<i>Traf6</i>-deficient primary cells show a higher sensitivity to TGF-β1.

<p><i>Traf6</i>-deficient primary cells show a higher sensitivity to TGF-β1.</p

TRAF6 mediates IL-1β or LPS-induced suppression of TGF-β1/Smad pathway.

<p>(<b>A</b>) HEK293 cells were treated with TGF-β1 (0.4 ng/ml) and/or IL-1β (2 ng/ml) as indicated. TGF-β-mediated Smad3 phosphorylation was demonstrated by anti-pSmad3 and total Smad3 antibodies. As a loading control, α-tubulin was used. (<b>B</b>) SBE-Luc assay was performed in HepG2 cells. These luciferase assays were normalized by the activities of co-transfected β-galactosidase. (<b>C</b>) TRAF6 or GFP was over-expressed in HEK293 cells by use of a lentiviral system. Cells were harvested after TGF-β1 addition for up to 6 hours followed by westernblotting to compare phospho-Smad2/3 levels. (<b>D</b>) TGF-β1 target genes, <i>CDKN2B</i>, <i>CDKN1A</i>, and <i>SMAD6</i>, were detected by quantitative RT-PCR using total RNA from vector-(GFP) or TRAF6-expressing HaCaT cells treated as indicated. Human <i>GAPDH</i> was used as a loading control. (<b>E</b>) FaO cells were infected with either control vector or Myc-TRAF6 on previous day and then treated with TGF-β alone or together with LPS up to 8 hours. Both floating and adherent cells were harvested to compare the induction of cleaved caspase-3. TGF-β-induced signal transduction was displayed by showing pSmad2 level. The results are representative of three independent experiments.</p

TRAF6 forms a complex with TβRIII in response to TGF-β1.

<p>(<b>A</b>) CAGA12-Luciferase assays were performed in HepG2 cells. The plasmids encoding HA-TβRIII, TRAF6, CAGA12-Luc, and Renilla-luc reporter gene were transfected as indicated and, on the next day, TGF-β1 (0.4 ng/ml) and/or IL-1β (20 ng/ml) was added for 16 hours. The obtained relative luciferase units(RLU) were normalized by renilla luciferase activities. (<b>B</b>) Using the control and TβRIII knock-down HaCaT cells, TGF-β1 (0.4 ng/ml) and/or IL-1β (20 ng/ml) were treated as shown for up to 3 hours. The level of total Smad3 and phospho-Smad3 protein was detected by immunoblotting. For the control of equal loading, β-actin was used. (<b>C</b>) HEK293 cells stably expressing HA-TβRIII were transfected with Myc-Traf6 plasmids and then treated with TGF-β1. Cells were harvested at various times and were subjected to immunoprecipitation with anti-HA antibody. Co-immunoprecipitated TRAF6 was detected with anti Myc antibody. (<b>D</b>) Complex formation ability between TβRIII and TRAF6 wild-type or the TRAF6 (C85A/H87A) E3 ligase mutant was compared after TGF-β stimulation for an hour. (<b>E</b>) According to the manufacturer's protocol, interaction was visualized by <i>in situ</i> proximity ligation assay (O-link) with proximity probes directed against TRAF6 and TβRIII using Alexa 555 labeling (red). Endogenous TRAF6 was co-localized with HA-TβRIII along the plasma membrane in the presence of TGF-β (red blobs). Bar = 2.5 µm. (<b>F</b>) Quantification of blobs per cell was carried out by semi-automated image analysis using the freeware software BlobFinder V 3.0. (<b>G</b>) HA-TβRIII-stably expressing 4T07 mouse mammary cancer cells were treated with TGF-β and LPS for one hour. Co-immunoprecipitation was carried out using anti-HA antibody to query interaction with endogenous TRAF6. The results are representative of three independent experiments.</p

Association of TRAF6/TβRIII is regulated by functional TβRII and TβRI.

<p>(<b>A</b>) Myc-TRAF6, HA-TβRIII, and/or Flag-TβRII were expressed in SNU638 human gastric cancer cells and treated with TGF-β1 for 60 min. Immunoprecipitation and immunoblotting were carried out to elucidate the requirement of TβRII for TRAF6/TβRIII interaction. (<b>B</b>) TRAF6 and TβRIII interaction in TβRI−/− MEFs in the presence or absence of exogenous TβRI expression. (<b>C</b>) TRAF6/TβRIII interaction in the presence of wild-type, kinase inactive (KR: K232R), and constitutively active (TD: T204D) TβRI in HEK293 cells. (<b>D</b>) Interaction between TRAF6 and TβRIII in the presence of IN-1130 (0.1 µM), a specific inhibitor of TβRI kinase. (<b>E</b>) Olink assay for TRAF6 and TβRIII interaction in the presence of TGF-β and/or IN-1130. Transfected cells were counterstained with Alexa 488-labeled HA antibody to HA-TβRIII (green), and the nuclei were stained with Hoechst 33342 (blue). Bar = 2.5 µm. (<b>F</b>) The number of <i>in situ</i> PLA signals (red blobs) per cell was counted by semi-automated image analysis. (<b>G</b>) Co-immunoprecipitation analysis was conducted to elucidate the function of TβRI in TRAF6/TβRIII interaction. TβRI mutants KR, TD, and TD (E161A) mutated in TRAF6 consensus binding motif in TβRI (TD) were used. The results are representative of three independent experiments.</p

TRAF6 enhances dissociation of TβRIII from TβRII/TβRI complex.

<p>(<b>A</b>) The interaction between TβRIII and TβRII was enhanced by TGF-β1, and interfered by TRAF6 expression in HEK293 cells. (<b>B</b>) Over-expression of TRAF6 in HEK293 HA-TβRIII-stable cells interfere the interaction of Flag-TβRII and HA-TβRIII in a dose-dependent manner. (<b>C</b>) HEK293 HA-TβRIII-stable cells were treated as indicated, and were subjected to immunoprecipitation. HA-TβRIII interacted with endogenous TβRII upon TGF-β1 treatment. The level of endogenous TβRII bound to HA-TβRIII was markedly decreased in TGF-β1/IL-1β co-treated cell, while the level of endogenous TRAF6 bound to HA-TβRIII was increased s. These results are representative of three independent experiments. (<b>D</b>) A proposed model of IL-1β/LPS-induced suppression of the TGF-β pathway.</p