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

Abstract

Transforming growth factor (TGF)-b, 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-b pathway, it is unknown how noncanonical TGF-b pathways are negatively regulated. Here we demonstrate that the inhibitory Smad Smad6, but not Smad7, negatively regulates TGF-b1-induced activation of the TRAF6-TAK1-p38 MAPK/JNK pathway, a noncanonical TGF-b pathway. TGF-b1-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-b1-TRAF6-TAK1-p38 MAPK/JNK pathway. Therefore, our findings provide insight into the molecular mechanisms underlying negative regulation of noncanonical TGF-b pathways.

A Structural View of Negative Regulation of the Toll-like Receptor-Mediated Inflammatory Pathway

“…However, this residue is not on the interface of TRAF6-A20 that we obtained. This result is expected because C103A mutant of A20 was shown to still bind to TRAF6 (59). The reason for the blockage of TRAF2-Ubc13 interaction may be that the C103 residue is involved in the interface of TRAF2-A20 complex; alternatively, mutation of this residue disrupts the stability of A20 and allosterically modifies its interaction sites.…”

“…Because these mutations cause loss of the domains that are required for CYLD-TRAF6 or CYLD-p62 interactions, the truncated CYLD can no longer associate with these proteins and deubiquitinate TRAF6. Nonsense and frameshift mutations predominantly constitute the mutations of CYLD in tumors (59,69).…”

MicroRNA-125a and -b inhibit A20 and MAVS to promote inflammation and impair antiviral response in COPD

“…Here, we show that these are the result of reduced induction of A20, leading to uncontrolled activation of NF-κB and subsequent induction of inflammatory cytokines. A20 is a pleiotropic protein involved in various ubiquitin-dependent pathways, including NF-κB (16) and MAPK pathway (45), and has also been shown to negatively regulate type I IFN inductions (43,46,47). Surprisingly, inhibition or ectopic expression of A20 did not affect IFN-β production.…”

Paeoniflorin suppresses TGF-β mediated epithelial-mesenchymal transition in pulmonary fibrosis through a Smad-dependent pathway

“…Data indicate that TGF-β1-induced Smad7 recruits Smad ubiquitin regulatory factor (Smurf) proteins with HECT type E3 ubiquitin ligase activity to suppress the canonical TGF-β pathway via the ubiquitination-mediated proteasomal degradation of ALK5. In addition, the non-canonical TGF-β pathway was blocked by Smad 6 but not Smad 7 through the recruitment of deubiquitinase A20 to TRAF6 [44][45][46] . Based on these results, we analyzed the effect of paeoniflorin on the levels of Smad7 and ALK5 in A549 cells.…”

“…45, dissolved in DMSO to a final concentration lower than 0.1%) was purchased from Nanjing Zelang Medical Technology Co, Ltd (Nanjing, China); prednisone acetate was purchased from Zhejiang Xianju Pharmaceutical Co, Ltd (Taizhou, China); bleomycin hydrochloride (BLM) was purchased from Nippon Kayaku (Tokyo, Japan); RPMI-1640 and fetal bovine serum (FBS) were purchased from HyClone (Logan, USA); recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, USA); E-cadherin, Smad2/3, p-Smad2 and p-Smad3 antibodies were purchased from Cell Signaling Technology (Boston, MA, USA); α-SMA antibodies were purchased from Epitomics (Burlingame, CA, USA); FSP-1, Smad7, ALK5 and vimentin antibodies were purchased from Bioworld Technology, Inc (Minneapolis, USA); Akt, p-Akt; JNK, p-JNK, ERK, p-ERK, p38, p-p38 and GAPDH antibodies were purchased from Kangchen Biotech (Shanghai, China); type I collagen ELISA kits were purchased from Abcam (Cambridge, UK); iScript cDNA synthesis kits and SsoFast EvaGreen Supermix were purchased from BioRad (Hercules, USA); and TRIzol reagent was purchased from TransGen Biotech (Beijing, China). All other chemicals and reagents used were of analytical grade.…”