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

Surface atherosclerotic lesions of the aorta in 21-week-old nondiabetic <i>Apoe</i>^{−<b>/</b>−} mice treated without/with vildagliptin infused with incretin receptor blockers.

<p>During the 4-week period of vildagliptin administration, the <i>Apoe</i>^−/− mice (17-weeks-old at the outset) were respectively infused with saline (vehicle), GLP-1 receptor blocker (Ex-9), the GIPR blocker (Pro³), and Ex-9+Pro³ by osmotic mini-pump. The entire aorta was stained with Oil Red O.</p

The atherosclerotic area and the macrophage infiltration area in the aortic root in 21-week-old nondiabetic <i>Apoe</i>^{−<b>/</b>−} mice treated without/with vildagliptin infused with incretin receptor blockers.

<p>The cross-sections of the aortic root were stained with Oil Red O. The area of macrophage infiltration was stained with MOMA-2.</p

Expressions of GLP-1R in various mouse tissues or cells.

<p>Expressions of GLP-1R in various mouse tissues or cells.</p

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

Expressions of GIPR in various mouse tissues or cells.

<p>Relative gene expressions of GLP-1R and GIP in various mouse tissues or cells determined by real-time PCR using 10 and 11 primers when the gene expression of the vasculature (aorta) is settled as 1.000.</p

Characteristics and laboratory data of diabetic <i>Apoe</i>^−/− mice.

<p>Mean ± SEM. ^a<i>P</i><0.05, ^b<i>P</i><0.01 vs. control. NEFA  =  non esterified fatty acids, NA  =  not assayed.</p

GIPR gene expression.

<p>GIPR mRNA levels in the pancreas and the exudate peritoneal macrophages obtained from <i>Apoe</i>^−/− mice, and J774A1 mouse macrophages were determined by real-time RT-PCR with 11 primers encoding different exons of the GIPR shown in Supplementary data. The amplification products visualized by gel electrophoresis had the expected lengths (bp).</p

The atherosclerotic area and the macrophage infiltration area in the aortic root in 21-week-old diabetic <i>Apoe</i>^{−<b>/</b>−} mice treated without/with vildagliptin infused with incretin receptor blockers.

<p>The cross-sections of the aortic root were stained with Oil Red O. The area of macrophage infiltration was stained with MOMA-2.</p

Foam cell formation in exudate peritoneal macrophages.

<p>Exudate peritoneal cells were isolated from the treated nondiabetic <i>Apoe</i>^−/− mice (a) or diabetic <i>Apoe</i>^−/− mice (b) at 21 weeks of age, or <i>db/db</i> diabetic mice (c) at the age of 13 weeks, 4 days after an intraperitoneal injection of thioglycolate. Adherent macrophages were incubated for 18 hours with the RPMI-1640 medium containing 10 μg/ml oxLDL in the presence of 0.1 mmol/l [³H]oleate conjugated with bovine serum albumin. Cellular lipids were extracted and the radioactivity of the cholesterol [³H]oleate was determined by thin-layer chromatography. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p