agents, including the myogenesis Differentiation Inhibitor discovered in this labora tory ( 17), that were initially described as having some other activity. At least five isoforms of TGF-f3 have been sequenced, and all members of the family consist of disulfide-linked dimers of 12,500 dalton peptide chains. TGF-f3 is present in relatively large quantities in the Q' granules of platelets, activated lymphocytes, and macrophages, and it has been found (at least in small quantities) in every cell line examined (64).
INHIBITION OF MYOBLAST DIFFERENTIATION
The initial report of inhibi
tion of terminal myogenic differentiation by a crude TGF-f3 preparation was
made by Evinger-Hodges et al ( 17), but the low level of secretion of this growth factor by the BRL cell line prevented its identification as TGF-f3.
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Three independent reports (27, 48, 56) identifying TGF-f3 as an inhibitor of myogenic differentiation appeared virtually simultaneously, and all showed essentially the same results in L6Al, L6E9, C2, and BC3Hl cell lines. The three reports agreed that TGF-f3 blocks all measured aspects of myogenic differentiation (fusion, elevation of creatine kinase activity, appearance of acetylcholine receptors, transition from f3- and y- to a-actin, and expression of other muscle-specific mRNAs and the corresponding proteins) in a con centration-dependent fashion. TGF-f3 has substantial effects on the morpholo gy of L6 myoblasts, inducing formation of stress fibers and foci with numer ous cells arrayed in a stelliform pattern (23). It also blocks the changes in ion transport associated with differentiation (8). The physiological significance of these observations with cell lines was demonstrated by the observation (1) that TGF-f3 also blocks differentiation of satellite cells prepared from adult rat skeletal muscle, as well as that of primary myoblast cultures from rat and quail embryos (17). TGF-f3 is a potent inhibitor, with half-maximal inhibition at 6 pM (0.15 ng/ml) and complete inhibition at 20 pM (0.5 ng/ml) in L6Al cells (27). Like FGF, it acts on a relatively early step (or steps) in the presumed cascade of events that lead to terminal differentiation; it is ineffective if added after the time required for commitment [defined by Nadal-Ginard (54) as attainment of a postmitotic state], and it apparently has no effect on subsequent steps in the pathway to expression of muscle-specific genes (27, 48, 56). We (D. Ewton, J. FIorini, unpublished) find this critical step at about 24 to 30 hr after IGF-I addition for L6Al cells and 18 hr for C2 cells. The inhibition of differentia tion by TGF-f3 is reversible upon removal of the inhibitor with a time course reasonably close to that observed following addition of "differentiation medium" to myoblasts (17, 24, 56). This suggests that TGF-f3 induces a transient signal that is readily reversible, and it does not cause irreversible damage to myoblasts. MECHANISM OF INHIBITION BY TGF- f3
One clue to the action of TGF-f3 is that its effects may be mediated by two oncogenes. In response to TGF-f3, BC3Hl cells showed a dramatic increase in junB and a more modest rise in c-jun mRNA (44), as was also found to occur (59) in nonmuscle cell lines. Induction of ras expression (via an adjacent glucocorticoid response element in stably transfected C2 cells treated with dexamethasone) blocked differenti ation (31), just as does TGF-f3. Activation of ras blocked developmental induction of muscle-specific proteins (a-actin and desmin) and suppressed nonmuscle proteins (f3- and y-actin, and vimentin) (55). This inhibition of differentiation by H-ras is associated with down-regulation of expression of the MyoDl gene (38). Thus all of these observations would be consistent with TGF-f3 acting by inducing expression of jun and/or ras. This may be an oversimplification. Cotransfection with H-ras and MyoDl
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gives at least partial restoration of differentiated functions, in contrast to TGF-/3 (and FGF), which inhibit differentiation in spite of high levels of MyoDI mRNA in aza-C3HlOTl /2-derived myoblasts (38). In C2C12 myo blasts, transfection with r'as decreased myogenin mRNA, but this decrease could be reversed by cotransfection with a constituitively expressed MyoDI (41). There were similar effects of expression of fos in aza-l0Tlf2 myoblasts. These results appear to indicate that either ras is not on the TGF-/3IFGF signaling pathway, or that these inhibtors act by several pathways and ras is involved in only one of them. This view is supported by the report (33) that pertussis toxin inhibited induction of expression of several oncogenes by TGF-/3, but had no effect on the stimulation of extracellular matrix protein gene expression in AKR-2B fibroblasts. Possible roles of the most widely studied intracellular modulators of hor mone actions have been eliminated by a number of investigations. For example, there was no change in intracellular cAMP levels in BC3Hl cells following treatment with either TGF-/3 or FGF (34). Tetrahydrophorbol acetate does not inhibit differentiation in BC3Hl cells (34) or C2 cells, nor do cyclooxygenase inhibitors (K. Magri, 1. FIorini, unpublished observations). There have been some indications of effects of TGF-J3 on established second messengers in other systems (64), but their relevance to muscle differentiation is by no means certain. The actions of TGF-f3 are so varied that is not likely to be useful to extrapolate from other systems, since there is no obvious reason to expect common mechanisms for such disparate actions.
Fibroblast Growth Factor CHEMISTRY AND ACTIONS There are two forms ofFGF, basic FGF (pI 9.6) and acidicFGF (pI 5.6) (reviewed in 29). Both are single peptide chains, with basic FGF composed of 146 amino acids and acidic FGF 140 amino acids. Basic FGF is the more widely distributed and (at least in the case of inhibition of myogenic differentiation) the more potent form. The FGFs are members of a family of related growth factors and have been described by at least 23 synonyms. They are mitogenic for cells of mesoderm- and neuroectoderm derived tissues and have pleiotypic actions similar to those described above for IGFs. Like the other growth factors considered here, basicFGF is secreted by many kinds of cells. FGF is a potent inhibitor of differentiation, with half-maximal inhibition by 0.05 ng/ml basic FGF or 1 ng/ml acidic FGF (9).
RELATIONSHIP BETWEEN MITOGENIC AND DIFFERENTIATION-INHIBITING
The initial suggestion (39) that mitogens block terminal differentiation was based on the observation that differentiation occurred more rapidly in conditioned medium, from which the mitogens had presum ably been metabolized. However, this point was not unambiguously demonACTIONS OF FGF
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strated until it was shown (72) that medium conditioning decreases FGF concentrations from inhibitory levels to barely detectable amounts. Initial reports on inhibition of differentiation by FGF implied and often specifically stated that the effect was associated with its mitogenic activity (implying that other mitogens would also be inhibitors), but it was shown rather early that FGF could block differentiation under conditions in which it was not mitogen ic, and this has been confirmed by several subsequent reports (9, 42, 43, 77). Thus, although the term mitogen removal is still used loosely in describing conditions used to promote myogenic differentiation, it is clear that there is no simple relationship between cell proliferation and differentiation to form postmitotic myotubes, except that the two are mutually exclusive. Indeed, except for two reports on effects of EGF on BC3Hl cells (l0, 82), FGF is the only purified mitogen that has been shown to inhibit myogenic differentiation, and one family of mitogens (the insulin-like growth factors) stimulates the process, as indicated above. FGF blocks an early step (or steps) in the events involved in terminal myogenic differentiation. Initial indications of this (46) have been followed by a report that removal of the growth factor for as little as two and one half hr (before readdition of FGF) allowed half of the MMl4 myoblasts to become committed to terminal differentiation (9). These experiments also show that FGF (like TGF-f3) has no effect on the subsequent expression of muscle-specific genes. It should be kept in mind that all of these studies on FGF actions have been done on cell lines; it has not yet been demonstrated that FGF acts on primary muscle cells by identical mechanisms, andFGF (unlike TGF,B) did not inhibit differentia tion of rat muscle satellite cells when they were stimulated by IGF-I (1).
STAGE OF DIFFERENTIATION INHIBITED BY FGF
MECHANISM(S) OF INHIBITION BY FGF There is no mechanism yet general ly accepted for the actions of FGF on myoblasts, and it has been suggested (75) that FGF actions are mediated by a novel signaling pathway not yet known. Lathrop et al (43) preincubated BC3Hl myoblasts with FGF (in the absence of serum) and showed that this preincubation significantly decreased the lag period before initiation of 3H-thymidine incorporation in response to serum. They concluded that FGF acted by "causing quiescent BC3HI cells to exit from the Go portion of the cell cycle, and to accumulate at a new restriction point 4 to 6 hours into the G1 portion of the cell cycle." It is difficult to define this new restriction point in terms of biochemical mechanisms. One attractive possibility is that increased expression of a cellular oncogene such as c-fos or c-myc in response toFGF (29) is involved in the inhibition of differentiation. However, elevation of c-myc did not repress expression of
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muscle-specific genes in myotubes (16), and persistent c-myc expression was not required for the inhibition of differentiation by FGF (or by TGF-,B) (76). The observation (77) that cycloheximide blocks the down-regulation of CK in differentiated BC3HI cells in response to FGF suggests that the inhibition of differentiation by this growth factor requires protein synthesis. It has been shown by several laboratories that FGF inhibits expression of the myogenesis determination genes MyoDI (81) and myogenin (E. N. Olson, personal communication; J. FIorini et aI, unpublished observations). Thus each of the growth factors considered here affects myogenin expression in a direction consonant with its effect on terminal myogenic differentiation.
OTHER GROWTH FACTORS Our literature searches have yielded few reports of actions of other growth fadors on myogenesis. Platelet-derived growth factor (PDGF) has been shown to have a number of effects on smooth muscle cells, but we (J. R. FIorini, unpublished observations) and C. D. Stiles (personal communication) could' detect no effect of this agent on skeletal muscle cells. It has been reported to be produced by rat skeletal myoblasts (73), but the physiological significance of this is not apparent. As mentioned above, there are two reports that EGF inhibits expression of some differentiated functions in BC3HI cells (10, 82), but it has no effect on primary myoblasts (30) or muscle cell lines (45,25). In many attempts, we have never observed any significant effects of EGF on L6 or C2 cells (25). CHANGES IN RECEPTORS WITH DIFFERENTIATION The finding that binding of EGF by mouse myoblasts decreases as these cells differentiate prompted Hauschka (45) to suggest that loss of growth factor receptors might account for the postmitotic state of myotubes. MM14 cells do not divide in response to EGF, so functional consequences of this decline in receptor number could not be demonstrated. Subsequently it was found that FGF receptors also disappear from the surfaces of differentiated MM l 4 cells (57). TGF-,B receptors also disappear from several muscle cell lines as they differentiate (21). The parallel decrease in TGF-,B-stimulated amino acid uptake in L6A1 myotubes showed that the receptors that disappeared were biologically functional. In contrast, there was only a 50% decrease in binding of TGF-f3 as BC3HI cells differentiated (21), and there was no decrease in FGF receptors (57), perhaps because these cells do not fuse. Uncoupling of biochemical and morphological differentiation by EGTA showed that the down-regulation of TGF-,B receptors is associated with fusion, but not induc tion of muscle-specific proteins (35),
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The generalization that mitogen receptors decrease with differentiation does not extend to the IGFs, which are very active mitogens for muscle cells. Under conditions in which TGF-fJ receptors virtually disappeared, we (21) detected little or no decrease in binding of 125I-IGF-I and no change in responses to IGF-1. Beguinot et al (5) reported a decrease (about 70%) in IGF-I receptors with differentiation in a different subline of L6 cells, but this may be a result of their expressing binding on the basis of protein (which continues to accumulate in myotubes) rather than DNA content of the cul tures. There is no decrease in binding of IGF-I and IGF-II to human muscle cells during differentiation (74), and there is a transient rise in IGF-I binding (78) and a dramatic increase in IGF-II binding (79) as C2 cells differentiate. Thus the decrease in FGF binding mentioned above is just one of many membrane changes that occur during differentiation, and it does not account for the postmitotic state of myotubes.
IN VIVO SIGNIFICANCE All three growth factors considered in detail here are secreted by skeletal muscle, so it is possible that they might act by autocrine/paracrine mecha nisms; current experiments (J. FIorini et aI, unpublished) indicate that IGF-II may play an important role in spontaneous differentiation of C2 cells incu bated in 2% horse serum. Both at the rnRNA and protein levels, genes for the IGFs have been shown to be expressed under conditions in which increased muscle growth occurs. These include regeneration (36) and hypertrophy induced by growth hormone administration (53). Allen & Boxhom (1) show ed that various combinations of IGF-I, TGF-fJ, and FGF could simulate the sequential phases of muscle regeneration. The TGF-fJ gene is expressed in mouse embryos at both the mRNA and protein (64) levels and (with FGF) is thought to play an important role in mesoderm formation in Xenopus oocytes (83). FGF levels in the chick embryo limb bud are high enough to inhibit differentiation of myoblasts (72), and FGF delays the onset of differentiation in muscle precursor cells from limb buds (71). Strohman's group (88) found that FGF is stored in the extracellular matrix of mature muscle and suggested that it plays a role in muscle hypertrophy. Although it is not yet possible to demonstrate conclusively that these agents affect normal growth and develop ment of muscle, it seems very likely that they do. SUMMARY AND CONCLUSIONS
Three families of growth factors/hormones have major effects on the differen tiation of skeletal muscle cells. Two (FGF and TGF-fJ) are potent inhibitors, and the third (IGF) exhibits a biphasic stimulatory action (but is not
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inhibitory even at high concentrations). All of these affect the expression of myogenin, one of the recently discovered family of myogenesis controlling genes, and FGF and TGF-f3 have been shown to inhibit the expression of MyoDI (and probably myf-5 and herculin) as well. These agents inhibit or stimulate (respectively) all measured aspects of myogenic differentiation fusion, expression of a set of muscle-specific genes, and attainment of a postmitotic state-in all cells that are capable of these responses, whether cell lines or primary muscle cell cultures. It now seems clear that the myogenesis
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controlling genes regulate the entire family of muscle-specific proteins. Therefore the demonstration that expression of these genes is controlled (both positively and negatively) by specific growth factors that are now available at high purity and in useful quantities offers the possibility of understanding myogenic differentiation at a level of molecular detail that is very exciting. ACKNOWLEDGMENTS
The preparation of this review was supported by grant HL11551 from the National Institutes of Health, United States Public Health Service. We thank the many colleagues who made manuscripts available to us prior to their publication. Literature Cited 1. Allen, R. E., Boxhorn, L. K. 1989.
Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta. in sulin-like growth factor-I, and fibroblast growth factor. J. Cell. Physiol. 138:
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53 9. Clegg, C. H., Linkhart, T. A . , Olwin , B. B., Hauschka, S. D. 1987. Growth
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23. Fiorini, J. R., Ewton, D. Z. 1988. Ac tions of transforming growth factor-/3 on muscle cells. J. Cell. Physiol. 135:3018 24. Fiorini, J. R., Ewton, D. Z., Evinger Hodges, M. J., Falen, S. L., Lau, R. L., et al. 1984. Stimulation and inhibition of myoblast differentiation by hormones. In Vitro 20:942-58 25. Fiorini, J. R., Ewton, D. Z., Falen, S. L., Van Wyk, J. J. 1986. Biphasic con centration dependency of the stimulation of myoblast differentiation by soma tomedins. Am. J. Physiol. 250:C771-78 26. Fiorini, J. R., Magri, K. A. 1989. Effects of growth factors on myogenic differentiation. Am. J. Physiol. 256: C701-11 27. Fiorini, J. R., Roberts, A. B., Ewton, D. Z., Falen, S. B., Flanders, K. C., Sporn, M. B. 1986. Transforming growth factor-/3. A very potent inhibitor of myoblast differentiation, identical to the differentiation inhibitor secreted by buffalo rat liver cells. J. Bioi. Chern. 261:16509-13 28. Froesch, E. R., Zapf, J. 1985. Insulin like growth factors and insulin: com parative aspects. Diabeto[ogia 28:48593 29. Gospodarowicz, D., Ferrara, N., Schweigerer, L., Neufeld, G. 1987. Structural characterization and biologi cal functions of fibroblast growth factor. Endocr. Rev. 8:95-114 30. Gospodarowicz, D., Mescher, A. L. 1977. A comparison of the responses of cultured myoblasts and chondrocytes to fibroblast and epidermal growth factors. J. Cell. Physiol. 93:117-28 31. Gossett, L. A., Zhang, W., Olson, E. N. 1988. Dexamethasone-dependent in hibition of differentiation of C2 rnyo blasts bearing steroid-inducible N-ras oncogenes. J. Cell Bioi. 106:2127-38 32. Hill, D. J., Clemmons, D. R., Wilson, S., Han, V. K., Strain, A. J., Milner, R. D. 1989. Immunological distribution of one form of insulin-like growth factor (lGF)-binding protein and IGF peptides in human fetal tissues. J. Mol. Endocri no!. 2:31-38 33. Howe, P. H., Cunningham, M. R., Leof, E. B. 1990. Distinct pathways regulate transforming growth factor {3l stimulated proto-oncogene and ex tracellular matrix gene expression. J. Cell. Physiol. 142:39-45 34. Hu, J. S., Olson, E. N. 1988. Regula tion of differentiation of the BC3H I muscle cell line through cAMP dependent and independent pathways. J. Bioi. Chern. 263:19670-77
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