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.