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10-31-2014, 06:49 AM
Follistatin complexes Myostatin and antagonises Myostatin-mediated inhibition of myogenesis

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Abstract

Follistatin is known to antagonise the function of several members of the TGF-β family of secreted signalling factors, including Myostatin, the most powerful inhibitor of muscle growth characterised to date. In this study, we compare the expression of Myostatin and Follistatin during chick development and show that they are expressed in the vicinity or in overlapping domains to suggest possible interaction during muscle development. We performed yeast and mammalian two-hybrid studies and show that Myostatin and Follistatin interact directly. We further show that single modules of the Follistatin protein cannot associate with Myostatin suggesting that the entire protein is required for the interaction. We analysed the interaction kinetics of the two proteins and found that Follistatin binds Myostatin with a high affinity of 5.84 × 10−10 M. We next tested whether Follistatin suppresses Myostatin activity during muscle development. We confirmed our previous observation that treatment of chick limb buds with Myostatin results in a severe decrease in the expression of two key myogenic regulatory genes Pax-3 and MyoD. However, in the presence of Follistatin, the Myostatin-mediated inhibition of Pax-3 and MyoD expression is blocked. We additionally show that Myostatin inhibits terminal differentiation of muscle cells in high-density cell cultures of limb mesenchyme (micromass) and that Follistatin rescues muscle differentiation in a concentration-dependent manner. In summary, our data suggest that Follistatin antagonises Myostatin by direct protein interaction, which prevents Myostatin from executing its inhibitory effect on muscle development.

Keywords



Follistatin;
Myostatin;
Myogenesis;
Chick;
Embryo;
Development;
Pax-3;
MyoD

Introduction

Myostatin, a member of the transforming growth factor-beta (TGF-β) family of signalling molecules, has been implicated in determining muscle size by restricting muscle growth McPherron and Lee, 1997 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB27) and McPherron et al., 1997 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB28). During development, Myostatin is expressed at the appropriate time and positions to locally decrease the rate of muscle growth without interfering with the establishment of the muscle pattern (Amthor et al., 2002b) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB5). Myogenic cells respond to Myostatin by down-regulating the expression of key transcriptional regulators of muscle development such as Pax-3, MyoD and Myf-5, which inhibit differentiation and further growth of muscle.Follistatin, a secreted glycoprotein, antagonises numerous members of the TGF-β superfamily including Myostatin Amthor et al., 2002a (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB4), Fainsod et al., 1997 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB9), Hemmati-Brivanlou et al., 1994 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB11), Iemura et al., 1998 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB14),Michel et al., 1993 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB29) and Zimmers et al., 2002 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB38). Follistatin and Myostatin are expressed in or near developing muscle Amthor et al., 1996 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB1), Amthor et al., 1999 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB3), Amthor et al., 2002a (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB4) and Amthor et al., 2002b (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB5). However, it has not yet been demonstrated whether Follistatin and Myostatin interact directly. Experimentally induced over-expression of Follistatin results in muscle enlargement, whereas the Follistatin−/− KO mouse displays muscle deficiency Lee and McPherron, 2001 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB21) and Matzuk et al., 1995 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB25). In the presence of Follistatin, Myostatin fails to bind its receptor, and Myostatin-induced muscle loss can be prevented Lee and McPherron, 2001 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB21) and Zimmers et al., 2002 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB38). Although both Myostatin and Follistatin are detected in serum, they do not associate in this medium. Instead, Myostatin circulates as a complex by associating either with the Myostatin propeptide, with FLRP, a Follistatin-related protein, or with GASP, which is a putative protease inhibitor that contains a Follistatin-like domain Hill et al., 2002 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB12) and Hill et al., 2003 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB13). This raises the question whether Follistatin and Myostatin interact directly or whether both proteins use independent signalling cascades.The Follistatin gene undergoes alternative splicing to yield either short or long forms of mRNAs. These are translated into pre-proteins and then modified to remove the signal sequence (reviewed by Patel, 1998 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB31)). The short isoform yields a protein composed of 288 amino acids (FS-288), which is 8–10 times more biologically active than the product of the long isoform (FS-315) (Inouye et al., 1991) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB15).Myostatin is synthesised as a precursor protein, which consists of a N-terminal propeptide domain that harbours the signal sequence and a C-terminal domain that forms a disulfide-linked dimer and functions as the active ligand McPherron et al., 1997 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB28) and Thomas et al., 2000 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB35). After cleavage of the propeptide, a large fraction of Myostatin is still non-covalently bound to its propeptide and requires release from the propeptide to attain biological activity Lee and McPherron, 2001 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB21) and Zimmers et al., 2002 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB38). Myostatin binds the Activin Receptor Type IIB, which leads to the intracellular phosphorylation of Smad3 Langley et al., 2002 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB20), Lee and McPherron, 2001 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB21), Massague and Chen, 2000 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB24) and McPherron and Lee, 1996 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB26). Phosphorylated Smad3 can bind other Smad proteins and these complexes translocate into the nucleus, where they regulate the transcription of target genes (Massague and Chen, 2000) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB24). Additionally, phosphorylated Smad3 binds and thereby inhibits the transcriptional activity of MyoD (Liu et al., 2001) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB23).In the first part of this study, we have compared the expression pattern of Myostatin and Follistatin with a view to detect evidence that these proteins may interact during muscle development. We next determined whether Follistatin binds Myostatin using yeast and mammalian two-hybrid systems. We analysed the kinetics of the Myostatin–Follistatin interaction using surface plasmon resonance. We have subsequently tested the biological relevance of this interaction by applying recombinant Myostatin and Follistatin to developing muscle of chick embryonic limb buds both in vitro and in vivo.Materials and methods

Yeast two-hybrid studies

The DupLEX-A™ yeast two-hybrid system (Origene) was used to test protein–protein interactions. The C-terminal coding region of mouse Myostatin (bp 905–1234; Genbank accession number NM010834 (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=NM010834[accn])) encoding the processed or mature portion of Myostatin was cloned into the pEG202 bait plasmid (carrying the HIS3gene) containing the Lex-A DNA binding domain using BamH1 restriction sites (LexA-MSTNmat). The portion of mouse Follistatin (bp 88–948; Genbank accession number NM08046 (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=NM08046[accn])) encoding the Follistatin was cloned into the pJG4-5 target plasmid (carrying the TRP1 gene) containing the B42 DNA activation domain using EcoR1 restriction sites (B42-FS-288). This portion of mouse Follistatin (1–287) is analogous to the active human Follistatin-288 and therefore is referred to in this manuscript as Follistatin-288. Interaction between the encoded fusion proteins was investigated by co-transforming the bait and target plasmids together with the reporter gene (lacZ) plasmid pJK103 (carrying the URA3 gene) into the yeast strain EGY194 (MATa trp1 his3 ura3 leu2:4 LexAop-LEU2). Transformed yeast cells were plated onto medium lacking histidine, uracil and tryptophan and grown at 30°C for 3 days to select for the presence of the three plasmids. Two independent colonies were then transferred to medium lacking histidine, uracil, tryptophan and leucine for 3 days at 30°C to select for positive interactions between mature Myostatin and Follistatin-288. Positives clones were then tested for expression of the second reporter gene, lacZ, by growth on medium containing X-gal and lacking histidine, uracil and tryptophan. In the DupLEX™ system, expression of the target-B42 activation domain fusion protein is galactose-inducible and therefore galactose growth-dependence was also tested. Finally, positives clones were tested against the negative bait control pEG202max to ensure specificity.To map the binding site of Follistatin to mature Myostatin, cDNA encoding truncations of Follistatin 1–63, 64–288, 1–86, 1–100, 1–136, 1–150, 1–200 and 1–250 were cloned into pJG4-5 to create target plasmids, which were tested against the mature Myostatin bait plasmid as described above.Mammalian two-hybrid studies

The TOPOŽ Tools Mammalian Two Hybrid Kit (Invitrogen) was used to verify yeast two-hybrid results. The C-terminal coding region of Myostatin (bp 905–1234; Genbank accession number as above) encoding the processed portion of Myostatin was TOPOŽ joined to the Psv40-GAL4 5′ element and SV40 pA 3′ element according to the manufacturer's instructions to create a linear DNA template for the bait protein (GAL4-MSTNmat). Similarly, the portion of Follistatin (bp 88–948; Genbank accession number as above) encoding the active Follistatin was TOPOŽ joined to the Psv40-VP16 5′ element and SV40 pA 3′ element to create a linear DNA template for the prey protein (VP16-FS-288). The linear DNA templates were PCR amplified using a proofreading polymerase and the following primers: 5′-TATGTATCATACACATACGATTTAGGT-3′ and 5′-GACTCAAAGGGAACTTGTTTATTGCAGCTTATAATG-3′ and PCR products were purified.

CHO cells (American Tissue Culture Collection), a Chinese hamster ovary cell line, were maintained in Dulbecco's modified Eagle medium (DMEM)/F12 (1:1) (Invitrogen) containing 10% fetal bovine serum (Sigma), 1 × 105 IU/l penicillin (Sigma) 100 mg/l streptomycin (Sigma) and 27.8 mM NaHCO3 (maintenance medium) at 37°C in a humidified atmosphere of 5% CO2.Thirty nanograms of the bait and prey linear constructs were co-transfected along with the reporter plasmid pGAL/lacZ into CHO cells seeded at 2 × 104 cells per well on a 96-well plate using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 24 h, the medium was changed to fresh maintenance medium. After a further 24 h, cells were fixed for in situ staining of β-galactosidase activity by hydrolysis of X-gal. For in situ staining, media was discarded from wells and the cells fixed with 0.05% glutaraldehyde for 15 min at room temperature. Cells were rinsed thoroughly with three washes of PBS and incubated for 1 h at 37°C with 1 mg/ml X-gal in 35 mM K3Fe(CN)6, 35 mM K4Fe(CN)6ˇ3H20 and 2 mM MgCl2.Control transfections were carried out to ensure specificity of interactions. For the background control, no DNA was transfected. Mature Myostatin and active Follistatin were also tested against the pCR2.1/LgT prey control plasmid and pCR2.1/p53 bait control plasmid, respectively, proteins with which they should not interact.Surface plasmon resonance

All plasmon surface resonance experiments were performed using the BIACORE 3000. Purified recombinant human Follistatin and recombinant mouse Myostatin were purchased from R&D System (USA). Follistatin was immobilised onto the surface of a CM5 sensor chip (600 resonance units) using amine-coupling chemistry. A range of Myostatin concentrations were injected over the sensor chip surface at a flow rate of 30 μl/min at 25°C. Hepes-buffered saline (HBS: 10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20 pH 7.4) was used as a running buffer and for sample dilution. For controls, Myostatin was run over a derivatised sensor chip, which lacked Follistatin. All Myostatin curves were corrected by subtraction of the blank run. Biacore evaluation software was used for the mathematical fitting of experimental data.Preparation of chick embryos

Fertilised chick eggs were incubated at 38°C, and the embryos were staged according to Hamburger and Hamilton (1992) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB10). Experiments were performed on embryos at stages 22 to 24, re-incubated between 6 and 8 h, sacrificed and processed for whole-mount in situ hybridisation.Myostatin and Follistatin bead preparation and application to limb buds

Recombinant Myostatin and Follistatin protein were purchased from R&D Systems. Myostatin was applied to 80- to 120-μm Affigel beads and Follistatin to Heparin beads of same size (both Sigma, UK). The proteins were loaded onto beads as described by Cohn et al. (1995) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB7). Myostatin and Follistatin were used at 1 mg/ml concentration. For control bead implantation, beads were soaked in PBS only. For bead implantation, the dorsal ectoderm and mesenchyme of the right wing were punctured with an electrolytically sharpened tungsten needle, and beads were inserted into the punctured mesenchyme using a blunt glass needle. Beads were implanted at HH-stages stated in the text.Whole-mount in situ hybridisation

All chick embryos were washed in PBS and then fixed overnight in 4% paraformaldehyde at 4°C. Anti-sense RNA probes were labelled with digoxygenin, and whole-mount in situ hybridisation was performed as described by Nieto et al. (1996) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB30). The following probes were used in this study: Follistatin, full-length fragment, 1.1 kb (gift from Dr. Anthony Graham); Myostatin, 1 kb fragment (gift from Professor Se Jin Lee);MyoD, clone CMD9 full 1.5 kb length fragment (gift from Professor Bruce Patterson) and Pax-3, 645 bp fragment corresponding to nucleotides 468–1113 (gift from Dr Martin Goulding). Whole-mount embryos were wax or cryo-sectioned at a thickness of 15 μm for histological examination.Chick limb bud micromass assay

Micromass assays were carried out as described in Swalla and Solursh (1986) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB34). Briefly, limb buds from HH stages 21–22 chick embryos were dissected and placed into a 0.05% Trypsin-EDTA solution (Invitrogen). The ectoderm was removed using tungsten needles and a single cell suspension of the limb bud mesenchyme was obtained. Micromass cultures were plated at 2 × 105 cells in 10 μl drops, allowed to adhere for 2 h and then treated with media (DMEM, 10% Fetal Calf Serum—Invitrogen) containing either Myostatin or Myostatin plus Follistatin at concentrations stated in the text (R&D Systems). Micromass cultures were then fixed in 4% PFA and processed for myosin heavy chain (MHC) immunocytochemistry. Cultures were dehydrated through a methanol series and treated with hydrogen peroxide to eliminate endogenous peroxidase activity. After rehydration, cultures were incubated with a monoclonal anti-PanMHC antibody (clone A4ˇ1025, gift from Dr Simon Hughes) in the presence of 10% horse serum. After washing in PBS, rabbit anti-mouse biotin secondary antibody (Dako) was applied, cultures again washed, then incubated with an avidin–biotin complex (Vector Laboratories) and stained using a nickel enhanced DAB/hydrogen peroxide reaction (Vector Laboratories) according to manufacturer's protocols.Results

Co-expression of Follistatin and Myostatin related to muscle development

We have previously published independent detailed expression patterns of Follistatin and Myostatin during chick embryonic development Amthor et al., 1996 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB1), Amthor et al., 1999 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB3), Amthor et al., 2002a (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB4) and Amthor et al., 2002b (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB5), which suggested an overlap in expression at sites of muscle development. Here, we directly compared the expression pattern of both genes during limb and trunk muscle development.Abutting or overlapping expression of Follistatin and Myostatin during wing bud development occurs for the first time at HH-stages 25–26. Follistatin is highly expressed in the proximal wing bud and fades distally (Fig. 1A) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG1). Myostatin is expressed in a central domain and the expression extends more distally compared toFollistatin (Fig. 1B) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG1). Transverse sections through the proximal part of the wing buds reveal expression ofFollistatin in the subectodermal mesenchyme and in a central domain of the dorsal premuscle mass (Fig. 1E) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG1). Myostatin is expressed in the mesenchymal core of the limb bud and also in a central domain of dorsal premuscle mass (Fig. 1F) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG1). However, at this stage, the expression of Follistatin and Myostatin does not encompass the entire premuscle mass as indicated by the expression of Pax-3 and MyoD (Figs. 1C and D) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG1).Follistatin expression is increased in the muscle masses up to HH-stage 31, whereas during these stages,Myostatin expression is only found in some subdomains of the developing muscle (Figs. 1G and H) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG1). Thereafter, at late embryonic stages, Follistatin is down-regulated in muscle, but expression resides in the connective tissue surrounding the muscles, whereas Myostatin is increasingly expressed in most of the muscles (data not shown, see also Amthor et al., 2002b (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB5)).<dl class="figure" id="FIG1" data-t="f" style="border: 1px solid rgb(215, 215, 215); margin-right: 0px; margin-bottom: 15px; margin-left: 0px; padding: 6px; vertical-align: baseline; border-top-left-radius: 3px; border-top-right-radius: 3px; border-bottom-right-radius: 3px; border-bottom-left-radius: 3px; "><dt class="autoScroll" data-style="height:996px;width:629px;" style="border: 0px; margin: 12px 0px 0px; padding: 0px; vertical-align: baseline; overflow-y: hidden; overflow-x: auto; ">http://ars.els-cdn.com/content/image/1-s2.0-S0012160604001186-gr1.jpg (http://www.sciencedirect.com/science/article/pii/S0012160604001186#gr1)</dt><dd id="labelCaptionFIG1" style="border: 0px; font-size: 0.8em; margin: 0px; padding: 0px; vertical-align: baseline; color: rgb(92, 92, 92); ">Fig. 1. Follistatin and Myostatin are expressed in or close to developing muscle. Comparison of Follistatin, Myostatin, Pax-3 andMyoD expression during wing and interlimb somite development. (A–D) HH-stage 26 wing buds, dorsal view. (E–H) Transverse sections of wing buds. (I–L) HH-stage 21 interlimb somites, lateral view and (M–P) corresponding frontal sections. (Q–T) Transverse sections of HH-stage 21 interlimb somites. (A) Strong proximal Follistatin expression, which fades distally in stage 26 wing bud. Section level indicated with broken green line. (B) Myostatin expression in a central domain of a stage 26 wing bud in contrast to the broader extension of the Pax-3-expressing (C) and MyoD-expressing (D) dorsal premuscle mass. (E) Transverse section of (A) shows Follistatin expression in a distinct location of the dorsal premuscle mass (arrow) and in the subectodermal mesenchyme (arrowhead). (F) Transverse section of (B) showsMyostatin expression in a distinct location of the dorsal premuscle mass (arrow) and in the mesenchymal core (arrowhead). (G and H) Follistatin and Myostatin expression in dorsal and ventral zeugopod muscles (arrows) at HH-stage 30. Follistatinexpression partly overlaps with Myostatin expression in distinct muscle subdomains (compare G and H, arrows). (I) StrongFollistatin expression at cranial and caudal somite edges and in a hypaxial domain in contrast to the high Myostatinexpression in the somite centre (J). Somite demarked by dotted line. (K) High Pax-3 expression in the hypaxial domain, moderate expression in the cranial and caudal domain of the dermomyotome (see also O) and no expression dorsomedially (see also S). (L) MyoD highlights the full extent of the myotome. (M) Frontal section shows high Follistatin expression in the cranial and caudal somite edges (arrowheads) and weak expression in the myotome. Brackets mark extent of somites. (N)Myostatin expression in the dermomyotomal and myotomal centre, but not at the cranial and caudal dermomyotomal edges nor in the dorsomedial and ventrolateral edges (R). (O) High Pax-3 expression in the cranial and caudal part of the dermomyotome (arrowheads). Intermediate expression in the dermomyotomal centre (arrow) and in the dorsal root ganglia (asterisks). (P) MyoD expression marks the cranio-caudal extent of the myotome. (Q) Transverse section reveals highFollistatin expression in the hypaxial domain of the somite with weak expression in the myotome. (R) Myostatin expression in the central part of the dermomyotome and myotome. (S) Highest level of Pax-3 expression in the hypaxial, moderate in the intermediate and no expression in the dorsomedial domain of the dermomyotome. Asterisk marks dorsal root ganglion. (T)MyoD expression marks the full extent of the myotome.
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Although Follistatin is expressed in interlimb somites as soon as they are formed, Myostatin is not up-regulated before HH-stage 19 (Amthor et al., 2002b) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB5). In stage 21 interlimb somites, Follistatin is strongly expressed at the cranial and caudal edges of the dermomyotome and in the hypaxial somite domain, whereas there is only a faint expression in the somite centre (Figs. 1I, M, Q) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG1). The expression of Myostatin is almost complementary to that of Follistatin as it is expressed in a central domain of the dermomyotome, but not at the somite edges or in the hypaxial domain (Figs. 1J, N, R) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG1). Highest expression level of Follistatincoincides with a high expression level of Pax-3 (Figs. 1K, O, S) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG1) but not of MyoD (Figs. 1L, P, T) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG1). At later stages, both Follistatin and Myostatin are predominantly expressed in hypaxial muscle (data not shown, see also Amthor et al., 2002b (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB5)).These data show that Follistatin and Myostatin are expressed in or close to developing muscle. They are expressed partly in overlapping domains and partly in abutting regions to each other. As both Myostatin and Follistatin are secreted signalling proteins, they appear to be expressed sufficiently close to enable protein–protein interaction.

Presser
10-31-2014, 06:49 AM
Two-hybrid investigations of Follistatin–Myostatin interactionOverexpression of Follistatin in mouse skeletal muscle results in a double-muscle phenotype that is similar to Myostatin knockout mice (Lee and McPherron, 2001) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB21). Follistatin has also been shown to inhibit Myostatin activity in a transcription-based reporter assay (Zimmers et al., 2002) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB38). Furthermore, systemic administration of Follistatin was shown to interfere with the activity of Myostatin produced at distant sites in vivo (Zimmers et al., 2002) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB38). We wanted to determine if Follistatin directly binds to Myostatin, rather than acting indirectly, and to identify the putative binding site of Follistatin. We used the yeast two-hybrid system to test for interaction between mature Myostatin and Follistatin-288 and then confirmed interactions using a mammalian two-hybrid system. Follistatin-288, was expressed as a target fusion protein to the B42 DNA activation domain (B42-FS-288). The fusion protein was tested for interaction in the yeast two-hybrid system with the mature portion of Myostatin expressed as a bait fusion protein to the LexA DNA binding domain (LexA-MSTNmat). Transactivation of the lacZ reporter occurred in yeast co-transformed with LexA-MSTNmat and B42-FS-288 only in the presence of galactose but not on medium containing glucose, consistent with the galactose dependency of the GAL1 promoter used to drive expression of the B42-FS-288 fusion protein (Fig. 2) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG2). Similarly, transactivation of LEU2 in yeast co-transformed with LexA-mature-Myostatin and B42-FS-288 expression plasmids grown on medium lacking leucine occurred only in the presence of galactose.<dl class="figure" id="FIG2" data-t="f" style="margin-right: 0px; margin-bottom: 15px; margin-left: 0px; padding: 6px; border: 1px solid rgb(215, 215, 215); vertical-align: baseline; border-top-left-radius: 3px; border-top-right-radius: 3px; border-bottom-right-radius: 3px; border-bottom-left-radius: 3px; "><dt class="autoScroll" data-style="height:319px;width:659px;" style="border: 0px; margin: 12px 0px 0px; padding: 0px; vertical-align: baseline; overflow-y: hidden; overflow-x: auto; ">http://ars.els-cdn.com/content/image/1-s2.0-S0012160604001186-gr2.jpg (http://www.sciencedirect.com/science/article/pii/S0012160604001186#gr2)</dt><dd id="labelCaptionFIG2" style="border: 0px; font-size: 0.8em; margin: 0px; padding: 0px; vertical-align: baseline; color: rgb(92, 92, 92); ">Fig. 2. Mature Myostatin interacts specifically with Follistatin-288. Lex A-Myostatin and B42-Follistatin fusion proteins were expressed in yeast, and yeast two-hybrid assays were performed with lacZ reporter gene and the LEU2 selection marker. Only expression of both mature Myostatin and full-length Follistatin (FS-288) transactivated the lacZ locus as visualised by X-gal staining. B42 was under the control of a galactose-dependent promotor and only medium containing galactose, but not glucose, resulted in transactivation of lacZ in the presence of mature Myostatin and Follistatin-288, which shows that Lex A-MSTNmat was not able to autoactivate lacZ. Likewise transactivation of LEU2, which enabled growth of yeast cells in absence of leucine, was only achieved in the presence of mature Myostatin and Follistatin-288. Again, Lex A was not able to autoactivate lacZ as cells grew only in presence of galactose, but not glucose. We also tested the ability of mature Myostatin to interact with various Follistatin domains (encoded by amino acids 1–63, 64–288, 1–100, 1–136, 1–150, 1–200 and 1–250) as well as the control vector pEG202-Max to interact with Follistatin-288, but all gave negative results.
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To determine which regions of Follistatin may be important for physical and functional interactions with mature Myostatin, we generated a series of Follistatin truncations as fusion proteins to the B42 activation domain. Initially, Follistatin-288 was divided into the N-terminal domain (aa 1–63) and the region composed of the three “Follistatin domains” (aa 64–288). Distinct and non-overlapping protein binding sites of Follistatin have previously been reported in these regions. A heparin sulphate-binding region is located within the first Follistatin domain: aa 75–86 Inouye et al., 1992 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB16) and Sumitomo et al., 1995 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB33). Two discontinuous sequences capable of binding Activin have also been identified within the 63-residue N-terminal domain: aa 3–26 and aa 46–59 (Wang et al., 2000) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB37). Neither of these domains interacted with mature Myostatin in the yeast two-hybrid system. Further Follistatin truncations, Follistatin 1–86, 1–100, 1–136, 1–150, 1–200 and 1–250, also failed to interact with mature Myostatin in the yeast two-hybrid system (Fig. 2) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG2).These results show that the N-terminal domain and the three Follistatin modules on their own are not sufficient to interact with Myostatin and suggest that these domains together constitute essential Myostatin-binding determinants. The interaction may either be mediated through several epitopes of the tertiary structure of Follistatin or deletion of any part of Follistatin may profoundly alter the conformation and thus prevent binding.After establishing a specific interaction between Follistatin-288 and mature Myostatin, we wanted to corroborate the result in mammalian cells. Mammalian proteins are more likely to retain their native confirmation in a mammalian cell. Bait and prey fusion constructs consisting of mature Myostatin and Follistatin-288, respectively, were co-transfected with the lacZ reporter plasmid into CHO cells. Identical results were obtained in the mammalian two-hybrid system as in the yeast two-hybrid system. Mature Myostatin and Follistatin-288 specifically interacted in CHO cells to induce expression of the reporter genelacZ as observed by in situ staining for β-galactosidase (Fig. 3) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG3). Mature Myostatin and Follistatin-288 did not, however, interact with control plasmids, verifying the specificity of the interaction.<dl class="figure" id="FIG3" data-t="f" style="margin-right: 0px; margin-bottom: 15px; margin-left: 0px; padding: 6px; border: 1px solid rgb(215, 215, 215); vertical-align: baseline; border-top-left-radius: 3px; border-top-right-radius: 3px; border-bottom-right-radius: 3px; border-bottom-left-radius: 3px; "><dt class="autoScroll" data-style="height:162px;width:667px;" style="border: 0px; margin: 12px 0px 0px; padding: 0px; vertical-align: baseline; overflow-y: hidden; overflow-x: auto; ">http://ars.els-cdn.com/content/image/1-s2.0-S0012160604001186-gr3.jpg (http://www.sciencedirect.com/science/article/pii/S0012160604001186#gr3)</dt><dd id="labelCaptionFIG3" style="border: 0px; font-size: 0.8em; margin: 0px; padding: 0px; vertical-align: baseline; color: rgb(92, 92, 92); ">Fig. 3. Mature Myostatin and Follistatin-288 interact in mammalian cells. The positive interaction between mature Myostatin and Follistatin-288 was confirmed in the mammalian two-hybrid system. Mature Myostatin as a fusion to GAL4 DNA binding domain and Follistatin-288 as a fusion to the VP16 activation domain together with a reporter plasmid containing the lacZgene were co-transfected into CHO cells, which were then assayed for β-galactosidase activity. CHO cells were fixed and stained in situ with X-gal. (A) Positive X-gal staining was seen in cells transfected with GAL4-MSTNmat, VP16-FS-288 and pGAL/lacZ, which indicates an interaction between Myostatin and Follistatin, but not in cells lacking either (B) Follistatin: GAL4-MSTNmat, pCR2.1/LgT prey control and pGAL/lacZ or (C) Myostatin: VP16-FS-288, pCR2.1p53 bait control and pGAL/lacZ. Magnification: 200×.
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Follistatin–Myostatin interaction kineticsOur studies using yeast and mammalian two-hybrid systems clearly show that Follistatin is able to interact with Myostatin. To determine the kinetics of this interaction, we employed a Surface Plasmon Resonance Biosensor (Biacore), which allows the affinity between two associating molecules to be determined. In a first step, Follistatin was irreversibly bound onto the surface of a dextran-coated sensory chip and subsequently exposed to differing concentrations of Myostatin. The interaction analysis consisted of two parts. In the first part of the measurement, Follistatin was exposed to its ligand Myostatin, which allowed protein complexes to be formed. In the second part, Myostatin was no longer present and the Follistatin coated sensor chip surface was washed to allow the dissociation of the protein complexes. The Follistatin–Myostatin interaction was measured in response units from which association and dissociation constants were calculated using the Biacore evaluation software.Follistatin–Myostatin interactions were studied at 6.25, 12.5, 25, 50 and 100 nM, and a reproducible series of concentration-dependent interaction curves were generated (Fig. 4) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG4). The gentle increase in the curves during the binding phase suggested a low association rate. More importantly, however, during the dissociation phase, the curves did not descend appreciably, which suggests that the Follistatin–Myostatin complex was very stable. The experimental data were fitted to the Langmuir (1:1) model of interaction, which resulted in an average association constant of (ka) 1.63 × 105 M−1 s−1 and a dissociation constant of (kd) 9.52 × 10−5 s−1. From these values, the affinity (kD) of Follistatin for Myostatin was calculated to be 5.84 × 10−10 M.<dl class="figure" id="FIG4" data-t="f" style="margin-right: 0px; margin-bottom: 15px; margin-left: 0px; padding: 6px; border: 1px solid rgb(215, 215, 215); vertical-align: baseline; border-top-left-radius: 3px; border-top-right-radius: 3px; border-bottom-right-radius: 3px; border-bottom-left-radius: 3px; "><dt class="autoScroll" data-style="height:147px;width:319px;" style="border: 0px; margin: 12px 0px 0px; padding: 0px; vertical-align: baseline; overflow-y: hidden; overflow-x: auto; ">http://ars.els-cdn.com/content/image/1-s2.0-S0012160604001186-gr4.gif (http://www.sciencedirect.com/science/article/pii/S0012160604001186#gr4)</dt><dd id="labelCaptionFIG4" style="border: 0px; font-size: 0.8em; margin: 0px; padding: 0px; vertical-align: baseline; color: rgb(92, 92, 92); ">Fig. 4. Surface plasmon resonance investigations of the Follistatin–Myostatin interaction. Association of Follistatin–Myostatin complexes was measured by increasing resonance units (RU) as the Follistatin coupled sensor chip was exposed to different concentrations of Myostatin for approximately 175 s. The level of dissociation of Follistatin is shown by the decrease in RU units for the different Myostatin concentrations after the exposure of the Follistatin-coupled sensor chip to Hepes-buffered saline for approximately 480 s.
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These results provide an independent additional means to show that Follistatin directly interacts with Myostatin. The kinetics of the interaction demonstrates that once Myostatin had bound Follistatin it did not readily dissociate from this coupling.

Follistatin prevents the Myostatin-mediated inhibition of embryonic limb muscle developmentWe recently reported that Myostatin retarded growth of limb muscle when applied to the developing wing bud of chick embryos (Amthor et al., 2002b) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB5). We showed that the inhibiting effect of Myostatin on muscle development was mediated by the down-regulation of Pax-3 and MyoD expression, and we found that the gene expression is altered within 6 h after exposure to Myostatin. Here, we tested whether the presence of Follistatin can prevent the effect of Myostatin on Pax-3 and MyoD expression. We used Affigel Blue beads and Heparin beads as protein carriers for Myostatin and Follistatin, respectively, and used the same proteins as for the Biacore kinetic evaluation. In previous studies, we found that experiments were most effective and reproducible when multiple beads were implanted (Amthor et al., 2002a) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB4). We, therefore, applied 8 to 11 Myostatin beads and additionally 8 to 11 Follistatin beads to HH-stages 22–24 wing buds, and in control experiments, the same number of beads soaked in PBS. Previously, we found that control beads never altered any gene expression, tissue architecture nor resulted in induced cell death (Amthor et al., 1998) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB2). Nevertheless, we performed a control experiment and tested the effect of Affigel Blue beads and Heparin beads, which had been soaked in PBS only, on the expression of Pax-3 and MyoD. At least eight Affigel Blue beads and at least eight Heparin beads were microsurgically placed into the dorsal subectodermal mesenchyme of HH-stages 22–24 wing buds and embryos were re-incubated for a further 6 to 8 h. Affigel Blue beads (blue colour) and Heparin beads (white colour) were alternately arranged (Fig. 5E) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG5). In each case, all beads were still well positioned and evenly spaced at the time of fixation. Wing buds neither changed in size nor in shape despite the high number of control beads. Furthermore, we found no alteration in the intensity or in the extent of Pax-3 or MyoD expression in the dorsal and ventral premuscle masses compared to unoperated wings (Pax-3, n = 11; MyoD, n = 7) (Figs. 5A–I) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG5). After transverse sectioning of such wing buds, we found that the control beads did not alter the tissue architecture or the expression of Pax-3 andMyoD when compared to unoperated wings.<dl class="figure" id="FIG5" data-t="f" style="margin-right: 0px; margin-bottom: 15px; margin-left: 0px; padding: 6px; border: 1px solid rgb(215, 215, 215); vertical-align: baseline; border-top-left-radius: 3px; border-top-right-radius: 3px; border-bottom-right-radius: 3px; border-bottom-left-radius: 3px; "><dt class="autoScroll" data-style="height:501px;width:667px;" style="border: 0px; margin: 12px 0px 0px; padding: 0px; vertical-align: baseline; overflow-y: hidden; overflow-x: auto; ">http://www.sciencedirect.com/sd/grey_pxl.gif (http://www.sciencedirect.com/science/article/pii/S0012160604001186#gr5)</dt><dd id="labelCaptionFIG5" style="border: 0px; font-size: 0.8em; margin: 0px; padding: 0px; vertical-align: baseline; color: rgb(92, 92, 92); ">Fig. 5. Follistatin antagonises Myostatin to block myogenesis during limb development. Dorsal view on HH-stage 24/25 chick wing buds and corresponding transverse section at mid-limb level after Follistatin and Myostatin bead implantation and a 6-h re-incubation period. Columns E, J, O, T show arrangement of beads in wing buds. Columns A, F, K, P, U show MyoDexpression in wing whole-mounts. Columns B, G, L, Q, V show transverse section of MyoD whole-mounts. Columns C, H, M, R, W show Pax-3 expression in wing whole-mounts. Columns D, I, N, S, X show transverse section of Pax-3 whole-mounts. Asterisks show beads. (A, B) Normal MyoD expression in dorsal and ventral premuscle masses in a non-manipulated wing bud. (C, D) Normal Pax-3 expression in dorsal and ventral premuscle masses in a non-manipulated wing bud. (E) Bead arrangement for control experiment (F–I). (F, G) Normal MyoD expression in dorsal and ventral premuscle masses after control bead implantation. (H, I) Normal Pax-3 expression in dorsal and ventral premuscle masses after control bead implantation. (J) Arrangement for Follistatin bead implantation (K–N). (K, L) Slight decrease in MyoD expression in the dorsal premuscle mass after Follistatin bead application is not visible in whole-mount, but after sectioning. (M, N) Normal expression of Pax-3 in dorsal and ventral premuscle masses after Follistatin bead implantation. (O) Arrangement for Myostatin bead implantation (P–S). (P, Q) Almost total loss of MyoD expression in the dorsal and moderate down-regulation in the ventral premuscle mass after Myostatin bead implantation. (R, S) Almost total loss of Pax-3 expression in the dorsal premuscle mass after Myostatin bead implantation. (T) Arrangement for concurrent Follistatin and Myostatin bead implantation (U–X). (U, V) Almost complete rescue of MyoD expression in dorsal and complete rescue in ventral premuscle masses after concurrent Follistatin and Myostatin bead implantation. (W, X) Complete rescue of Pax-3 expression in dorsal and ventral premuscle masses after concurrent Follistatin and Myostatin bead implantation.
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We previously found that Follistatin moderately increased the expression of Pax-3 and decreased the expression of MyoD (Amthor et al., 2002a) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB4). However, we had not examined the short-term effect of Follistatin as early as 6 h after protein application. Here, we placed 8–11 Heparin beads soaked in Follistatin and additionally 8–11 Affigel Blue beads soaked in PBS (which in subsequent experiments have been replaced with Myostatin beads) in the developing wing buds (Fig. 5J) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG5). Six to 7 h after such operation, we found no obvious alteration of Pax-3 expression compared to control bead implantation (n = 7) ( Figs. 5M (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG5)–N and compare to Figs. 5H (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG5)–I). When the effect of Follistatin on MyoD expression was examined, we observed in approximately 50% of cases a faint decrease in MyoD expression compared to the effect of control beads (n= 9) ( Figs. 5K (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG5)–L and compare to Figs. 5F (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG5)–G).To test for the effect of Myostatin on the expression of Pax-3 and MyoD, we placed 8–11 Affigel Blue beads soaked in Myostatin and additionally 8–11 Heparin beads soaked in PBS (which in the subsequent experiments have been replaced with Follistatin beads) in the developing wing buds (Fig. 5O) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG5). Six to 8 h after Myostatin exposure, we found a drastic down-regulation of Pax-3 as well as MyoD expression in the dorsal and to a lesser extent in the ventral premuscle masses compared to control experiments (Pax-3, n = 12;MyoD, n = 8). We occasionally found operated wings with residual Pax-3 and MyoD expression, which was in the centre of the dorsal side of the wing buds, whereas others lacked Pax-3 and MyoD expression completely (Figs. 5P–S) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG5).Finally, we tested whether Follistatin, when simultaneously applied with Myostatin, can inhibit the effect of Myostatin. We placed 8–11 Affigel Blue beads soaked in Myostatin and immediately afterwards an additional 8–11 Heparin beads soaked in Follistatin in the developing wing buds (Fig. 5T) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG5). In all cases, the Pax-3expression in the dorsal premuscle masses was rescued in three out of eight cases completely. Pax-3expression in the ventral premuscle masses was completely rescued in all cases (n = 8) (Figs. 5W–X) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG5). In cases of incomplete rescue, Pax-3 expression was observed immediately adjacent to or between the Follistatin beads, whereas the expression was not found near the Myostatin beads. Examination of MyoDexpression after combined Myostatin/Follistatin treatment revealed in two out of eight cases a nearly complete rescue of MyoD expression in the dorsal premuscle masses (Figs. 5U–V) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG5). Ventral premuscle masses showed a complete rescue of MyoD expression in six of eight cases. In cases of incomplete rescue,MyoD expression was near the Follistatin beads, but not near the Myostatin beads.These data show that Follistatin prevents the ability of Myostatin to down-regulate key transcription factors of muscle development. The antagonising effect of Follistatin appears to be concentration-dependent because the rescue of Pax-3 and MyoD expression was highest next to the source of Follistatin and lowest next to the source of Myostatin.Chick limb bud cultures confirm the inhibitory effect of Follistatin on MyostatinTo determine whether Follistatin can block the inhibitory effect of Myostatin on terminally differentiated muscle, we tested the effect of both proteins on micromass cultures of dissociated chick limb bud mesenchyme cells. In these cultures, limb bud cells differentiate into muscle and cartilage when plated at high density (Swalla and Solursh, 1986) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB34).Control micromass cultures (non-Myostatin treated) form terminally differentiated muscle cells, which are immunoreactive against Myosin Heavy Chain (MHC), after a 24-h culture period (Fig. 6A (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG6), n = 10). Supplementing the micromass cultures with 125 ng/ml Myostatin resulted in an almost complete lack of MHC-positive cells in all cases ( Fig. 6B (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG6), n = 10). Concurrent exposure of micromass cultures to 125 ng/ml Myostatin and Follistatin at concentrations of 156, 312 and 625 ng/ml rescued the formation of MHC-positive cells (for each concentration, n = 4, Figs. 6C, D, E (http://www.sciencedirect.com/science/article/pii/S0012160604001186#FIG6)). The concurrent addition of Follistatin with Myostatin, however, rescued this effect in a concentration-dependent manner, as increased concentrations of Follistatin led to an increased formation of MHC-positive cells in 100% of cases. Supplementing with Follistatin alone at concentrations of 156, 312 and 625 ng/ml moderately decreased the formation of MHC-positive cells (data not shown; for each concentration, n = 4).<dl class="figure" id="FIG6" data-t="f" style="margin-right: 0px; margin-bottom: 15px; margin-left: 0px; padding: 6px; border: 1px solid rgb(215, 215, 215); vertical-align: baseline; border-top-left-radius: 3px; border-top-right-radius: 3px; border-bottom-right-radius: 3px; border-bottom-left-radius: 3px; "><dt class="autoScroll" data-style="height:429px;width:170px;" style="border: 0px; margin: 12px 0px 0px; padding: 0px; vertical-align: baseline; overflow-y: hidden; overflow-x: auto; ">http://www.sciencedirect.com/sd/grey_pxl.gif (http://www.sciencedirect.com/science/article/pii/S0012160604001186#gr6)</dt><dd id="labelCaptionFIG6" style="border: 0px; font-size: 0.8em; margin: 0px; padding: 0px; vertical-align: baseline; color: rgb(92, 92, 92); ">Fig. 6. Follistatin antagonises the ability of Myostatin to block terminal differentiation of muscle in micromass cultures. Differentiated muscle is shown by expression of Myosin heavy chain (MHC) after immunohistochemistry. Chick limb bud mesenchyme was cultured as micromasses. Cultures were untreated (A), supplemented with 125 ng/ml Myostatin (B), 125 ng/ml Myostatin plus 156 ng/ml Follistatin (C), 125 ng/ml Myostatin plus 312 ng/ml Follistatin (D) or 125 ng/ml Myostatin plus 625 ng/ml Follistatin (E). Myostatin alone significantly reduced the number of MHC-positive cells compared to controls (B compare with A). This effect was reversed by concurrent addition of Follistatin, in a concentration-dependent manner (C–E).
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These data show that Follistatin blocked the inhibitory effect of Myostatin on the formation of differentiated muscle in a concentration-dependent manner.

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10-31-2014, 06:50 AM
DiscussionMyostatin is the most powerful inhibitor of muscle growth identified to date Kambadur et al., 1997 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB19),McPherron and Lee, 1997 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB27) and McPherron et al., 1997 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB28). Our data suggest that Follistatin antagonises the ability of Myostatin to inhibit muscle development. This suggestion is based on following observations: we have shown that Follistatin and Myostatin are expressed in or very close to developing muscle. They are expressed in overlapping or in very closely located domains, which suggests that they can interact as they both are secreted signalling molecules. Our in vitro experiments show that the proteins can interact directly with high affinity. We finally have shown that Follistatin prevents the inhibiting effect of Myostatin on muscle development both in vivo and in vitro.

The hypertrophy and hyperplasia of muscle, found in the absence of Myostatin, clearly demonstrates that excessive muscle growth can be a default pathway Kambadur et al., 1997 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB19), McPherron and Lee, 1997 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB27) and McPherron et al., 1997 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB28). We suggest that the implementation of Myostatin during evolution created a simple mechanism to regulate muscle growth whenever required by simply up- or down-regulatingMyostatin gene expression or by blocking the Myostatin protein. The developmental need to accurately tune the effect of Myostatin is nicely demonstrated during somite development. From HH-stage 20 onwards, the expression of Myostatin in the centre of the dermomyotome and myotome is framed by the expression ofFollistatin in the lips of the dermomyotome. The highest expression of Pax-3 and, significantly, the generation of muscle cells are found in the dermomyotomal lips but not in the dermomyotomal centre Kahane et al., 1998 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB17) and Kahane et al., 2001 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB18). This distribution of Myostatin and Follistatin expression therefore follows their predicted functions as observed in the knockout animals: the Myostatin−/− mouse results in excessive muscle growth, whereas the Follistatin−/− mouse displays muscle hypotrophy Matzuk et al., 1995 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB25) and McPherron et al., 1997 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB28). Myostatin executes its effect via down-regulating the expression of key transcription factors that control muscle development, which inhibited further muscle growth (Amthor et al., 2002b) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB5). We demonstrated the rapidity of Myostatin to inhibit the expression of MyoD and Pax-3 within a matter of a few hours. We show here that Follistatin can almost completely prevent the effect of Myostatin onPax-3 and MyoD expression. Furthermore, we also show that the ability of Myostatin to inhibit terminal muscle differentiation is blocked by Follistatin in a concentration-dependent manner.One question, which needed to be answered, was whether Follistatin acts directly or indirectly to antagonise Myostatin. Firstly, we established that Follistatin interacts with Myostatin directly by using three different methods: the yeast two-hybrid system, the mammalian two-hybrid system and the surface plasmon resonance biosensor. It may be significant that interactions between Myostatin and Follistatin were detected using both yeast and mammalian two-hybrid systems. This suggests that the post-translational modifications found solely in higher organism are not required in order for these two proteins to interact. Next, we attempted to establish which of the domains of Follistatin were essential or sufficient for binding to Myostatin. Follistatin consists of four domains, which have been previously shown to harbour specific protein binding sites, such as for Heparin and Activin Inouye et al., 1992 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB16), Sidis et al., 2001 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB32), Sumitomo et al., 1995 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB33) and Wang et al., 2000 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB37). We generated different Follistatin truncations, but none of the tested Follistatin domains was able to bind Myostatin on their own. This suggests that all Follistatin domains together are important for effective Myostatin binding. However, our experiments did not determine whether internal deletions of the Follistatin protein could maintain its ability to bind Myostatin. We analysed the kinetics of the binding and calculated an affinity of Follistatin to bind Myostatin of 5.84 × 10−10 M. The affinity of Activin to bind the Activin Type II receptor was estimated to range between 2 and 7 × 10−9 M, which demonstrates that Follistatin is likely to compete with the binding of Myostatin to its receptor (Donaldson et al., 1999) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB8). Although Follistatin associates with Myostatin at a low rate compared to the association constant of the Follistatin-BMP interaction (1.63 × 105 M−1 s−1 for Follistatin–Myostatin compared to 2.06 × 106 M−1 s−1 for Follistatin-BMP-7, Amthor et al., 2002a (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB4) and Iemura et al., 1998 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB14), the dissociation constant suggests that once the Follistatin–Myostatin complex has formed, they do not readily dissociate. This results in the strong affinity of Follistatin for Myostatin. From a pharmacological perspective, these data predict that the efficiency of Follistatin to antagonise Myostatin could still be drastically enhanced if its structure would be altered to enable a more rapid association.Follistatin is expressed as soon as somites are formed, whereas Myostatin expression in trunk somites is not found before HH-stage 20 Amthor et al., 1996 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB1) and Amthor et al., 2002b (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB5). This clearly indicates additional functions of Follistatin during muscle development apart from modulating Myostatin activity. In fact, overexpression of Follistatin results in an excessive muscle growth, which by far exceeds the gain of muscle seen in the Myostatin knockout (Lee and McPherron, 2001) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB21). This strengthens the view that Follistatin additionally antagonises the inhibitory effects of other TGF-βs such as BMP-2, -4, -7 and Activin on muscle development Amthor et al., 2002a (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB4) and Link and Nishi, 1997 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB22). In our micromass experiments, we show that the Follistatin blockade of Myostatin rescues the differentiation of muscle. However, Follistatin on its own inhibits muscle differentiation initially (first 24 h of micromass culture), but eventually, the number of muscle cells is increased (unpublished observations). This is in line with the observation that Follistatin increases proliferation and delays differentiation of muscle precursors, dependent upon the presence of BMPs (Amthor et al., 2002a) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB4). Thus, Follistatin supports muscle growth via different signalling mechanisms, which largely depend on which TGF-β is present. It is noteworthy that the concentration of Myostatin capable of halting muscle development in the micromass cultures was considerably lower than that used to soak the beads for implantation studies. In fact, concentrations as low as 10 ng/ml Myostatin protein inhibited myogenesis in vitro whereas beads soaked in less than 1 mg/ml of the same protein will have little effect if at all in vivo (Amthor et al., 2002b (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB5); Amthor, unpublished). However, the concentration of Myostatin protein in the soak solution is not a true reflection of the concentration of protein delivered to the limb bud. Therefore, no conclusions can be drawn regarding the levels required in vivo to mediate the effect.The capacity of Follistatin to completely antagonise the effect of Myostatin is in stark contrast to its interaction with BMP-7 (Amthor et al., 2002a) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB4). BMPs act dose-dependently on muscle with high concentrations inducing muscle loss and low concentrations promoting muscle growth (Amthor et al., 1998) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB2). Follistatin converts the muscle growth-inhibiting effect of BMP-7 into a strong stimulant of muscle growth, which suggests that the role of Follistatin in this case is to alter the absolute levels of BMP-7 available to bind to its receptor (Amthor et al., 2002a) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB4). In the case of Myostatin, Follistatin seems to completely prevent receptor activation because Myostatin, whilst bound to Follistatin, is unable to induce the phosphorylation of Smad3 (G. Nicholas, unpublished). Such differences in biological activity can be well explained by biochemical interaction data, as the dissociation rate of the Follistatin–BMP-7 interaction is much higher than that of the Follistatin–Myostatin interaction (1.66 × 10−3 s−1 for Follistatin–BMP-7 compared to 9.52 × 10−5 s−1 for Follistatin–Myostatin) (Amthor et al., 2002a) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB4).Although Follistatin and Myostatin can be detected in serum, they do not form complexes. Instead, most Myostatin is retained in a latent form by binding to its propeptide, to GASP and to the protein of the Follistatin related gene (FLRG) Hill et al., 2002 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB12) and Hill et al., 2003 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB13). Gene deletion has demonstrated that Follistatin undoubtedly antagonises molecules that inhibit muscle development (Matzuk et al., 1995) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB25). Furthermore, one of the major sites of Follistatin expression during embryogenesis is in tissues, which develop to muscle(Amthor et al., 1996) (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB1). We suggest that Follistatin acts locally at site of production to antagonise molecules like Myostatin. In fact, Follistatin but not FLRG harbours heparin-binding epitopes, which facilitates interactions with the extracellular matrix and therefore is likely to restrict diffusion Inouye et al., 1992 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB16), Sidis et al., 2001 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB32) and Sumitomo et al., 1995 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB33).Recently, it was demonstrated that blockade of Myostatin leads to a functional and histological improvement of dystrophic muscle Bogdanovich et al., 2002 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB6) and Wagner et al., 2002 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB36). Although our study focused on the antagonistic effect of Follistatin on Myostatin during muscle development, there is ample evidence that the activity of Follistatin is not stage restricted Lee and McPherron, 2001 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB21) and Zimmers et al., 2002 (http://www.sciencedirect.com/science/article/pii/S0012160604001186#BIB38). In fact, we have observed high levels of Follistatin and Myostatin expression in dystrophic muscle of the Dystrophin-deficient mouse (mdx mouse) (Amthor, unpublished). Additionally, the capacity of Follistatin not only to block Myostatin but to modulate other inhibitors of muscle development, such as BMPs and Activin, emphasises that Follistatin could be a very potent molecule to combat muscle loss during dystrophies, muscle ageing, disuse or denervation.AcknowledgementsThis work was supported by a grant from the Deutsche Forschungsgemeinschaft (Am 151/2-1) to H. A.; by a grant from the Wellcome Trust (061425) to I.M. We are also thankful to FRST (New Zealand) and Marsden for funding support.

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