Are fibroblast growth factors regulators of myogenesis in vivo?

Pergamon

ARE FIBROBLAST GROWTH REGULATORS OF MYOGENESIS Bradiey

B. Olwin, * Kevin

Hannon

FACTORS

and Arthur

IN

VIVO?

J. Kudla

Department of Biochemistry Purdue University West Lafayette, IN 47907, U.S.A.

Recent advances in understanding of skeletal muscle d$%erentiation implicate fibroblast growth factors (FGFs) as regulators of myogenesis; however, the identity and actions offactors that repress myogenesis in vivo remain to be established. This review willfocus on thefibroblast growthfactorfamily and the evidence for its role in regulating myogenesis in culture and in vivo. Keywords: FGF, myogenesis,

OVERVIEW

skeletal muscle, myoD, receptors. differentiation

OF SKELETAL

MUSCLE

DIFFERENTIATION

Skeletal muscle is one of the few tissues that undergoes terminal differentiation. Skeletal muscle is derived from mesoderm that arises from the somites. The first skeletal muscle cells to appear in the developing embryo are those destined to form the somitic myotomal muscle [ 11.The factors that control the proliferation, migration and differentiation of the myotomal skeletal muscle cells have not yet been identified. All other skeletal muscle cells are thought to originate from migrating cells present at the lip of the somitic dermamyotome [2-51; these cells migrate to form the axial muscles, including the muscles of the limbs. Skeletal muscle fibers are a true syncitium of cells formed from the fusion and organization of hundreds of mononucleated cells into a tubular structure called a myotube. A number of myotubes then organize to form a structural unit (myofiber) that participates in contraction. While advances have been made in understanding the conversion of proliferating myoblasts into terminally differentiated myotubes [6-81, little is known concerning the organization of myotubes into functional fibers. Although skeletal muscle cells are terminally differentiated, their regenerative capacity is retained throughout adult life in the form of specialized stem cells called satellite cells, which are closely juxtaposed to the muscle fiber [9]. Satellite cells arise from an unknown precursor [IO, 111, escape the differentiation process and remain throughout adult life as quiescent, undifferentiated myoblasts that retain the capacity for self-renewal and muscle fiber regeneration [9]. *To whom correspondence should be addressed. 14s

B. B. Olwin et al.

146 TABLE

1. Factors

Factor

Skeletal

FGF-I FGF-2

MM14 MM14

muscle

that control

myogenesis

in tissue culture

cell type

[13], primary [13], C2Cl2

morose [14], and primary chick [IS] [16], primary mouse [14], primary rat [17], primary

chick

I151 FGF-4 FGF-6 TGF-/?I IGF-I IGF-II PDGF-BB Proliferin TNF Integrin receptor

ligand

FIBROBLAST

MM14 c2c12 C2Cl2 L6 [24] L6 [24] L6 [25], L6 [27] C2Cl2 primary

[18] [19] [20]. L6 [21-231.

primary

primary

[26]

[28] chick

GROWTH

mouse

rat [17]

[29]

FACTORS

AND

THEIR

RECEPTORS

At least I1 factors have been reported to affect myogenic differentiation (Table 1). A large number of these factors are not characterized enough to determine their contribution to regulation of myogenesis in vivo. Moreover, little attempt has been made to determine if these factors are acting directly or indirectly. Because the differentiation of skeletal muscle in vivo is asynchronous and occurs over a long developmental time period, it is likely that several different factors are involved. Two distinct categories of factors have been identified. The first and largest category comprises factors that repress myogenic differentiation: their removal from the medium is required before myoblasts commit to terminal differentiation [12]. The second group of factors, IGF- 1 and IGF-2, can either augment or inhibit differentiation depending on the concentrations used [8]. Fibroblast

Growth

Factors

Of the nine characterized FGFs [30-321, six have been examined for their capacity to repress differentiation in skeletal muscle cell lines [ 181. FGF-1 , FGF-2, FGF4 and FGF-6 [19] are potent repressors of myogenesis, while FGF-5 and FGF-7 are inactive. The hallmarks of the FGF family include: (i) high affinity for heparin or heparan sulfate; (ii) two invariant conserved cysteine residues; and (iii) an overall homology of 30%. A number of properties separate this family of growth factors from others. Three FGFs, FGF- 1, FGF-2 and FGF-9 do not possess signal secretory peptides yet all three are present extracellularly. In addition, a number of FGFs have been reported to localize to the nucleus. Alternate upstream translational initiation of FGF-2 and FGF-3 may be involved in their nuclear localization [33-351. An intracellular role for FGF action in FGF signal transduction has been proposed [36, 371 but is not yet established. Fibroblast

Growth

Factor Receptors

The actions of FGFs are mediated through binding to high affinity sites on the cell surface. Three distinct classes of FGF receptors have been characterized: (i) a family

FGFs us Regulators

qf Myogenesis

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14’

Signal Transduction FIGURE 1. High affinity binding and signalling by FGF-2. Heparan sulfate chains are depicted by thick dark lines and are attached to a transmembrane HSPG. Two glycosaminoglycan sites, site ‘A’ and site ‘B’, are required for signal transduction. Site A binds specifically to FGF-2. The minimal oligosaccbaride sequence that binds FGF-2, shown boxed, is five carbohydrate units with one idurouosyl 2-O-sulfate group. Site B interacts with FGFR-1 and is not precisely defined, but requires both 2-O-sulfated and 6-O-sulfated carbohydrate units. The formation of a ternary complex between FCF-2, specific heparan sulfate glycosaminoglycan sites A and B and FGFR-1 are required for high affinity binding and signal transduction. (Adapted from 1451.)

of at least four integral membrane protein tyrosine kinases (FGFR-I to FGFR4) [38. 391; (ii) a large family of cell surface heparan sulfate proteoglycans (HSPGs) [40,41]; and (iii) a unique cysteine-rich FGF receptor (CFR) of unknown function [42]. The tyrosine kinase receptors do not appear capable of binding FGF directly but require interaction with cell surface heparan sulfate proteoglycans to form a high affinity FGF-binding site [43, 441. At least two heparan sulfate binding sites appear to be required for FGF signaling [45]; one binding site is present on the growth factor and one binding site is present on the kinase receptor [46]. A two-site model has been proposed [45] whereby a single heparan sulfate giycosaminoglycan chain must interact with both binding sites for receptor activation (Fig. 1). The mechanisms governing specificity for FGF binding are complex and not well understood. They are important as most FGFRs appear capable of binding several FGFs [38, 391. At least two mechanisms are involved; it is not known if they are independent. The first mechanism involves alternate splicing of the extracellular domains of FGFR-1, FGFR-2 and FGFR-3 [38, 391. Until recently, this was thought to be the only mechanism for generation of FGF specificity. However, differentially sulfated heparin fragments promote mitogenesis by different FGFs in the same cell type [45]. These data suggest the specificity of FGF interactions with FGF tyrosine kinase receptors is determined through alternate splicing of the receptor mRNA and variability of rare heparan sulfate microdomains present in the cell surface HSPGs. CFR is an integral membrane protein that contains a high cysteine content (9%) and an intracellular domain of 13 amino acids [42]. The primary structure is not homologous to any known protein. Two additional proteins of 70 and 45 kDa are

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likely to be critical for CFR function as they bind CFR near the carboxy terminus and are intracellular ([47] and manuscript in preparation). The function of CFR in FGF signaling is not clear but it may be involved in processes distinct from those mediated by the tyrosine kinase receptors. LOCALIZATION

OF FGFs AND FGF RECEPTORS SKELETAL MUSCLE Localization

IN DEVELOPING

in Somites

Both FGF-2 mRNA and antisense message have been localized to the somite myotome and dermamyotome in chickens at Hamburger and Hamilton [48] stages 19 to 26 [49]. FGF-2 protein has been immunolocalized to the myotome and dermamyotome of chicks at stages 16 and 18, but by stage 22 the protein is exclusively localized in the myotome [50]. Intense FGF-2 immunostaining was also observed in the differentiated muscle of 6 day chick myotomes [51]. In the mouse, FGF-4 mRNA is not expressed in somites until the sclerotome, dermatome and myotome are distinct, and then intense expression is observed in the myotome [52]. Later, at 14.5 d pc, no FGF-4 mRNA can be detected in the somites or developing skeletal muscle [52]. Expression of FGF-5 [53] and FGF-6 [I 91 has also been localized to developing myotomal skeletal muscle. For both. the expression of the growth factor mRNA is concurrent with, or occurs following, expression of a muscle-specific marker (cwcardiac actin), indicating that these mRNAs are present in differentiated skeletal muscle. FGF-1 has been immunolocalized to the myotome regions of embryonic rats at 11 d pc. This intense immunoreactivity remains until approximately 19-20 d pc when it decreases in intensity [54]. A common denominator in the FGF expression and immunolocalization patterns in myotomal muscle identifies the highest level of expression in differentiated muscle rather than skeletal muscle precursors. FGF receptor expression has also been examined during somitigenesis. In 8.5 d pc mice FGFR-2 expression is observed in the somites, while FGFR-1 expression was reported absent in one study [55], but present in another where it was expressed in both the dermatome and sclerotome of 8.5 and 9.5 d pc mice [56]. In IO-day chick embryos, FGFR-1 was expressed in skeletal muscle at a much higher intensity than FGFR-3 or FGFR-2 [57]. FGFR4 message in 9.5 d pc mouse embryos has been detected exclusively within the myotome region [58]. In contrast, intense CFR staining was observed in the epithelial cells at the dorsal lip of the dermamyotome [59]. These cells most likely represent the myotomal muscle precursors [I]. CFR staining was maintained in the myotome until differentiation of the skeletal muscle cells [59]. Localization

in the Developing

Limb

Biological assays have identified FGF-2 activity in the developing chick limb as early as stage 18. The levels of FGF and their receptors decrease in the limb prior to hatching [60, 611. FGF-2 mRNA and an endogenous FGF-2 antisense message is detectable prior to and following the formation of the chick limb bud between stages 16 and 25 [49]. The pattern of FGF-2 immunochemical staining was consistent with

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149

the pattern of FGF-2 mRNA expression [50]. Both the protein and the mRNA for FGF-2 were found in the apical ectodermal ridge and the subjacent mesenchyme. As opposed to the somites, there is no antisenseexpression of FGF-2 in the limb muscle mass[49]. Later in limb development, after stage 28, FGF-2 mRNA and protein were present in differentiating muscle masses[49, 501. In the mouse, FGF-4 messageis expressed at high levels in the apical ectodermal ridge, but not in the limb mesenchymal tissue [62]. FGF-6 mRNA is also detected in the differentiating muscle massesof the mouse limb and persistsafter the muscle tissue has become terminally differentiated [63]. The expression pattern of the FGFRs in the developing limb muscle is not as extensively investigated as their expression patterns in somites. In the 11.5 d pc mouse limb bud, FGFR-2 expression was localized to mesenchymal aggregatescorresponding to future bones, while FGFR-I expression appeared to be much more diffusely distributed in the mesenchyme [55]. FGFR-4 expression in the developing limb has not been examined. When developing mouse limb buds first appear at 9 d pc. syndecan (an FGF-binding heparan sulfate proteoglycan) protein has been localized in the mesenchymal region [64]. By 13 dpc, staining for syndecan is lost in the regions destined for myogenesis [64]. CFR is present in the undifferentiated mesenchymal cells of the limb and is lost from differentiated skeletal musclefibers, but appears to be maintained in the satellite cells [59]. Localization of these FGFRs does not imply that signalling pathways are active, as signal transduction requires a ternary complex comprised of a specific heparan sulfate sequenceon HSPG, the appropriate mRNA splice variant of the FGFR and the corresponding ligand (seeFig. 1). Further studies are obviously needed to elucidate the role of the FGFs in skeletal muscle development. MECHANISMS

OF FGF-MEDIATED

REPRESSION

OF MYOGENESIS

Regulation of skeletal muscle differentiation by environmental factors has been studied using primarily skeletal muscle cell lines [8]. A few reports have examined the effects of environmental factors on primary cultures. A large number of factors have been shown to affect skeletal muscledifferentiation in culture (Table 1). Although this representsa tremendous diversity in response,the effects of only a few of these factors have been examined in primary cultures or in viw.

Temporal Sequence qf Mdvogenesis in Culture A temporal sequencedefining distinct events occurring during myogenesis in cell culture has been established [65]. Removal of FGFs from most cell lines initiates a permanent withdrawal from the cell cycle that is termed commitment to a postmitotic phenotype [14, 661. This is the first detectable phenotypic change in the myogenic program, occurs from the G 1-phase of the cell cycle, and is irreversible [66. 671. As FGFs prevent the acquisition of all phenotypes associated with skeletal muscle differentiation, it is thought that FGFs repressall aspectsof myogenesisin cell culture [ 141. The ability of FGF to inhibit differentiation could be due to a mitogenic effect of FGF on myoblasts that prevents exit from the cell cycle or a direct effect that

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represses acquisition of the myogenic phenotype. The latter hypothesis is strongly suggested by two independent experiments. First, in the presence of FGF, skeletal muscle cells can be reversibly withdrawn from the cell cycle if the serum concentration is lowered [ 141. If serum is added back, the cells will re-enter the cell cycle. Second, in a cell line that expresses skeletal muscle genes but is defective for fusion and thus, can undergo reversible differentiation, FGF induces de-differentiation without causing the cells to re-enter the cell cycle (manuscript in preparation). Regulation

of Skeletal Muscle Regulatory

Transcription

Factors by FGF

FGF-2 regulates the activity of four transcriptional activators of skeletal musclespecific genes: myoD, myf-5, myogenin and MRF-4 [6, 71. These MRFs (skeletal muscle regulatory transcription factors) heterodimerize with ubiquitous factors (E 12 and E47) and then bind to an E-box consensus sequence (CANNTG) to activate transcription [6, 71. In addition to regulating the transcription of skeletal musclespecific genes, they have the unique ability to convert fibroblasts into the myogenic lineage when their expression is forced [6, 71. Repression of skeletal muscle differentiation by FGF via the MRFs is not understood; however, two mechanisms have been proposed. The first suggests that FGF signalling increases the level of an inhibitor interacting stoichiometrically with the MRFs or their heterodimer partners, thereby preventing activation of MRFdependent genes [6, 681. In support of these data are the observations that a number of intracellular factors affect skeletal muscle myogenesis. Among these are a number of oncogenes including myc andfos [69-771. Other factors proposed to bind directly to myoD in viva include c-jun and Id (77-80). The ratio of activated .fos and presumably myc, Id, and c-jun to myoD is critical for their inhibitory activities since the forced expression of myoD or myogenin will reverse their block of myogenesis [69, 701. The second proposed regulatory mechanism relies on the following observations: (i) myoD and myf-5 are often expressed in proliferating myoblasts yet are biologically inert; and (ii) high levels of transfected myoD, myf-5 myogenin or MRF4 do not activate the myogenic program in the presence of exogenous FGF-2. These data argue against a stoichiometric interaction of the MRFs with an inhibitor such as Id. cfos, c-jun, or c-myc as the primary means of repression [6,68, 8 11. Instead, these data suggest post-translational control of the MRFs or associated coregulators. This is consistent with the observations that overexpression of PKA, PKC and Ha-ras inhibit myogenesis [74, 82-871. A recent study examining myogenin phosphorylation demonstrates that FGF increases the phosphorylation of a critical thr residue present in the basic DNA binding site [88]. The phosphorylation occurs in vitro and in COS cells overexpressing myogenin and PKC. As all MRFs possess a thr residue at a similar position in the basic DNA binding region, phosphorylation of this thr residue was hypothesized to be a universal mechanism for FGF-mediated repression of myogenesis [88]. The COS cells used in this study did not convert to the myogenic phenotype upon forced expression of MRFs and thus, the effect of thr phosphorylation on the inhibition of myogenesis was not directly addressed. A second manuscript examined the phosphorylation of MRF4 in C3HlOT1/2 cells [87]. In contrast to the data in the first manuscript, data in this manuscript show that the corresponding MRF thr residue is not phosphorylated when MRF4 is overexpressed in C3HlOT1/2

FGFs us Regulators

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cells in the presence of exogenous FGF. Removal of FGF converts these cells to a myogenic phenotype since they overexpress MRF4 [89]. Moreover, this group has performed a series of experiments demonstrating that phosphorylation of MRF4 is not responsible for regulation of myogenesis by FGFs, PKC or PKA [87]. Although the inhibitory mechanisms involved may differ between the four transcriptional activators, correlation of regulatory mechanisms with the differentiated phenotype is essential; much work is still required before the signalling pathways involved are understood. Regulation of the Cell Cycle by MyoD and FGF

In addition to its ability to transcriptionally activate skeletal muscle-specific genes, myoD inhibits mitogenesis in nonmyogenic cell types and can reduce or block proliferation in transformed cells [90. 911. The two activities of myoD are independent, as mutations that abolish myogenic activity do not block the ability of myoD to arrest cell growth. MyoD may thus play an important role in the withdrawal of myoblasts from the cell cycle elicited by FGF removal. This hypothesis has been strengthened by an elegant series of experiments demonstrating that the block to proliferation may occur via a direct interaction of myoD with the retinoblastoma protein (pRB) or a related protein [92]. Inactivation of pRB by binding to T antigen. phosphorylation or mutation blocks myogenic differentiation [92]. The interaction of myoD and pRB appears necessary for the induction of myogenesis and for withdrawal from the cell cycle. FGF withdrawal would result in dephosphorylation of pRB followed by binding of pRB to myoD, forming a complex that locks the cell in a nonproliferative state and activates myoD-dependent transcription. In addition, pRB may be required for myoD-dependent transcriptional activation of skeletal musclespecific genes. A major challenge is to determine the signalling pathway involved in growth regulation and its relationship to signal transduction pathways regulating activation of skeletal muscle-specific genes. ROLES

FOR FGF REGULATION

OF MYOGENESIS

IN VW0

All proposed roles for FGF involvement in the regulation of myogenesis in vivo are derived from effects of exogenous FGF added to primary cultures, or localization of FGFs and their receptors via immunohistochemistry or in situ hybridization. Efects

qf FGF in Primary Myoblast Cultures

Primary myogenic cells from the chick embryo and satellite cells from the mouse and rat are repressed from differentiation by FGF-2 [ 15, 17, 93, 941, suggesting these cells are repressed by FGF in vivo. Approximately 50% of the primary chick cells are responsive to FGF-2; half of the myogenic cell population is not dependent on FGF for repression of terminal differentiation. Perhaps these differences in growth factor responsiveness reflect different skeletal muscle myoblast lineages and allow greater flexibility in the control of myogenic differentiation in vivo. A second effect of FGF was observed in primary embryonic chick skeletal muscle cultures. A significantly greater number of cells acquired a myogenic phenotype if exposed to FGF [15]. These

1.52

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cells would normally form non-myogenic colonies unless exposed to FGF during culture. Therefore. in addition to its ability to repress myogenesis, FGFs may function to alter the number of cells entering the myogenic lineage. This is consistent with FGF-mediated regulation of the MRFs, which have been proposed to play a role in determination of the myogenic phenotype. Actions qf FGF in Experiments

Peyformed In Vivo

Experiments beginning to examine the role(s) of FGFs in vivo or in explant cultures have been recently published. In one study, an avian replication-defective retrovirus was constructed to express FGF-2 and used to infect developing limb buds in vivo [95]. Interestingly, ectopic overexpression of FGF-2 did not grossly affect skeletal muscle development but instead caused duplications of both proximal and distal limb elements [95]. Thus, FGF-2 may play an important role in limb outgrowth during limb development. This hypothesis is further strengthened by the localization of FGF-2 protein and FGF-2 mRNA to the apical ectodermal ridge and the subjacent mesenchyme [49, 501, which play important roles in limb growth and patterning. Similar conclusions have been reached from the observed FGF-induced limb outgrowth in explanted mouse embryo trunk and limbs [62]. Evidence,for

Regulation of Myogenesis

by FGF In Vivo

The involvement of FGF in regulating myogenesis appears indisputable when one examines experiments performed with primary cultures and skeletal muscle cell lines. However, close examination of the expression patterns of FGF-2 in the chick [49, 50, 961 and FGF-4 [97], FGF-5 [53] and FGF-6 [19] in the mouse, reveals that the majority of FGF expression is present in dzyerentiated skeletal muscle cells or in skeletal muscle tissue that is undergoing extensive differentiation. Based on the culture data, it is expected that FGFs should be disappearing in differentiated tissues as their repressive activities are no longer needed. This conundrum can be explained in one of three ways. First, members of the FGF family may not be involved in regulation of myogenesis and the tissue culture data are artifactual. This is highly unlikely as primary cultures, a number of independently isolated myogenic cell lines and other cell types converted to myogenic cells by forced expression of MRFs all show similar regulation by FGF-2. Second, the FGF family member that regulates myogenesis may not yet be identified. FGFs detected in differentiated skeletal muscle cells may be involved in processes other than regulation of skeletal muscle cell differentiation. As FGF-5 does not repress the differentiation of cultured skeletal muscle cells, its presence in skeletal muscle tissue may not directly affect myogenic differentiation [18]. Consistent with this hypothesis, FGF-5 has been shown to be a potent skeletal muscle-derived survival factor for cultured chick motoneurons [98]. Production and release of FGF-5 may be essential for the guidance and continued survival of motoneurons that innervate the skeletal muscle fiber. Perhaps FGF-6 also plays an important role in differentiated rather than proliferating skeletal muscle tissue. A third explanation is that more than one member of the FGF family may be involved in regulating myogenesis. As the skeletal muscle precursor cells are migrat-

FGFS us Regulutors r$ MyrJgenesis In Viw

153

ing from the dermamyotome to their destinations in the limb bud, one FGF family member may regulate their proliferative state. Upon arriving at their destination in the limb bud, a different FGF family member may regulate skeletal muscle precursor cell proliferation. As skeletal muscle differentiation is highly asynchronous, the differentiated cells may then produce a third FGF that would continue to repress differentiation in the proliferating population, ensuring that a large population of these cells is maintained for continued skeletal muscle development.

PERSPECTIVES

Aside from the obvious reasons for understanding the molecular mechanisms involved in skeletal muscle differentiation, understanding the factors regulating skeletal muscle growth may play a major role in the development of therapies to cure or treat patients with Duchenne Muscular Dystrophy. Myoblast transfer involves injecting myoblasts expressing an intact dystrophin gene into skeletal muscle tissue [99, 1001. Large numbers of myoblasts must be injected as these cells, once removed from the tissue and cultured, are incapable of proliferation in viva [lOl-1031. Moreover, other genetic defects may be treatable using myoblast transfer therapies [ 104, 1051. If these experimental approaches are to provide long term cures for genetic diseases, we must understand the factors responsible for controlling the growth and differentiation of myoblasts, in particular satellite cells. Manipulation of the proliferative capacity of these cells should aid and enhance the current paradigms used in myoblast transfer therapies. A second disease specific to skeletal muscle where growth control appears aberrant is the rhabdomyosarcoma, a common solid tumor in children. Rhabdomyosarcoma cells exhibit unusual properties in their growth regulation and differentiation [106, 1071. The limited amount of data available suggest that the mechanisms involved in normal myoblast signalling have been disrupted and may account for the phenotype of this tumor. The combination of a mechanistic approach in cell culture coupled with in viro experiments that manipulate the FGF response are likely to soon yield an understanding of the role(s) of FGF in the regulation of skeletal muscle development.

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