SMAD2 ubiquitination through PY motif regulates skeletal muscle mass and fibrotic degeneration

Transforming growth factorβ (TGFβ) signaling regulates diverse aspects of vertebrate skeletal muscle tissue including differentiation, homeostasis, regeneration and pathogenic degeneration. Ubiquitination of SMAD2, an intracellular transducer of TGFβ signaling, is a well-studied negative feedback regulation of the signaling pathway in the field of cell biology, but it's relevance in skeletal muscle tissue has been elusive. In this study, to elucidate the in vivo role of SMAD2 ubiquitination, we generated Smad2dPY mutant mice in which a 15 bp sequence encoding the PY motif of SMAD2 protein is deleted from Smad2 gene. By removing this motif, the SMAD2 protein escapes from protein-protein interaction with NEDD4 family E3 ligases and thus is devoid of ubiquitination-dependent negative regulation. Smad2dPY mice showed no obvious abnormality in development, growth or fertility, indicating that SMAD2 ubiquitination through PY motif is dispensable for these processes. The skeletal muscle of Smad2dPY mice demonstrated reduced weight and myofiber size reduction at 12 months old. SMAD2 protein level was increased in the skeletal muscle of Smad2dPY mice while SMAD2 ubiquitination was reduced. Primary myoblasts of Smad2dPY mice displayed higher TGFβ responsiveness and suppressed terminal differentiation, which may explain the reduced muscle mass. The TGFβ responsiveness of the interstitial fibroblast population was also increased. Fibrotic tissue remodeling triggered by cardiotoxin injection was exacerbated in Smad2dPY mice. Altogether, our study identified SMAD2 ubiquitination through PY motif as an important regulatory mechanism operating in skeletal muscle tissue to maintain the TGFβ signaling pathway at the desired level in homeostasis and tissue remodeling.