TGF-β1 decreases RECK levels through a Smad and JNK dependent pathway.

(A) NIH3T3 fibroblasts were pre-treated for 30 minutes with different inhibitors: TGF-β-RI kinase inhibitor SB525334, PI3K inhibitor LY294002, MEK1 inhibitor PD98059, JNK inhibitor SB600125, and p38 inhibitor SB203580. After the pre-treatment, fibroblasts were treated with 5ng/ml of TGF-β1 for 24 hours or untreated to serve as controls. Western blot analysis of cell extracts were performed to determine the levels of RECK and FN. tubulin levels were used as a loading control. (B) NIH3T3 fibroblasts were transiently transfected with a pool of siRNA against Smad-2 and Smad-3 or with a scrambled siRNA as a control. 24 h post-transfection, cells were treated with 5 ng/ml of TGF-β1 for 24 h, or left untreated to serve as a control. Western blot analysis of cell extracts were performed to determine the levels of RECK, Smad-3 and β1-Integrin. β-tubulin levels were used as a loading control. (C) NIH3T3 fibroblasts were pre-treated for 30 minutes with SIS3, a specific inhibitor of Smad-3 activation, and the JNK inhibitor SB600125; cells were treated either alone or in combination. Afterwards, fibroblasts were treated with 5 ng/ml of TGF-β1, or left untreated as a control, for 24h. Western blot analysis of cell extracts were performed to determine the levels of RECK and β1-Integrin. β-tubulin levels were used as a loading control. The quantifications shown in A, B and C are from two independent. Statistical significance was assessed using two-way ANOVA and a Bonferroni multiple-comparison post hoc test. &, P<0.05 relative to TGF-β1 untreated fibroblasts.