Oncogenic Ras-induced proliferation requires autocrine fibroblast growth factor 2 signaling in skeletal muscle cells.

Constitutively activated Ras proteins are associated with a large number of human cancers, including those originating from skeletal muscle tissue. In this study, we show that ectopic expression of oncogenic Ras stimulates proliferation of the MM14 skeletal muscle satellite cell line in the absence of exogenously added fibroblast growth factors (FGFs). MM14 cells express FGF-1, -2, -6, and -7 and produce FGF protein, yet they are dependent on exogenously supplied FGFs to both maintain proliferation and repress terminal differentiation. Thus, the FGFs produced by these cells are either inaccessible or inactive, since the endogenous FGFs elicit no detectable biological response. Oncogenic Ras-induced proliferation is abolished by addition of an anti-FGF-2 blocking antibody, suramin, or treatment with either sodium chlorate or heparitinase, demonstrating an autocrine requirement for FGF-2. Oncogenic Ras does not appear to alter cellular export rates of FGF-2, which does not possess an NH(2)-terminal or internal signal peptide. However, oncogenic Ras does appear to be involved in releasing or activating inactive, extracellularly sequestered FGF-2. Surprisingly, inhibiting the autocrine FGF-2 required for proliferation has no effect on oncogenic Ras-mediated repression of muscle-specific gene expression. We conclude that oncogenic Ras-induced proliferation of skeletal muscle cells is mediated via a unique and novel mechanism that is distinct from Ras-induced repression of terminal differentiation and involves activation of extracellularly localized, inactive FGF-2.

ERK1/2 is required for myoblast proliferation but is dispensable for muscle gene expression and cell fusion.

Skeletal muscle satellite cells, which are found between the muscle fiber and the basal lamina, remain quiescent and undifferentiated unless stimulated to remodel skeletal muscle or repair injured skeletal muscle tissue. Quiescent satellite cells express c-met and fibroblast growth factor receptors (FGFR) 1 and 4, suggesting these receptors are involved in maintaining the undifferentiated quiescent state or involved in satellite cell activation. Although the signaling pathways involved are poorly understood, the mitogen activated protein kinase (MAPK) cascade has been implicated in the regulation of skeletal muscle growth and differentiation by FGFs. In this study, we investigated if activation of the Raf-MKK1/2-ERK1/2 signaling cascade plays a role in FGF-dependent repression of differentiation and proliferation of MM14 cells, a skeletal muscle satellite cell line. Inactivation ofthe Raf-MKK1/2-ERK1/2 pathway in myoblasts through the overexpression of dominant negative mutants of Raf-1 blocks ERK1/2 activity and prevents myoblast proliferation. Additionally, inhibition of MKK1/2 by treatment with pharmacological inhibitors also blocks FGF-mediated stimulation of ERK1/2 and blocks the G1 to S phase transition of myoblasts. Unexpectedly, we found that inactivation of the Raf-ERK pathway does not activate a muscle reporter, nor does inactivation of this pathway promote myogenic differentiation. We conclude that FGF-stimulated ERK1/2 signaling is required during the G1 phase of the cell cycle for commitment of myoblasts to DNA synthesis but is not required for mitosis once cells have entered the S-phase. Moreover, ERK1/2 signaling is not required either to repress differentiation, to promote skeletal muscle gene expression, or to promote myoblast fusion.

Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.

MyoD(-/-) satellite cells in single-fiber culture are differentiation defective and MRF4 deficient.

MyoD-deficient mice are without obvious deleterious muscle phenotype during embryogenesis and fetal development, and adults in the laboratory have grossly normal skeletal muscle and life span. However, a previous study showed that in the context of muscle degeneration on a mdx (dystrophin null) genetic background, animals lacking MyoD have a greatly intensified disease phenotype leading to lethality not otherwise seen in mdx mice. Here we have examined MyoD(-/-) adult muscle fibers and their associated satellite cells in single myofiber cultures and describe major phenotypic differences found at the tissue, cellular, and molecular levels. The steady-state number of satellite cells on freshly isolated MyoD(-/-) fibers was elevated and abnormal branched fiber morphologies were observed, the latter suggesting chronic muscle regeneration in vivo. Single-cell RNA coexpression analyses were performed for c-met, m-cadherin, and the four myogenic regulatory factors (MRFs.) Most mutant satellite cells entered the cell cycle and upregulated expression of myf5, both characteristic early steps in satellite cell maturation. However, they later failed to normally upregulate MRF4, displayed a major deficit in m-cadherin expression, and showed a significant diminution in myogenin-positive status compared with wildtype. MyoD(-/-) satellite cells formed unusual aggregate structures, failed to fuse efficiently, and showed greater than 90% reduction in differentiation efficiency relative to wildtype. A further survey of RNAs encoding regulators of growth and differentiation, cell cycle progression, and cell signaling revealed similar or identical expression profiles for most genes as well as several noteworthy differences. Among these, GDF8 and Msx1 were identified as potentially important regulators of the quiescent state whose expression profile differs between mutant and wildtype. Considered together, these data suggest that activated MyoD(-/-) satellite cells assume a phenotype that resembles in some ways a developmentally "stalled" cell compared to wildtype. However, the MyoD(-/-) cells are not merely developmentally immature, as they also display novel molecular and cellular characteristics that differ from any observed in wild-type muscle precursor counterparts of any stage.

Cysteine-rich fibroblast growth factor receptor alters secretion and intracellular routing of fibroblast growth factor 3.

Expression of the cysteine-rich fibroblast growth factor (FGF) receptor (CFR) in COS-1 cells strongly inhibits the secretion of co-expressed FGF3. By using a column retention assay and affinity chromatography, we demonstrate that at physiological salt concentrations FGF3 binds with strong affinity to CFR in vivo and in vitro. Furthermore, to show that FGF3 binds to CFR in vivo, truncation mutants of CFR with changed subcellular distributions were shown to cause a similar redistribution of FGF3. Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the Golgi apparatus, we show here that in COS-1 cells a substantial proportion of CFR is secreted. This is due to a carboxyl-terminal proteolytic cleavage that releases the intraluminal portion of the protein for secretion. However, the apparent size of the integral membrane and secreted CFR appears similar, since the loss of protein mass is balanced by a gain of complex carbohydrates. The released CFR is associated with the extracellular matrix through its affinity for glycosaminoglycans. These findings show that CFR can modulate the secretion of FGF3 and may control its biological activity by regulating its secretion.

Loss of FGF receptor 1 signaling reduces skeletal muscle mass and disrupts myofiber organization in the developing limb.

The identities of extracellular growth factors that regulate skeletal muscle development in vivo are largely unknown. We asked if FGFs, which act as repressors of myogenesis in culture, play a similar role in vivo by ectopically expressing in the developing limb a truncated FGF receptor 1 (dnFGFR1) that acts as a dominant negative mutant. Hind limbs and the adjacent somites of Hamburger and Hamilton (HH) stage 17 chickens were infected with a replication-competent RCAS virus encoding dnFGFR1. By ED5, the virus had spread extensively within the limb and the adjacent somites with little rostral or caudal expansion of the infection along the axial midline. Viral infection and mutant receptor expression were coincident as revealed by the distribution of a viral coat protein and an HA epitope tag present on the carboxy terminus of dnFGFR1. Within 48 h following injection of dnFGFR1, we could detect no obvious changes in skeletal muscle precursor cell migration into the hind limb as compared to control limbs infected with an empty RCAN virus. However, by 3 days following infection of RCAS-dnFGFR1 virus, the level of skeletal muscle-specific myosin heavy chain was decreased and the expression pattern altered, suggesting disruption of skeletal muscle development. Two striking muscular phenotypes were observed in dnFGFR1-expressing limbs, including an average loss of 30% in skeletal muscle wet weight and a 50% decrease in myofiber density. At all ages examined the loss of skeletal muscle mass was accompanied by a loss of myoblasts and an unexpected concomitant loss of fibroblasts. Consistent with these observations, explants of infected cells revealed a reduction in the number of myonuclei in myotubes. Although the myofiber density per unit area was decreased over 50% compared to controls there were no detectable effects on myofiber diameter. The loss in myofiber density was, however, accompanied by an increase in the space surrounding individual myofibers and a generalized loss of myofiber integrity. It is noteworthy that long-bone development was unaffected by RCAS-dnFGFR1 infection, suggesting that FGFR2 and FGFR3 signaling was not disrupted. Our data provide conclusive evidence that FGFR1 signaling is necessary to maintain myoblast number and plays a role in myofiber organization.

Regulation of myogenesis by fibroblast growth factors requires beta-gamma subunits of pertussis toxin-sensitive G proteins.

Terminal differentiation of skeletal muscle cells in culture is inhibited by a number of different growth factors whose subsequent intracellular signaling events are poorly understood. In this study, we have investigated the role of heterotrimeric G proteins in mediating fibroblast growth factor (FGF)-dependent signals that regulate myogenic differentiation. Pertussis toxin, which ADP-ribosylates and inactivates susceptible G proteins, promotes terminal differentiation in the presence of FGF-2, suggesting that Galpha or Gbeta gamma subunits or both are involved in transducing the FGF-dependent signal(s) that inhibits myogenesis. We found that Gbetagamma subunits are likely to be involved since the expression of the C terminus of beta-adrenergic receptor kinase 1, a Gbetagamma subunit-sequestering agent, promotes differentiation in the presence of FGF-2, and expression of the free Gbeta gamma dimer can replace FGF-2, rescuing cells from pertussis toxin-induced differentiation. Addition of pertussis toxin also blocked FGF-2-mediated activation of mitogen-activated protein kinases (MAPKs). Ectopic expression of dominant active mutants in the Ras/MAPK pathway rescued cells from pertussis toxin-induced terminal differentiation, suggesting that the Gbeta gamma subunits act upstream of the Ras/MAPK pathway. It is unlikely that the pertussis toxin-sensitive pathway is activated by other, as yet unidentified FGF receptors since PDGF (platelet-derived growth factor)-stimulated MM14 cells expressing a chimeric receptor containing the FGF receptor-1 intracellular domain and the PDGF receptor extracellular domain were sensitive to pertussis toxin. Our data suggest that FGF-mediated signals involved in repression of myogenic differentiation are transduced by a pertussis toxin-sensitive G-protein-coupled mechanism. This signaling pathway requires the action of Gbeta gamma subunits and activation of MAPKs to repress skeletal muscle differentiation.

The FGF receptor-1 tyrosine kinase domain regulates myogenesis but is not sufficient to stimulate proliferation.

Ligand-stimulated activation of FGF receptors (FGFRs) in skeletal muscle cells represses terminal myogenic differentiation. Skeletal muscle cell lines and subsets of primary cells are dependent on FGFs to repress myogenesis and maintain growth. To understand the intracellular events that transduce these signals, MM14 skeletal muscle cells were transfected with expression vectors encoding chimeric receptors. The chimeras are comprised of the PDGF beta receptor (PDGFbetaR) extracellular domain, the FGFR-1 intracellular domain, and either the PDGFbetaR or FGFR-1 transmembrane domain. The chimeric receptors were autophosphorylated upon PDGF-BB stimulation and are capable of stimulating mitogen-activated protein kinase activity. Activation of the tyrosine kinase domain of either chimera repressed myogenesis, suggesting intracellular responses regulating skeletal muscle differentiation are transduced by activation of the FGFR-1 tyrosine kinase. Unexpectedly, we found that activation of either chimeric receptor failed to stimulate cellular proliferation. Thus, it appears that regulation of skeletal muscle differentiation by FGFs requires only activation of the FGFR tyrosine kinase. In contrast, stimulation of proliferation may require additional, as yet unidentified, signals involving the receptor ectodomain, the FGF ligand, and heparan sulfate either alone, or in combination.

Cysteine-rich FGF receptor regulates intracellular FGF-1 and FGF-2 levels.

The cysteine-rich FGF receptor (CFR) is a 150-kD membrane-associated glycoprotein that specifically binds FGFs. CFR protein is not detectable at the cell surface and immunocytochemistry with anti-CFR antibodies demonstrates that CFR is concentrated in the Golgi apparatus. These data suggest CFR does not function as a plasma membrane FGF receptor. CFR expressed in chinese hamster ovary cells reduces the intracellular accumulation of exogenously applied FGF-1 and FGF-2. A mutant CFR lacking the juxtamembrane, transmembrane and intracellular domains is unable to alter intracellular FGF levels. Mutant CFR is detected throughout the cell, indicating that the domains absent in mutant CFR are required for appropriate subcellular localization and the regulation of intracellular FGF levels. Although the activation of plasma membrane receptors is necessary for cellular responses to FGFs, a requirement for intracellular FGF has also been proposed. The subcellular localization of CFR and its ability to regulate the levels of intracellular FGFs suggests that CFR may be involved in intracellular FGF trafficking and the regulation of cellular responses to FGFs.

Identification and characterization of a fibroblast growth factor (FGF) binding domain in the cysteine-rich FGF receptor.

Three distinct transmembrane glycoproteins bind fibroblast growth factor (FGF) family members. These include heparan sulfate proteoglycans, the tyrosine kinase-containing FGF receptors (FGFRs), and a cysteine-rich FGF receptor (CFR). The four FGFRs are thought to mediate FGF-signaling events but require the participation of the heparan sulfate proteoglycans to bind FGFs and transduce intracellular signals. However, a number of groups have proposed that FGF action requires events independent of FGFR activation. CFR, a high affinity FGF-binding protein, was first isolated from chicken embryos. To better understand the interactions between CFR and FGFs, we have constructed a series of CFR deletion mutants and CFR fragments. Analysis of these has identified a approximately 200-amino acid domain that constitutes a CFR FGF binding site. A CFR fragment of 450 residues, CFR290-740, binds FGF-2 with an affinity indistinguishable from the full-length molecule, whereas smaller fragments display greatly reduced FGF binding. Although CFR binds heparin with high affinity, an analysis of the heparin-CFR interaction failed to identify a linear sequence containing a heparin binding site. Two types of FGF binding sites were identified: an ionic strength and heparin-independent site that represents FGF binding to CFR290-740 and an additional FGF binding site that is heparan sulfate-dependent and sensitive to high ionic strength. This latter site is likely to bind FGF indirectly via heparan sulfate binding to CFR. FGF-2 peptides that encompass a sequence implicated in FGF-2 binding to FGFRs also block FGF-2 binding to CFR. Our data suggest that binding of FGFs to CFR and FGFRs is mutually exclusive, since the CFR FGF binding site does not require heparan sulfate, and similar regions on FGF-2 interact with both FGFRs and CFR.

Differentially expressed fibroblast growth factors regulate skeletal muscle development through autocrine and paracrine mechanisms.

Several FGF family members are expressed in skeletal muscle; however, the roles of these factors in skeletal muscle development are unclear. We examined the RNA expression, protein levels, and biological activities of the FGF family in the MM14 mouse skeletal muscle cell line. Proliferating skeletal muscle cells express FGF-1, FGF-2, FGF-6, and FGF-7 mRNA. Differentiated myofibers express FGF-5, FGF-7, and reduced levels of FGF-6 mRNA. FGF-3, FGF-4, and FGF-8 were not detectable by RT-PCR in either proliferating or differentiated skeletal muscle cells. FGF-I and FGF-2 proteins were present in proliferating skeletal muscle cells, but undetectable after terminal differentiation. We show that transfection of expression constructs encoding FGF-1 or FGF-2 mimics the effects of exogenously applied FGFs, inhibiting skeletal muscle cell differentiation and stimulating DNA synthesis. These effects require activation of an FGF tyrosine kinase receptor as they are blocked by transfection of a dominant negative mutant FGF receptor. Transient transfection of cells with FGF-1 or FGF-2 expression constructs exerted a global effect on myoblast DNA synthesis, as greater than 50% of the nontransfected cells responded by initiating DNA synthesis. The global effect of cultures transfected with FGF-2 expression vectors was blocked by an anti-FGF-2 monoclonal antibody, suggesting that FGF-2 was exported from the transfected cells. Despite the fact that both FGF-l and FGF-2 lack secretory signal sequences, when expressed intracellularly, they regulate skeletal muscle development. Thus, production of FGF-1 and FGF-2 by skeletal muscle cells may act as a paracrine and autocrine regulator of skeletal muscle development in vivo.

Conservation of ligand specificity between the mammalian and amphibian fibroblast growth factor receptors.

We have previously cloned and sequenced a newt keratinocyte growth factor receptor (KGFR) cDNA which exhibited a unique spatial and temporal expression pattern in the regenerating newt limb. In this report, we further characterize the biochemical and functional properties of this newt KGFR. A stable Chinese hamster ovary transfectant overexpressing the newt KGFR was capable of binding both 125I-fibroblast growth factor-1 (FGF-1) and 125I-FGF-7 but not 125I-FGF-2, indistinguishable from the human KGFR. Scatchard analysis and cross-linking studies further support the conclusion that FGF-1 and FGF-7 are the ligands for the newt KGFR. In addition to their ability to bind to FGFs, both the human and the newt KGFR are also capable of repressing differentiation in mouse MM14 myoblasts. MM14 cells express FGFR1 and are repressed from differentiation by FGF-1, FGF-2, and FGF-4 but not FGF-7. Co-transfection of MM14 cells with either a human or newt KGFR expression construct conferred a response to FGF-7 as determined by a human alpha-cardiac actin/luciferase reporter construct. The response to FGF-7 was similar to the endogenous FGF response as FGF-7 prevented MM14 myoblasts from undergoing terminal differentiation. Thus, both the human and the newt KGFRs transduce signals similar to those transduced via the endogenous mouse FGFR1. Together these data indicate that this newly isolated newt KGFR is a functional receptor as it binds two FGF family members with high affinity and mediates signaling in skeletal muscle myoblasts. Because the binding pattern of the newt KGFR is similar to the pattern observed for its mammalian counterpart, it emphasizes the strict conservation that this ligand/receptor system has undergone through evolution.

Changes in the expression of fibroblast growth factor receptors mark distinct stages of chondrogenesis in vitro and during chick limb skeletal patterning.

Members of the fibroblast growth factor (FGF) family of growth factors are key regulators of limb skeletal patterning and growth. Abnormal expression of FGFs or mutations in their receptors (fgfrs) result in skeletal disorders. Here we show that changes in the expression of fgfrs are intrinsic properties of differentiating cartilage. In mesenchymal micromass cultures differentiating into cartilage, as in ovo, fgfr 1 mRNA was found predominantly in undifferentiated, proliferating mesenchyme, fgfr 2 in precartilage cell aggregates, and fgfr 3 in differentiating cartilage nodules. Thus, our data suggest that switches in the expression of fgfr 1, 2, and 3 mRNAs are associated with phases of cartilage patterning both in vitro and in ovo, and mark distinct stages in the development of the limb skeleton.

A requirement for fibroblast growth factor in regulation of skeletal muscle growth and differentiation cannot be replaced by activation of platelet-derived growth factor signaling pathways.

The distinct effects of cytokines on cellular growth and differentiation suggest that specific signaling pathways mediate these diverse biological activities. Fibroblast growth factors (FGFs) are well-established inhibitors of skeletal muscle differentiation and may operate via activation of specific signaling pathways distinct from recently identified mitogen signaling pathways. We examined whether platelet-derived growth factor (PDGF)-activated signaling pathways are sufficient to mediate FGF-dependent repression of myogenesis by introducing the PDGF beta receptor into a mouse skeletal muscle cell line. Addition of PDGF-BB to cells expressing the PDGF beta receptor activated the PDGF beta receptor tyrosine kinase, stimulated mitogen-activated protein (MAP) kinase, and increased the steady-state levels of junB and c-fos mRNAs. Despite the activation of these intracellular signaling molecules, PDGF beta receptor activation elicited no detectable effect on cell proliferation or differentiation. In contrast to PDGF-BB, addition of FGF-2 to myoblasts activated signaling pathways that resulted in DNA synthesis and repression of differentiation. Because of the low number of endogenous FGF receptors expressed, FGF-stimulated signaling events, including tyrosine phosphorylation and activation of MAP kinase, could be detected only in cells expressing higher levels of a transfected FGF receptor cDNA. As the PDGF beta receptor- and FGF receptor-stimulated signaling pathways yield different biological responses in these skeletal muscle cells, we hypothesize that FGF-mediated repression of skeletal muscle differentiation activates signaling pathways distinct from those activated by the PDGF beta receptor. Activation of PDGF beta receptor tyrosine kinase activity, stimulation of MAP kinase, and upregulation of immediate-early gene expression are not sufficient to repress skeletal muscle differentiation.

Genesis of olfactory receptor neurons in vitro: regulation of progenitor cell divisions by fibroblast growth factors.

Olfactory receptor neurons are produced continuously in mammalian olfactory epithelium in vivo, but in explant cultures neurogenesis ceases abruptly. We show that in vitro neurogenesis is prolonged by fibroblast growth factors (FGFs), which act in two ways. FGFs increase the likelihood that immediate neuronal precursors (INPs) divide twice, rather than once, before generating neurons; this action requires exposure of INPs to FGFs by early G1. FGFs also cause a distinct subpopulation of explants to generate large numbers of neurons continually for at least several days. The data suggest that FGFs delay differentiation of a committed neuronal transit amplifying cell (the INP) and support proliferation or survival of a rare cell, possibly a stem cell, that acts as a progenitor to INPs.

Role of FGFs in skeletal muscle and limb development.

Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR-1 through FGFR-4); (ii) a cysteine-rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR-1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling. We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF-2 or FGF-2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF-2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF-2 plays an important role in limb outgrowth. We propose that FGF-2 is an apical ectodermal ridge-derived factor that participates in limb outgrowth and patterning.

FGF-2: apical ectodermal ridge growth signal for chick limb development.

The apical ectodermal ridge permits growth and elongation of amniote limb buds; removal causes rapid changes in mesodermal gene expression, patterned cell death, and truncation of the limb. Ectopic fibroblast growth factor (FGF)-2 supplied to the chick apical bud mesoderm after ridge removal will sustain normal gene expression and cell viability, and allow relatively normal limb development. A bioassay for FGFs demonstrated that FGF-2 was the only detectable FGF in chick limb bud extracts. By distribution and bioactivity, FGF-2 is the prime candidate for the chick limb bud apical ridge growth signal.

Regulation by heparan sulfate in fibroblast growth factor signaling.

The integral role of heparan sulfate proteoglycans in FGF signaling provides a potential means of regulating FGF activity. This regulation may be used by the cell, where the modification of heparan sulfate glycosaminoglycans during their synthesis in the Golgi can produce cell type- and potentially ligand-specific sulfation sequences. The description of these sequences will not only provide information on how this regulation is achieved, perhaps lending insight into other heparan sulfate-ligand interactions, but may also discern sulfated mimetics that can be used to disrupt or alter FGF signaling. These mimetics may be useful in the treatment disrupt or alter FGF signaling. These mimetics may be useful in the treatment of disease, or in understanding how FGF signaling via discrete pathways within the cell leads to specific cellular responses, such as activation of mitogenic signaling pathways, calcium fluxes, and cellular differentiation.

Are fibroblast growth factors regulators of myogenesis in vivo?

Recent advances in understanding of skeletal muscle differentiation implicate fibroblast growth factors (FGFs) as regulators of myogenesis; however, the identity and actions of factors that repress myogenesis in vivo remain to be established. This review will focus on the fibroblast growth factor family and the evidence for its role in regulating myogenesis in culture and in vivo.

Activating and inhibitory heparin sequences for FGF-2 (basic FGF). Distinct requirements for FGF-1, FGF-2, and FGF-4.

Chlorate-treated Swiss 3T3 fibroblasts, with impaired synthesis of heparan sulfate proteoglycan, were used as target cells in assessing the ability of exogenous heparin-derived saccharides to promote the mitogenic activity of basic fibroblast growth factor 2 (FGF-2). Full-size native heparin (carrying iduronosyl 2-O-sulfate and glucosaminyl 6-O-sulfate groups), as well as a dodecasaccharide fraction isolated after limited deaminative cleavage of heparin, were efficient promoters, whereas the corresponding decasaccharides, or smaller oligosaccharides, were inactive. Neither selectively 2-O-desulfated nor preferentially 6-O-desulfated heparin were active. However, the latter derivative competed with native heparin for binding to FGF-2 and thus blocked the ability of native heparin to promote the mitogenic activity of FGF-2. The 6-O-desulfated heparin also prevented the ability of FGF-2 to suppress myogenic differentiation in MM14 mouse myoblasts. The binding region for FGF-2 has been identified as a pentasaccharide sequence containing a single essential O-sulfate group, at C2 of iduronic acid (1). It is proposed that the dodecasaccharide sequence required to promote receptor signaling by FGF-2 encompasses this pentasaccharide region, which binds the growth factor, and a site interacting with the receptor that contains essential 2-O- and 6-O-sulfate groups. Similar studies involving the related growth factors, FGF-1 and FGF-4, revealed differential effects of saccharides. The mitogenic effect induced by FGF-1 thus was not blocked by either the 2-O- or the 6-O-desulfated heparins. However, both of these derivatives, at high concentrations, promote mitogenic activity of FGF-4. It is concluded that specific saccharide sequences within heparan sulfate glycosaminoglycan chains favor the signaling by distinct members of the FGF family.