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INHIBITION OF MYOSTATIN WITH EMPHASIS ON FOLLISTATIN AS A THERAPY FOR MUSCLE DISEASE
LOUISE R. RODINO-KLAPAC, PhD,[SUP]1,[/SUP][SUP]2[/SUP] AMANDA M. HAIDET, BS,[SUP]1,[/SUP][SUP]2[/SUP] JANAIAH KOTA, PhD,[SUP]1,[/SUP][SUP]2[/SUP] CHALONDA HANDY, BS,[SUP]1,[/SUP][SUP]2[/SUP] BRIAN K. KASPAR, PhD,[SUP]1,[/SUP][SUP]2[/SUP] and JERRY R. MENDELL, MD[SUP]1,[/SUP][SUP]3[/SUP]
Author information ► Copyright and License information ►
The publisher's final edited version of this article is available at Muscle Nerve
See other articles in PMC that cite the published article.
Abstract
In most cases, pharmacologic strategies to treat genetic muscle disorders and certain acquired disorders, such as sporadic inclusion body myositis, have produced modest clinical benefits. In these conditions, inhibition of the myostatin pathway represents an alternative strategy to improve functional outcomes. Preclinical data that support this approach clearly demonstrate the potential for blocking the myostatin pathway. Follistatin has emerged as a powerful antagonist of myostatin that can increase muscle mass and strength. Follistatin was first isolated from the ovary and is known to suppress follicle-stimulating hormone. This raises concerns for potential adverse effects on the hypothalamic–pituitary–gonadal axis and possible reproductive capabilities. In this review we demonstrate a strategy to bypass off-target effects using an alternatively spliced cDNA of follistatin (FS344) delivered by adeno-associated virus (AAV) to muscle. The transgene product is a peptide of 315 amino acids that is secreted from the muscle and circulates in the serum, thus avoiding cell-surface binding sites. Using this approach our translational studies show increased muscle size and strength in species ranging from mice to monkeys. Adverse effects are avoided, and no organ system pathology or change in reproductive capabilities has been seen. These findings provide the impetus to move toward gene therapy clinical trials with delivery of AAV-FS344 to increase size and function of muscle in patients with neuromuscular disease.
Keywords: follistatin, myostatin inhibition, muscle disease, muscle enhancement
Strategies to increase muscle size and strength through inhibition of the myostatin pathway show promise for clinical application.[SUP]34[/SUP] Follistatin is a potent antagonist of myostatin that takes advantage of its ability to hinder access to signaling receptors on skeletal muscle. The muscle-building properties of follistatin are well demonstrated,[SUP]36[/SUP] but because it is a peptide with multiple functions, concerns have been raised regarding off-target effects when considering its appropriateness for treatment of muscle disease. The goal of this review is to thoroughly discuss these complex interactions and demonstrate a strategy that takes advantage of known follistatin properties that can be harnessed to promote efficacy to increase muscle mass and muscle strength in the absence of adverse clinical effects.
Emphasis on myostatin inhibition emerges because treating muscle disorders by most pharmacologic approaches has been disappointing. Androgen steroids, popular among athletes, pose long-term risks[SUP]66[/SUP]including: (1) endocrine (gonadal atrophy and sterility)[SUP]28[/SUP]; (2) somatic (changes in blood lipid profiles and cardiac hypertrophy)[SUP]3[/SUP][SUP],[/SUP][SUP]30[/SUP][SUP],[/SUP][SUP]37[/SUP]; and (3) neuropsychiatric (anxiety, depression, hostility, paranoia)[SUP]57[/SUP]; and attempts to treat muscle disorders have been disappointing.[SUP]5[/SUP] Glucocorticosteroids, the only beneficial drug treatment for muscular dystrophy, are virtually entirely targeted toward the Duchenne muscular dystrophy (DMD) population.[SUP]46[/SUP][SUP],[/SUP][SUP]50[/SUP] Even in this patient group the mechanism of benefit is poorly understood, and the evidence that muscle mass is increased is meager.[SUP]46[/SUP] For genetic muscle diseases, gene manipulation strategies are on the horizon, including gene replacement,[SUP]12[/SUP][SUP],[/SUP][SUP]19[/SUP][SUP],[/SUP][SUP]20[/SUP][SUP],[/SUP][SUP]62[/SUP] exon skipping,[SUP]1[/SUP][SUP],[/SUP][SUP]44[/SUP] and mutation suppression.[SUP]7[/SUP][SUP],[/SUP][SUP]23[/SUP]Despite enthusiasm, experimental studies suggest that these approaches usually fall short of returning function to normal.[SUP]40[/SUP] Combinational approaches that include partial correction of the underlying defect (i.e., micro-dystrophin) combined with increasing muscle size and strength appear to offer more.[SUP]2[/SUP] For muscle diseases where correction of the underlying defect might not be an option, increasing muscle size and strength may be opportune for both genetic and acquired muscle diseases where treatment options are limited. Examples include some forms of muscular dystrophy where gene manipulation strategies are not yet applicable (e.g., facioscapulohumeral dystrophy, FSHD), acquired disorders such as sporadic inclusion body myositis, where pharmacologic treatment failures predominate, or cachectic disorders related to cancer or aging that may be ideally suited for a muscle-enhancing approach.
MYOSTATIN PATHWAY
The potential for follistatin as a therapeutic agent for muscle disease cannot be fully understood without knowledge of the myostatin pathway. Myostatin is a member of the transforming growth factor-beta (TGF-β) superfamily of signal peptides. It is expressed specifically in developing and adult skeletal muscle.[SUP]45[/SUP] During development, myostatin expression limits the size of the muscle in concert with multiple factors that sculpt the limbs in relation to skeletal, vascular, and ectodermal patterns of growth.[SUP]4[/SUP] Myogenic cells respond to myostatin by downregulating the expression of Pax-3 and Myf-5, important transcriptional regulators of myogenic cell proliferation, and Myo-D, an early marker of muscle differentiation. In their sentinel report in 1997, McPherron et al.[SUP]45[/SUP] demonstrated the biological effect of targeted disruption of growth and differentiation factor-8 (GDF-8) gene in the mouse. GDF-8 null mice were significantly larger in size than wildtype animals, and there was widespread increase in skeletal muscle mass (Fig. 1). Individual muscles of mutant mice weighed 2−3 times more than those of wildtype animals. The increase in mass was the result of a combination of muscle hypertrophy and hyperplasia. These experiments established the GDF-8 peptide as a major player for inhibiting muscle growth, with the designated name “myostatin.”
FIGURE 1
Myostatin null animals exhibit increased muscle mass. Adult myostatin null mice demonstrating increased size (right) as compared to wildtype (left) animals. Reprinted with permission from Lee SJ, McPherron AC. Curr Opin Genet Dev 1999:5:604−607. ...
MYOSTATIN SYNTHESIS
The human myostatin gene (MSTN) maps to chromosome 2q32.2.[SUP]67[/SUP] The gene contains three exons and three putative transcription start sites that encode a 376-amino acid precursor protein composed of a signal peptide, an N-terminal propeptide domain and a C-terminal domain that gives rise to the active peptide (Fig. 2). Myostatin activation requires stepwise proteolytic cleavages of the precursor protein. Initially, furin family enzymes remove the signal peptide (24-amino acid). A second cleavage event at amino acid sites 240−243 leaves two fragments: an N-terminal propeptide domain of 27,640 Da and C-terminal domain of 12,400 Da destined to become the active myostatin protein.[SUP]34[/SUP] Parallel fragments of the myostatin C-terminal are linked through a disulfide bond, referred to as the myostatin C-terminal dimer that remains noncovalently complexed to the N-terminal propeptide.[SUP]33[/SUP][SUP],[/SUP][SUP]71[/SUP] This noncovalent complex circulates in the blood and maintains the myostatin C-terminal dimer in a latent, inactive state.[SUP]25[/SUP][SUP],[/SUP][SUP]33[/SUP] A third cleavage at amino acid 76 is required for the myostatin C-terminal to become active.[SUP]79[/SUP] This occurs via a different enzyme group, a metalloproteinase that belongs to the bone morphogenic protein (BMP)-1/tolloid (TLD) family.
FIGURE 2
Blocking the myostatin pathway. Myostatin (M) activation requires stepwise proteolytic cleavages of the precursor protein. After the signal peptide (SP) is removed, a second cleavage event leaves two fragments: an N-terminal propeptide domain of ≈28 ...
Myostatin signaling acts through the activin receptor type IIB (ActRIIB) on skeletal muscle by setting in motion an intracellular cascade of events. First, there is presumed recruitment of a type I co-receptor.[SUP]34[/SUP]Activin receptor-like kinases 4 and/or 5 (ALK-4, ALK-5) represent candidate coreceptors that are phosphorylated by ActRIIB.[SUP]59[/SUP] This in turn leads to phosphorylation of TGF-β specific Smads 2 and 3 that form a complex with Smad 4. The Smad 2/3/4 complex is translocated to the nucleus to regulate expression of targeted genes such as MyoD and myogenic regulatory factors (MRFs) (Fig. 2).[SUP]32[/SUP][SUP],[/SUP][SUP]33[/SUP][SUP],[/SUP][SUP]42[/SUP]
Apart from an essential role in muscle growth, recent evidence indicates that myostatin has a regulatory role in skeletal muscle fibrosis. Li et al.[SUP]38[/SUP] revealed that myostatin and the ActRIIB receptor are expressed on muscle fibroblasts, thus inducing their proliferation and the production of extracellular matrix proteins. This proliferation leads to the induction of the canonical Smad signaling pathway in fibroblasts by Smad3 phosphorylation and downstream p38 MAPK and Akt pathways.[SUP]38[/SUP] This enhances the therapeutic potential for myostatin inhibition that could lead to muscle enlargement while at the same time decreasing muscle fibrosis. In many muscle disorders, active fibrosis leads to the irreversibility of the condition, be it inherited or acquired.
FOLLISTATIN SYNTHESIS, ISOFORMS, AND PHYSIOLOGIC ROLE
Follistatin, secreted as a glycoprotein, was originally identified in porcine ovarian follicular fluid and received its name because it suppresses synthesis and secretion of follicle-stimulating hormone (FSH) from the pituitary gland.[SUP]56[/SUP] It is highly conserved, with overall species homology of 83% and 95% in mammals. Two groups isolated and published their results in 1987. One coined the term follistatin,[SUP]14[/SUP] and the other named it FSH-suppressing protein (FSP).[SUP]61[/SUP] With time, follistatin became the popular designation, but the name hardly does justice to a peptide with functions that extend beyond FSH suppression.
The follistatin gene localizes to chromosome 5q11.2. It is composed of a relatively small 6-kb genomic DNA consisting of six exons. There is an alternative splice site that generates two major species, a full-length version that encodes a 344-amino acid preprotein differing by a 27-amino acid sequence from its carboxy-shortened version of the 317-amino acid form missing exon 6 (Fig. 3).[SUP]64[/SUP][SUP],[/SUP][SUP]65[/SUP] Prior to activation, follistatin, like myostatin, undergoes further posttranslational modification to lose another 29 amino acids by removal of the signal peptide that results in polypeptides of 315 (FS315), often referred to as the long isoform and 288 (FS288), called the short isoform. There is also evidence to suggest that FS315 can be proteolytically cleaved in vivo at the carboxy-terminal to give an intermediate isoform of 303 amino acids.[SUP]69[/SUP]
FIGURE 3
The follistatin gene consists of six exons. Alternative splicing generates two isoforms, FS317 and FS344. Alternative splicing occurs at the 3′ end of the gene between exon 5 and exon 6. Splicing out of intron 5 generates a stop codon immediately ...
At the time follistatin was first isolated, little was known of its mechanism of action. In a major breakthrough, follistatin was found to be an activin-binding protein.[SUP]52[/SUP] An important function of follistatin is its collaborative role in reproductive physiology with other TGF-β superfamily members, activin and inhibins. These TGF-βfamily peptides have overlapping autocrine/paracrine functions. All three were initially purified from gonadal fluids and characterized based on their ability to modulate FSH. In addition to gonadal sites of production (ovary/testes), these peptides are all produced by cells in the hypothalamic–pituitary axis (gonadatropes and folliculostellate cells). Follistatin binds activin and attenuates the release of FSH. Activin is secreted by the follicle of the ovary and serves to enhance FSH secretion. Inhibins, which are secreted in two forms (A and B), inhibit the release of FSH at the hypothalamic–pituitary level. In addition, it is well documented that follistatin can abrogate the effects of GnRH in stimulating FSH secretion.[SUP]77[/SUP][SUP],[/SUP][SUP]78[/SUP] This is also due in part to the blocking of transcriptional activation of the GnRH receptor gene by activin.[SUP]16[/SUP]
This complex interaction of follistatin in relation to pituitary and gonadal function has raised concerns about its potential use as a therapeutic agent in the clinic. However, potential recombinant products can take advantage of differences between the isoforms in their ability to bind heparin sulfate. A well-recognized follistatin heparin-binding site is present at residues 72−86, which is a region rich in basic amino acids.[SUP]73[/SUP] In contrast, the carboxy-terminal 27 amino acid sequence of FS-315, composed of 44% acidic amino acids, interferes with the heparin site.[SUP]70[/SUP] These considerations take on a novel perspective with regard to gene therapy considering potential transgene products. FS-288, the shorter alternatively spliced product has an ≈10-fold higher affinity to activin compared to FS-315.[SUP]24[/SUP][SUP],[/SUP][SUP]69[/SUP][SUP],[/SUP][SUP]70[/SUP] In addition, FS-288 targets heparin sulfate proteoglycan binding sites at cell surfaces, while FS-315 represents a soluble serum-based or circulating follistatin isoform.[SUP]63[/SUP] In developing a gene therapy product for clinical use, we have taken advantage of this property. In our preclinical research studies, adeno-associated (AAV) virus that carries cDNA FS-344 delivers a gene therapy product (FS-315) without interruption in reproductive capabilities in either males or females in species ranging from mice to monkeys. Our results support prior observations that impairment of activin binding is more closely allied with FS-288 and its cell surface-binding properties mediated by heparan sulfate proteoglycans. This strategy greatly enhances the margin of safety for clinical trials because the FS-315 isoform has a limited effect on activin modulation by protecting the pituitary–gonadal axis from unwanted alterations. The same can be said for avoiding off-target effects mediated by cell surface binding of follistatin, including functions related to cellular differentiation, repair, and apoptosis.[SUP]5[/SUP]
The origin of follistatin under normal physiologic conditions is not entirely understood. Clearly, follistatin is produced locally in the pituitary gland and in gonads, ovaries, and testes. Overall, measurements of follistatin during the menstrual cycle show few changes.[SUP]15[/SUP][SUP],[/SUP][SUP]18[/SUP][SUP],[/SUP][SUP]29[/SUP] However, a notable exception is during pregnancy, when follistatin concentrations rise toward term in parallel with activin.[SUP]15[/SUP][SUP],[/SUP][SUP]76[/SUP][SUP],[/SUP][SUP]79[/SUP] Follistatin is widely distributed throughout multiple organs, and the majority of follistatin found in the circulation is likely secreted from the walls of blood vessels.
GENETICALLY ALTERED MICE OVER- AND UNDEREXPRESSING FOLLISTATIN
A component of understanding the functional role of follistatin can be gleaned from studies of genetically modified mice. Studies that evaluate the overexpression of the follistatin gene through genetically induced gain-of-function mutations are worth study to examine the potential for off-target effects. However, information derived from such models requires cautious interpretation because of species differences and influences of overexpression during development that are not clinically relevant. Despite caveats, the findings in a transgenic model in which the follistatin gene was introduced under control of a muscle-specific myosin light chain promoter are encouraging.[SUP]33[/SUP] Muscle mass increase was significantly greater than observed in the myostatin null mutant mouse. These results suggest that at least part of the effect of follistatin results from impact on another pathway independent of myostatin inhibition. This hypothesis is reinforced by additional studies in which mice that overexpress transgenic follistatin were crossed with myostatin null animals.[SUP]36[/SUP] The resulting phenotype appeared to be additive, with a quadrupling of muscle mass in follistatin[SUP]+[/SUP]/myostatin[SUP]−/−[/SUP]mice, outstripping the effects of either myostatin nulls or follistatin-overexpressing animals alone. These findings emphasize that other signaling pathways could be exploited to increase muscle size and strength.[SUP]36[/SUP]
In another gain-of-function mutant mouse line, the metallothionein (MT)-1 promoter was placed upstream of the follistatin gene.[SUP]21[/SUP] Observations on gonadal changes in this model are thought-provoking, but they may not be directly related to clinical translational considerations. On a positive note, the MT-follistatin transgenic offspring were viable and developed to adults. In addition, no deleterious effects were seen in any organ system other than gonadal tissue. In males testes size was decreased, with variable Leydig cell hyperplasia, an arrest in spermatogenesis and seminiferous tubular degeneration leading to infertility. Females had thin uteri and small ovaries, and many became infertile. Interpreting these results in the context of what we would anticipate in a clinical trial of gene therapy requires caution. In the patient, our concern would be that high serum levels of follistatin would bind activin and result in reduced serum FSH levels leading to gonadal dysfunction. Instead, the follistatin-overexpressing mice had normal FSH levels. This implies that in the transgenic mouse model, follistatin overexpression exerted its effects through gene expression in the matrix of the end organ, disrupting local regulation. This would not parallel the gene therapy paradigm, where the transgene product, FS344, is nontissue bound, and has no effect on reproductive function.
On the opposite side of the spectrum, a loss-of-function mutant mouse was created by a targeted deletion of the follistatin gene.[SUP]43[/SUP] The mutant mice survived until birth but died within hours of delivery. Defects included growth-retardation and shiny, taut skin. There was poor whisker development, hyperkeratosis of skin, abnormal tooth development, defects in the hard palate, and reduced size of intercostal and diaphragm muscles. The central and peripheral nervous systems, however, were intact. At best, this short-lived model has little relevance to our goals of studying follistatin as a potential therapeutic agent, but it does reinforce that follistatin may mediate effects through pathways of the TGF-β family other than myostatin.
INHIBITION OF MYOSTATIN WITH EMPHASIS ON FOLLISTATIN AS A THERAPY FOR MUSCLE DISEASE
LOUISE R. RODINO-KLAPAC, PhD,[SUP]1,[/SUP][SUP]2[/SUP] AMANDA M. HAIDET, BS,[SUP]1,[/SUP][SUP]2[/SUP] JANAIAH KOTA, PhD,[SUP]1,[/SUP][SUP]2[/SUP] CHALONDA HANDY, BS,[SUP]1,[/SUP][SUP]2[/SUP] BRIAN K. KASPAR, PhD,[SUP]1,[/SUP][SUP]2[/SUP] and JERRY R. MENDELL, MD[SUP]1,[/SUP][SUP]3[/SUP]
Author information ► Copyright and License information ►
The publisher's final edited version of this article is available at Muscle Nerve
See other articles in PMC that cite the published article.
Abstract
In most cases, pharmacologic strategies to treat genetic muscle disorders and certain acquired disorders, such as sporadic inclusion body myositis, have produced modest clinical benefits. In these conditions, inhibition of the myostatin pathway represents an alternative strategy to improve functional outcomes. Preclinical data that support this approach clearly demonstrate the potential for blocking the myostatin pathway. Follistatin has emerged as a powerful antagonist of myostatin that can increase muscle mass and strength. Follistatin was first isolated from the ovary and is known to suppress follicle-stimulating hormone. This raises concerns for potential adverse effects on the hypothalamic–pituitary–gonadal axis and possible reproductive capabilities. In this review we demonstrate a strategy to bypass off-target effects using an alternatively spliced cDNA of follistatin (FS344) delivered by adeno-associated virus (AAV) to muscle. The transgene product is a peptide of 315 amino acids that is secreted from the muscle and circulates in the serum, thus avoiding cell-surface binding sites. Using this approach our translational studies show increased muscle size and strength in species ranging from mice to monkeys. Adverse effects are avoided, and no organ system pathology or change in reproductive capabilities has been seen. These findings provide the impetus to move toward gene therapy clinical trials with delivery of AAV-FS344 to increase size and function of muscle in patients with neuromuscular disease.
Keywords: follistatin, myostatin inhibition, muscle disease, muscle enhancement
Strategies to increase muscle size and strength through inhibition of the myostatin pathway show promise for clinical application.[SUP]34[/SUP] Follistatin is a potent antagonist of myostatin that takes advantage of its ability to hinder access to signaling receptors on skeletal muscle. The muscle-building properties of follistatin are well demonstrated,[SUP]36[/SUP] but because it is a peptide with multiple functions, concerns have been raised regarding off-target effects when considering its appropriateness for treatment of muscle disease. The goal of this review is to thoroughly discuss these complex interactions and demonstrate a strategy that takes advantage of known follistatin properties that can be harnessed to promote efficacy to increase muscle mass and muscle strength in the absence of adverse clinical effects.
Emphasis on myostatin inhibition emerges because treating muscle disorders by most pharmacologic approaches has been disappointing. Androgen steroids, popular among athletes, pose long-term risks[SUP]66[/SUP]including: (1) endocrine (gonadal atrophy and sterility)[SUP]28[/SUP]; (2) somatic (changes in blood lipid profiles and cardiac hypertrophy)[SUP]3[/SUP][SUP],[/SUP][SUP]30[/SUP][SUP],[/SUP][SUP]37[/SUP]; and (3) neuropsychiatric (anxiety, depression, hostility, paranoia)[SUP]57[/SUP]; and attempts to treat muscle disorders have been disappointing.[SUP]5[/SUP] Glucocorticosteroids, the only beneficial drug treatment for muscular dystrophy, are virtually entirely targeted toward the Duchenne muscular dystrophy (DMD) population.[SUP]46[/SUP][SUP],[/SUP][SUP]50[/SUP] Even in this patient group the mechanism of benefit is poorly understood, and the evidence that muscle mass is increased is meager.[SUP]46[/SUP] For genetic muscle diseases, gene manipulation strategies are on the horizon, including gene replacement,[SUP]12[/SUP][SUP],[/SUP][SUP]19[/SUP][SUP],[/SUP][SUP]20[/SUP][SUP],[/SUP][SUP]62[/SUP] exon skipping,[SUP]1[/SUP][SUP],[/SUP][SUP]44[/SUP] and mutation suppression.[SUP]7[/SUP][SUP],[/SUP][SUP]23[/SUP]Despite enthusiasm, experimental studies suggest that these approaches usually fall short of returning function to normal.[SUP]40[/SUP] Combinational approaches that include partial correction of the underlying defect (i.e., micro-dystrophin) combined with increasing muscle size and strength appear to offer more.[SUP]2[/SUP] For muscle diseases where correction of the underlying defect might not be an option, increasing muscle size and strength may be opportune for both genetic and acquired muscle diseases where treatment options are limited. Examples include some forms of muscular dystrophy where gene manipulation strategies are not yet applicable (e.g., facioscapulohumeral dystrophy, FSHD), acquired disorders such as sporadic inclusion body myositis, where pharmacologic treatment failures predominate, or cachectic disorders related to cancer or aging that may be ideally suited for a muscle-enhancing approach.
MYOSTATIN PATHWAY
The potential for follistatin as a therapeutic agent for muscle disease cannot be fully understood without knowledge of the myostatin pathway. Myostatin is a member of the transforming growth factor-beta (TGF-β) superfamily of signal peptides. It is expressed specifically in developing and adult skeletal muscle.[SUP]45[/SUP] During development, myostatin expression limits the size of the muscle in concert with multiple factors that sculpt the limbs in relation to skeletal, vascular, and ectodermal patterns of growth.[SUP]4[/SUP] Myogenic cells respond to myostatin by downregulating the expression of Pax-3 and Myf-5, important transcriptional regulators of myogenic cell proliferation, and Myo-D, an early marker of muscle differentiation. In their sentinel report in 1997, McPherron et al.[SUP]45[/SUP] demonstrated the biological effect of targeted disruption of growth and differentiation factor-8 (GDF-8) gene in the mouse. GDF-8 null mice were significantly larger in size than wildtype animals, and there was widespread increase in skeletal muscle mass (Fig. 1). Individual muscles of mutant mice weighed 2−3 times more than those of wildtype animals. The increase in mass was the result of a combination of muscle hypertrophy and hyperplasia. These experiments established the GDF-8 peptide as a major player for inhibiting muscle growth, with the designated name “myostatin.”
FIGURE 1Myostatin null animals exhibit increased muscle mass. Adult myostatin null mice demonstrating increased size (right) as compared to wildtype (left) animals. Reprinted with permission from Lee SJ, McPherron AC. Curr Opin Genet Dev 1999:5:604−607. ...
MYOSTATIN SYNTHESIS
The human myostatin gene (MSTN) maps to chromosome 2q32.2.[SUP]67[/SUP] The gene contains three exons and three putative transcription start sites that encode a 376-amino acid precursor protein composed of a signal peptide, an N-terminal propeptide domain and a C-terminal domain that gives rise to the active peptide (Fig. 2). Myostatin activation requires stepwise proteolytic cleavages of the precursor protein. Initially, furin family enzymes remove the signal peptide (24-amino acid). A second cleavage event at amino acid sites 240−243 leaves two fragments: an N-terminal propeptide domain of 27,640 Da and C-terminal domain of 12,400 Da destined to become the active myostatin protein.[SUP]34[/SUP] Parallel fragments of the myostatin C-terminal are linked through a disulfide bond, referred to as the myostatin C-terminal dimer that remains noncovalently complexed to the N-terminal propeptide.[SUP]33[/SUP][SUP],[/SUP][SUP]71[/SUP] This noncovalent complex circulates in the blood and maintains the myostatin C-terminal dimer in a latent, inactive state.[SUP]25[/SUP][SUP],[/SUP][SUP]33[/SUP] A third cleavage at amino acid 76 is required for the myostatin C-terminal to become active.[SUP]79[/SUP] This occurs via a different enzyme group, a metalloproteinase that belongs to the bone morphogenic protein (BMP)-1/tolloid (TLD) family.
FIGURE 2Blocking the myostatin pathway. Myostatin (M) activation requires stepwise proteolytic cleavages of the precursor protein. After the signal peptide (SP) is removed, a second cleavage event leaves two fragments: an N-terminal propeptide domain of ≈28 ...
Myostatin signaling acts through the activin receptor type IIB (ActRIIB) on skeletal muscle by setting in motion an intracellular cascade of events. First, there is presumed recruitment of a type I co-receptor.[SUP]34[/SUP]Activin receptor-like kinases 4 and/or 5 (ALK-4, ALK-5) represent candidate coreceptors that are phosphorylated by ActRIIB.[SUP]59[/SUP] This in turn leads to phosphorylation of TGF-β specific Smads 2 and 3 that form a complex with Smad 4. The Smad 2/3/4 complex is translocated to the nucleus to regulate expression of targeted genes such as MyoD and myogenic regulatory factors (MRFs) (Fig. 2).[SUP]32[/SUP][SUP],[/SUP][SUP]33[/SUP][SUP],[/SUP][SUP]42[/SUP]
Apart from an essential role in muscle growth, recent evidence indicates that myostatin has a regulatory role in skeletal muscle fibrosis. Li et al.[SUP]38[/SUP] revealed that myostatin and the ActRIIB receptor are expressed on muscle fibroblasts, thus inducing their proliferation and the production of extracellular matrix proteins. This proliferation leads to the induction of the canonical Smad signaling pathway in fibroblasts by Smad3 phosphorylation and downstream p38 MAPK and Akt pathways.[SUP]38[/SUP] This enhances the therapeutic potential for myostatin inhibition that could lead to muscle enlargement while at the same time decreasing muscle fibrosis. In many muscle disorders, active fibrosis leads to the irreversibility of the condition, be it inherited or acquired.
FOLLISTATIN SYNTHESIS, ISOFORMS, AND PHYSIOLOGIC ROLE
Follistatin, secreted as a glycoprotein, was originally identified in porcine ovarian follicular fluid and received its name because it suppresses synthesis and secretion of follicle-stimulating hormone (FSH) from the pituitary gland.[SUP]56[/SUP] It is highly conserved, with overall species homology of 83% and 95% in mammals. Two groups isolated and published their results in 1987. One coined the term follistatin,[SUP]14[/SUP] and the other named it FSH-suppressing protein (FSP).[SUP]61[/SUP] With time, follistatin became the popular designation, but the name hardly does justice to a peptide with functions that extend beyond FSH suppression.
The follistatin gene localizes to chromosome 5q11.2. It is composed of a relatively small 6-kb genomic DNA consisting of six exons. There is an alternative splice site that generates two major species, a full-length version that encodes a 344-amino acid preprotein differing by a 27-amino acid sequence from its carboxy-shortened version of the 317-amino acid form missing exon 6 (Fig. 3).[SUP]64[/SUP][SUP],[/SUP][SUP]65[/SUP] Prior to activation, follistatin, like myostatin, undergoes further posttranslational modification to lose another 29 amino acids by removal of the signal peptide that results in polypeptides of 315 (FS315), often referred to as the long isoform and 288 (FS288), called the short isoform. There is also evidence to suggest that FS315 can be proteolytically cleaved in vivo at the carboxy-terminal to give an intermediate isoform of 303 amino acids.[SUP]69[/SUP]
FIGURE 3The follistatin gene consists of six exons. Alternative splicing generates two isoforms, FS317 and FS344. Alternative splicing occurs at the 3′ end of the gene between exon 5 and exon 6. Splicing out of intron 5 generates a stop codon immediately ...
At the time follistatin was first isolated, little was known of its mechanism of action. In a major breakthrough, follistatin was found to be an activin-binding protein.[SUP]52[/SUP] An important function of follistatin is its collaborative role in reproductive physiology with other TGF-β superfamily members, activin and inhibins. These TGF-βfamily peptides have overlapping autocrine/paracrine functions. All three were initially purified from gonadal fluids and characterized based on their ability to modulate FSH. In addition to gonadal sites of production (ovary/testes), these peptides are all produced by cells in the hypothalamic–pituitary axis (gonadatropes and folliculostellate cells). Follistatin binds activin and attenuates the release of FSH. Activin is secreted by the follicle of the ovary and serves to enhance FSH secretion. Inhibins, which are secreted in two forms (A and B), inhibit the release of FSH at the hypothalamic–pituitary level. In addition, it is well documented that follistatin can abrogate the effects of GnRH in stimulating FSH secretion.[SUP]77[/SUP][SUP],[/SUP][SUP]78[/SUP] This is also due in part to the blocking of transcriptional activation of the GnRH receptor gene by activin.[SUP]16[/SUP]
This complex interaction of follistatin in relation to pituitary and gonadal function has raised concerns about its potential use as a therapeutic agent in the clinic. However, potential recombinant products can take advantage of differences between the isoforms in their ability to bind heparin sulfate. A well-recognized follistatin heparin-binding site is present at residues 72−86, which is a region rich in basic amino acids.[SUP]73[/SUP] In contrast, the carboxy-terminal 27 amino acid sequence of FS-315, composed of 44% acidic amino acids, interferes with the heparin site.[SUP]70[/SUP] These considerations take on a novel perspective with regard to gene therapy considering potential transgene products. FS-288, the shorter alternatively spliced product has an ≈10-fold higher affinity to activin compared to FS-315.[SUP]24[/SUP][SUP],[/SUP][SUP]69[/SUP][SUP],[/SUP][SUP]70[/SUP] In addition, FS-288 targets heparin sulfate proteoglycan binding sites at cell surfaces, while FS-315 represents a soluble serum-based or circulating follistatin isoform.[SUP]63[/SUP] In developing a gene therapy product for clinical use, we have taken advantage of this property. In our preclinical research studies, adeno-associated (AAV) virus that carries cDNA FS-344 delivers a gene therapy product (FS-315) without interruption in reproductive capabilities in either males or females in species ranging from mice to monkeys. Our results support prior observations that impairment of activin binding is more closely allied with FS-288 and its cell surface-binding properties mediated by heparan sulfate proteoglycans. This strategy greatly enhances the margin of safety for clinical trials because the FS-315 isoform has a limited effect on activin modulation by protecting the pituitary–gonadal axis from unwanted alterations. The same can be said for avoiding off-target effects mediated by cell surface binding of follistatin, including functions related to cellular differentiation, repair, and apoptosis.[SUP]5[/SUP]
The origin of follistatin under normal physiologic conditions is not entirely understood. Clearly, follistatin is produced locally in the pituitary gland and in gonads, ovaries, and testes. Overall, measurements of follistatin during the menstrual cycle show few changes.[SUP]15[/SUP][SUP],[/SUP][SUP]18[/SUP][SUP],[/SUP][SUP]29[/SUP] However, a notable exception is during pregnancy, when follistatin concentrations rise toward term in parallel with activin.[SUP]15[/SUP][SUP],[/SUP][SUP]76[/SUP][SUP],[/SUP][SUP]79[/SUP] Follistatin is widely distributed throughout multiple organs, and the majority of follistatin found in the circulation is likely secreted from the walls of blood vessels.
GENETICALLY ALTERED MICE OVER- AND UNDEREXPRESSING FOLLISTATIN
A component of understanding the functional role of follistatin can be gleaned from studies of genetically modified mice. Studies that evaluate the overexpression of the follistatin gene through genetically induced gain-of-function mutations are worth study to examine the potential for off-target effects. However, information derived from such models requires cautious interpretation because of species differences and influences of overexpression during development that are not clinically relevant. Despite caveats, the findings in a transgenic model in which the follistatin gene was introduced under control of a muscle-specific myosin light chain promoter are encouraging.[SUP]33[/SUP] Muscle mass increase was significantly greater than observed in the myostatin null mutant mouse. These results suggest that at least part of the effect of follistatin results from impact on another pathway independent of myostatin inhibition. This hypothesis is reinforced by additional studies in which mice that overexpress transgenic follistatin were crossed with myostatin null animals.[SUP]36[/SUP] The resulting phenotype appeared to be additive, with a quadrupling of muscle mass in follistatin[SUP]+[/SUP]/myostatin[SUP]−/−[/SUP]mice, outstripping the effects of either myostatin nulls or follistatin-overexpressing animals alone. These findings emphasize that other signaling pathways could be exploited to increase muscle size and strength.[SUP]36[/SUP]
In another gain-of-function mutant mouse line, the metallothionein (MT)-1 promoter was placed upstream of the follistatin gene.[SUP]21[/SUP] Observations on gonadal changes in this model are thought-provoking, but they may not be directly related to clinical translational considerations. On a positive note, the MT-follistatin transgenic offspring were viable and developed to adults. In addition, no deleterious effects were seen in any organ system other than gonadal tissue. In males testes size was decreased, with variable Leydig cell hyperplasia, an arrest in spermatogenesis and seminiferous tubular degeneration leading to infertility. Females had thin uteri and small ovaries, and many became infertile. Interpreting these results in the context of what we would anticipate in a clinical trial of gene therapy requires caution. In the patient, our concern would be that high serum levels of follistatin would bind activin and result in reduced serum FSH levels leading to gonadal dysfunction. Instead, the follistatin-overexpressing mice had normal FSH levels. This implies that in the transgenic mouse model, follistatin overexpression exerted its effects through gene expression in the matrix of the end organ, disrupting local regulation. This would not parallel the gene therapy paradigm, where the transgene product, FS344, is nontissue bound, and has no effect on reproductive function.
On the opposite side of the spectrum, a loss-of-function mutant mouse was created by a targeted deletion of the follistatin gene.[SUP]43[/SUP] The mutant mice survived until birth but died within hours of delivery. Defects included growth-retardation and shiny, taut skin. There was poor whisker development, hyperkeratosis of skin, abnormal tooth development, defects in the hard palate, and reduced size of intercostal and diaphragm muscles. The central and peripheral nervous systems, however, were intact. At best, this short-lived model has little relevance to our goals of studying follistatin as a potential therapeutic agent, but it does reinforce that follistatin may mediate effects through pathways of the TGF-β family other than myostatin.
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