Smad6 inhibits non-canonical TGF-β1 signalling by recruiting the deubiquitinase A20 to TRAF6.

Transforming growth factor (TGF)-β, a pivotal cytokine involved in a variety of cellular functions, transmits signals through Smad-dependent canonical and Smad-independent noncanonical pathways. In contrast to the canonical TGF-β pathway, it is unknown how noncanonical TGF-β pathways are negatively regulated. Here we demonstrate that the inhibitory Smad Smad6, but not Smad7, negatively regulates TGF-β1-induced activation of the TRAF6-TAK1-p38 MAPK/JNK pathway, a noncanonical TGF-β pathway. TGF-β1-induced Smad6 abolishes K63-linked polyubiquitination of TRAF6 by recruiting the A20 deubiquitinating enzyme in AML-12 mouse liver cells and primary hepatocytes. In addition, the knockdown of Smad6 or A20 in an animal model or cell culture system maintains TAK1 and p38 MAPK/JNK phosphorylation and increases apoptosis, emphasizing the crucial role of the Smad6-A20 axis in negative regulation of the TGF-β1-TRAF6-TAK1-p38 MAPK/JNK pathway. Therefore, our findings provide insight into the molecular mechanisms underlying negative regulation of noncanonical TGF-β pathways.