Fig. 6

Overexpression of JUNB promotes TGFB2 signaling-induced EMT, cell invasion, and TGFB2 protein synthesis. A Prolonged exposure to TGFB2 ligand exerts less antiproliferative effects. The numbers of asynchronously growing UTA6-Control and UTA6-JUNB cells were quantified, in the absence of tetracycline and in response to increasing time of exogenous TGFB2 ligand treatment. Data are shown as means with SEM from 3 independent experiments using two-way ANOVA with Tukey’s multiple comparisons test (*p<0.05, **p<0.01). B Overexpression of JUNB promotes the expression of mesenchymal proteins in cells exposed to TGFB2 ligand. Immunoblots show the levels of JUNB, SMAD2, pSMAD2 (Ser465/467), and mesenchymal markers (SNAI1, ZEB1, Fibronectin, Integrin α5 and β2, and vimentin) in UTA6-Control and UTA6-JUNB cells asynchronously growing in the absence of tetracycline and treated with 8 ng/ml of recombinant TGFβ2 for the indicated times. HSP90 is used as a loading control. C JUNB promotes a mesenchymal-like phenotype upon treatment with TGFB2 ligand. Representative images of UTA6-Control and UTA6-JUNB cells stained with phalloidin after treatment with TGFB2 ligand for 72 h. Scale bars, 20 and 10 μm are indicated in the original and zoom images, respectively. Arrows indicate migratory cells with mesenchymal phenotype. D Overexpression of JUNB increased TGFB2 ligand-induced cell migration and invasion in Matrigel invasion assays. UTA6-Control and UTA6-JUNB cells that migrated through the membrane were stained with crystal violet, and representative fields were photographed (upper panel). Scale bars: 100 μm. Cell invasion was quantified by counting the number of cells passing through the membrane normalized to total cell number, from eleven random fields (lower panel). Data are shown as means with SEM from 3 technical replicates of 2 independent experiments. Statistical analyses were performed using two-way ANOVA with Tukey’s multiple comparisons test (*p<0.05, ****p<0.0001). E Overexpression of JUNB decreases TGFB2 mRNA levels in UTA6 cells exposed to exogenous TGFB2 ligand. Relative TGFB2 mRNA levels in UTA6-Control and UTA6-JUNB cells treated with 8 ng/ml of recombinant TGFB2 for the indicated times were analyzed by RT-qPCR. Data are shown as means with SEM from 3 independent experiments. Statistical analyses were performed using two-way ANOVA with Tukey’s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001). F Overexpression of JUNB in cells treated with TGFB2 ligand leads to an increase in endogenous TGFB2 protein. Protein abundance of JUNB, TGFB2, and TGFB2 monomers in asynchronously growing cells in the absence of tetracycline treated with 8 ng/ml of exogenous TGFB2 were analyzed by immunoblotting. HSP90 was used as a loading control. G Overexpression of JUNB induces TGFB2 mRNA association to polysome-enriched microsomal fraction, under stimulation with TGFB2 ligand. UTA6-Control and UTA6-JUNB cells treated or not with TGFB2 ligand for 48 h were fractionated as described in the “Materials and methods.” Relative TGFB2 mRNA steady-state levels were analyzed by RT-qPCR on total RNA isolated from the whole cell extract (Input) or the polysome-enriched microsomal fraction. Data are shown as means with SEM from 3 independent experiments. Statistical analyses were performed with two-way ANOVA using Tukey’s multiple comparisons test (*p<0.05, **p<0.01; ns: non-significant). H Proposed model of JUNB as a promoter of cell proliferation and cell invasion. In response to mitogenic factors, JUNB protein is expressed during G1/S to G2/M to regulate cell cycle progression in proliferating cells. After long exposure to exogenous TGFB2, which are conditions found in a number of solid tumors, JUNB expression is increased and promotes TGFB2 signaling and EMT by enhancing TGFB2 protein level. This largely occurs via increasing the association of TGFB2 mRNA to polysomes leading to increased endogenous TGFB2 production. This mechanism most probably creates a positive autocrine loop increasing TGFB signaling promoting EMT, cell migration, and invasion