The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy

<div><p>Myostatin, a muscle-specific transforming growth factor-β (TGF-β), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex. However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain. This inhibitory core resides near the N-terminus of the prodomain and includes an α-helix that is evolutionarily conserved among other TGF-β family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors. Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin. A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-β1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency. Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand. This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings.</p></div

p29 restores the reduced myotube formation resulting from LGMD1C-causing mutant caveolin 3 (CAV3^P104L).

<p>(<b>A</b>) Wright-Giemsa-stained C2C12 cells expressing LGMD1C-causing Pro104Leu mutant caveolin 3 (CAV3^P104L) at 7 days after differentiation with (+) or without (–)1 μM p29 (<b>left</b>). Scale bar, 100 μm. Fusion indices of these cells following addition of 1 μM of p29 were calculated in triplicate as the percentage of the total nuclei in myotubes/mm² (<b>right</b>). Values are the means ± SD (<i>n</i> = 5). *<i>P</i> < 0.05. (<b>B</b>) (<b>C</b>) Phase-contrast (<b>left</b>) and fluorescence (<b>right</b>) images of MyHC in C2C12 myoblasts expressing the empty vector (mock) or Pro104Leu mutant caveolin 3 at 7 days after differentiation with (+) or without (–) 1 μM p29. Scale bar, 100 μm. (<b>C</b>) Immunoblot analysis of MyHC and β-actin in C2C12 cells expressing the empty vector (mock) or Pro104Leu mutant caveolin 3 (CAV3^P104L) at 7 days after differentiation with (+) or without (–) 1 μM p29 (<b>left</b>). Densitometric analysis (<b>right</b>). Values are mean ± SD fold increases compared with untreated C2C12 cells expressing the empty vector (mock) (<i>n</i> = 5). *<i>P</i> < 0.05.</p

The identified inhibitory core of the myostatin prodomain specifically suppresses myostatin and its analog, GDF11, and includes an AH that is evolutionarily conserved among several other TGF-β family members.

<p>(<b>A</b>) The full-length myostatin prodomain (f-Pro) and its inhibitory core (Pro11) inhibited the transcriptional activities of myostatin and GDF11, but not of TGF-β1 or activin A, in HEK293 cells. (<b>B, C</b>) Sequence alignment of the prodomains of myostatin in nine species (<b>B</b>) and nine TGF-β family members (<b>C</b>). Red indicates the identified inhibitory core of the myostatin prodomain, consisting of 29 amino acids. The AH structure (blue) of the TGF-β1 prodomain has been shown to bind to both its ligand and TSP-1* [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133713#pone.0133713.ref006" target="_blank">6</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133713#pone.0133713.ref018" target="_blank">18</a>]. Crystallographic analyses of TGF-β1 and its receptors have predicted that the random coiled structure (RC, green) and the AH are located closely to its type I receptor, whereas the latency lasso structure (LL, brown) is located close to its type II receptor** [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133713#pone.0133713.ref019" target="_blank">19</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133713#pone.0133713.ref020" target="_blank">20</a>].</p

Identification of the inhibitory core of the myostatin prodomain.

<p>(<b>A</b>) Truncation and deletion constructs of human myostatin prodomain:human Fc fusion proteins (<b>left</b>). Percentage inhibitory effect of each construct on myostatin activity in comparison with the full-length prodomain (f-Pro, <b>right</b>). (<b>B</b>) Recombinant myostatin-induced transcriptional activity in HEK293 human embryonic kidney cells co-transfected with a pGL3-(CAGA)₁₂-luciferase reporter gene, pCMV-β-Gal, and various prodomain region:Fc fusion constructs. Values are the mean ± SD (<i>n</i> = 6). RLU, relative luminescence units.</p

Intramuscular injection of p29 rescues muscle atrophy and weakness in caveolin 3-deficient LGMD1C model mice.

<p>(<b>A</b>) Effect of p29 on <i>in vitro</i> myostatin activity in the HEK293-(CAGA)₁₂-luciferase system. Cells were stimulated with 10-ng/ml myostatin and simultaneously exposed to increasing concentrations (2, 20, 200, or 2000 nM) of p29 or albumin (control). All experiments were performed triplicate, repeatedly twice. (<b>B</b>) Appearance of TA muscles at 28 days after local injection of p29 (20 nmol) or albumin (C, control) into the ipsilateral and contralateral TA muscles of wild-type and CAV3^P104L Tg mice. (<b>C</b>) Weights of TA muscles injected with 20 nmol p29 or albumin in wild-type and CAV3^P104L Tg mice (<i>n</i> = 10). <i>*P</i> < 0.05. (<b>D</b>) Weights of caveolin 3-deficient TA muscles injected with different amounts of p29 or albumin (<b>right</b>, <i>n</i> = 10). <i>*P</i> < 0.05. (<b>E</b>) Specific force of the TA muscle in wild-type (<b>left</b>) and CAV3^P104L Tg (<b>right</b>) mice treated with p29 or albumin. <i>*P</i> < 0.05. Values are the means ± SD (<i>n</i> = 10).</p

Local injection of p29 alleviates the reduction in myofiber size by restoration of the decreased numbers of satellite cells.

<p>(<b>A</b>) Histological analysis of TA muscles treated with p29 or albumin (control) in wild-type (<b>upper</b>) and CAV3^P104L (<b>lower</b>) mice. Scale bar, 50 μm (<b>left</b>). Distribution of SMAs in TA muscles of mice treated with p29 or albumin (<b>right</b>; <i>n</i> = 7; 250 myofibers were assessed in each mouse). (<b>B</b>) Immunohistochemical analysis of M-cadherin-positive satellite cells (green, arrows) in TA muscles of wild-type (<b>upper</b>) and CAV3^P104L (<b>lower</b>) mice (<b>left)</b> treated with p29 or albumin. Red indicates laminin α2 and gray indicates nuclei. Numbers of satellite cells per 100 myonuclei in TA muscles (<b>right</b>). One-thousand myonuclei were assessed in each muscle (<i>n</i> = 7). <i>*P</i> < 0.05. (<b>C</b>) Fluorescence images of satellite cells attached to single myofibers isolated from the TA muscles of wild-type (Wild) and CAV3^P104L mice treated with (+) or without (–) p29 (<b>left</b>). Mouse caveolin 1 (CAV1) was used as a marker of satellite cells (green). Nuclei were counterstained with DAPI (blue). The white arrow indicates (satellite cells). Quantification of the number of satellite cells attached to single myofibers (<b>right</b>). Numbers of satellite cells per 100 myonuclei (right). Data are expressed as the mean ± SD (<i>n</i> = 5). *<i>P</i> < 0.05.</p

p29 enhances myogenesis suppressed by myostatin and GDF11, but not activin or TGF-β1.

<p>(<b>A</b>) C2C12 myoblasts were maintained in growth medium. Mononucleated myoblasts differentiate into multinucleated myotubes in differentiation medium with (+) or without (–) 1 μM p29 for 7 days. Phase-contrast and fluorescence images of cells stained for myotube markers MyHC, myogenin, and CK. Scale bar, 100 μm. (<b>B</b>) The protein analysis of MyHC in C2C12 cells expressing in growth or differentiation media with (+) or without (–) 1 μM p29. (<b>C</b>) Wright-Giemsa-stained C2C12 cells expressing an empty vector in growth or differentiation media with (+) or without (–) 1 μM p29 (<b>left</b>). Scale bar, 100 μm. Fusion indices were calculated in triplicate as the percentage of the total nuclei in myotubes/mm² (<b>right</b>). Values are the means ± SD (<i>n</i> = 5). *<i>P</i> < 0.05. (<b>D</b>) Phase-contrast (<b>left)</b> and fluorescence (<b>right</b>) images of MyHC in C2C12 myoblasts expressing the empty vector (mock), myostatin, GDF11, activin A, or TGF-β1 at 7 days after differentiation with (+) or without (–) 1 μM p29. Scale bar, 100 μm. (<b>E</b>) Immunoblot analysis of MyHC protein in C2C12 cells at 7 days after differentiation with (+) or without (–) 1 μM p29 (<b>upper</b>). Densitometric analysis (<b>lower</b>). Values are the mean ± SD fold increases compared with untreated C2C12 cells expressing the empty vector (mock) (<i>n</i> = 5). *<i>P</i> < 0.05.</p

Interaction of the inhibitory core of myostatin with its ligand and receptors.

<p>Co-localization (<b>Upper</b>) and co-immunoprecipitation (<b>Lower</b>) of the inhibitory core (IC) of the myostatin prodomain and its ligand (<b>A</b>), its type I receptors (ALK4 and ALK5, <b>B</b>), and its type II receptors (ActRIIA and ActRIIB, <b>C</b>) in COS-7 embryonic kidney cells expressing FLAG-tagged IC and V5- or HA-tagged ligand or receptors. Scale bar, 20 μm. Whole cell extracts (WCE) were immunoprecipitated with anti-FLAG, anti-V5, or anti-HA agarose and then immunoblotted using anti-FLAG, anti-V5, or anti-HA antibodies, respectively.</p

p29 inhibits activation of intramuscular myostatin signaling in caveolin 3-deficient LGMD1C model mice.

<p>Immunoblot (<i>n</i> = 5). (<b>A</b>) and northern blot (<i>n</i> = 7). (<b>B</b>) analyses of TA muscles treated with or without p29 (<b>upper</b>). Densitometric analyses (<b>lower</b>). Values are mean ± SD fold increases compared with untreated wild-type muscles. <i>*P</i> < 0.05.</p