Biological Characterization of Differential Function and Activation Between Closely-Related TGFβ Family Ligands

Abstract

Growth differentiation factor 11 (GDF11) and GDF8 (also known as myostatin/MSTN) are closely-related members of the transforming growth factor β (TGFβ) family of extracellular ligands. Due to the high sequence identity of their functional mature domains, the two ligands were long believed to serve similar or redundant roles in mammalian skeletal and cardiac muscle development and regulation. However, GDF11 has been shown to be more potent in activating the SMAD2/3 pathway than GDF8 in vitro, and in the myocardium in vivo, likely due to more efficient utilization of key signaling receptors. Crystal structure resolution of the GDF11:FS288 complex, apo-GDF11, and apo-GDF8 further revealed unique structural properties of the proteins, whereby substitution of GDF11 amino acid residues into GDF8 enhanced the activity of the resulting chimeric protein, compared to native GDF8. Building upon these previous investigations into the signaling activity and structural features of mature GDF11 and GDF8, we interrogated the potentially distinct endogenous functions of these ligands via genetic modifications that specifically target regions of their respective mature signaling domains. We have shown that key amino acids in the fingertip region of the GDF11 mature domain are specifically required for early-stage skeletal development. Substituting these amino acids with the corresponding residues from GDF8 results in unique prenatal axial skeletal transformations, without apparent perturbation of skeletal or cardiac muscle development or homeostasis. Additionally, we have characterized significant differences in skeletal muscle growth during early-stage development in chimeric mice with gain in potency of GDF8. These data support the notion that GDF11 and GDF8 are biologically distinct proteins during development in vivo. With this foundation, we have begun generation of new chimeric mice with genetic modifications targeting the GDF11 and GDF8 prodomains to determine whether molecular differences in their respective prodomains translate to differential functional outcomes or distinct pathway activation in vivo. Defining the roles of these ligands is crucial to the study of aging effects and to the potential development of therapeutics to reduce skeletal and cardiac muscle deterioration.