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

Binding affinities (<i>K</i>_d) of Ang II, olmesartan, R239470 and R794847 to AT₁ wild-type (WT) and mutant receptors.

<p>Binding affinities (<i>K</i>_d) of Ang II, olmesartan, R239470 and R794847 to AT₁ wild-type (WT) and mutant receptors.</p

Binding affinities (<i>K</i>_d) of Ang II and R compounds to AT₁ wild-type (WT) and mutant receptors.

<p>Binding affinities (<i>K</i>_d) of Ang II and R compounds to AT₁ wild-type (WT) and mutant receptors.</p

Putative binding mode of various ligands in AT₁ receptor.

<p>Transmenbrane (TM)1–7 are shown as colored ribbons: blue (TM1), cyan (TM2), green (TM3), lime green (TM4), yellow (TM5), orange (TM6) and red (TM7). Sidechains of the focused residues are shown as gray lines, and ligands are represented as magenta sticks. The hydrogen bonds and electrostatic interactions are indicated by yellow dotted lines. The distance between Leu¹¹² and the hydroxyphenyl moiety of R794847 is represented as a green dotted line. The unit of distance was Å. (a) without ligand, (b) olmesartan, (c) R239470, (d) R794847.</p

Binding affinities (<i>K</i>_d) of Ang II, olmesartan, R239470 and R794847 to AT₁ wild-type (WT) and mutant receptors.

<p>Binding affinities (<i>K</i>_d) of Ang II, olmesartan, R239470 and R794847 to AT₁ wild-type (WT) and mutant receptors.</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

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