Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases

Abstract

Myostatin is a transforming growth factor beta family member that acts as a negative regulator of skeletal muscle growth. Myostatin circulates in the blood of adult mice in a noncovalently held complex with other proteins, including its propeptide, which maintain the C-terminal dimer in a latent, inactive state. This latent form of myostatin can be activated in vitro by treatment with acid; however, the mechanisms by which latent myostatin is activated in vivo are unknown. Here, we show that members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteinases can cleave the myostatin propeptide in this complex and can thereby activate latent myostatin. Furthermore, we show that a mutant form of the propeptide resistant to cleavage by BMP-1/TLD proteinases can cause significant increases in muscle mass when injected into adult mice. These findings raise the possibility that members of the BMP-1/TLD family may be involved in activating latent myostatin in vivo and that molecules capable of inhibiting these proteinases may be effective agents for increasing muscle mass for both human therapeutic and agricultural applications.

Fig. 1.

Cleavage of the myostatin propeptide by the BMP-1/TLD family of proteinases. (A and B) Detection of a propeptide degradation product in CHO cell-conditioned media. Conditioned media prepared from CHO cells expressing the propeptide (A) or WT and mutant forms of propeptide/Fc fusion proteins (B) were analyzed by SDS/PAGE followed by Western blot analysis using antibodies directed against either the myostatin propeptide (A) or IgG (B). Note that mutation of D76 to A resulted in the absence of the degradation product. (C) Purification of WT and mutant propeptide/C-terminal dimer complexes. Protein complexes were purified as described (9) and analyzed by SDS/PAGE in the presence or absence of 2-mercaptoethanol followed by Western blot analysis, as indicated. Note that, like the WT propeptide, the D76A mutant propeptide purified in a complex with the C-terminal dimer. The propeptide degradation product did not copurify with the C-terminal dimer and was thus not part of the complex. Bands denoted by * indicate misfolded myostatin species, which were evident under nonreducing conditions. (D and E) Cleavage of the propeptide by BMP-1/TLD proteinases. WT and mutant complexes were incubated with purified proteinases (14) and subjected to SDS/PAGE followed by Western blot analysis using antibodies directed against the propeptide. Incubations were carried out with 1 μg of latent complex and 250 ng of proteinase for 16 h at 37°C, except that in D the samples were incubated with an additional 250 ng of BMP-1 for 4 h more. In E, lanes labeled “no enzyme” indicate samples incubated for 16 h at 37°C in the absence of enzyme. Note that all enzymes were capable of generating the cleavage product and that the D76A mutant protein was completely resistant to cleavage.

Fig. 2.

Activation of latent myostatin activity by BMP-1/TLD proteinases. (A) Activation of pGL3-(CAGA)₁₂-luciferase reporter gene activity by purified myostatin C-terminal dimer. (B) Activation of the myostatin propeptide/C-terminal dimer latent complex by heat treatment. (C and D) Activation of the myostatin (MSTN) propeptide/C-terminal dimer latent complex by BMP-1/TLD proteinases. A standard curve using purified myostatin C-terminal dimer was generated for each set of assays to quantify myostatin activity. The samples used for the reporter assays in C and D are the same samples shown in Fig. 1 D and E, respectively. In B-D, black bars represent WT, and pink bars represent D76A mutant complexes. Note that, although heat treatment activated both the WT and mutant complexes, each proteinase was capable of activating only the WT complex. *, P < 0.05; **, P < 0.01. RLU, relative light units.

Fig. 3.

Muscle growth induced by injection of the D76A mutant propeptide/Fc fusion protein into mice. (A) Inhibition of reporter gene activity by WT and mutant propeptide/Fc fusion proteins in vitro. A204 cells transfected with the reporter construct were incubated with 10 ng/ml purified myostatin (MSTN) C-terminal dimer and various concentrations of WT (black) or D76A mutant (pink) propeptide/Fc fusion protein. Note that the WT and mutant proteins were equally effective in blocking myostatin activity. (B) Increased muscle mass in mice injected with mutant propeptide/Fc fusion protein. Numbers indicate percent increase in muscle mass compared with PBS-injected animals. Actual numbers used for these calculations are shown in Table 1. Only D76A at 10 mg/kg and JA16 at 60 mg/kg gave statistically significant increases in muscle mass (see Table 1). Brown, pectoralis; red, triceps; blue, quadriceps; green, gastrocnemius; purple, tibialis.